Samuel Leung

PURPOSE To improve on current standards for breast cancer prognosis and prediction of chemotherapy benefit by developing a risk model that incorporates the gene expression-based "intrinsic" subtypes luminal A, luminal B, HER2-enriched, and basal-like. METHODS A 50-gene subtype predictor was developed using microarray and quantitative reverse transcriptase polymerase chain reaction data from 189 prototype samples. Test sets from 761 patients (no systemic therapy) were evaluated for prognosis, and 133 patients were evaluated for prediction of pathologic complete response (pCR) to a taxane and anthracycline regimen.The intrinsic subtypes as discrete entities showed prognostic significance (P = 2.26E-12) and remained significant in multivariable analyses that incorporated standard parameters (estrogen receptor status, histologic grade, tumor size, and node status). A prognostic model for node-negative breast cancer was built using intrinsic subtype and clinical information. The C-index estimate for the combined model (subtype and tumor size) was a significant improvement on either the clinicopathologic model or subtype model alone. The intrinsic subtype model predicted neoadjuvant chemotherapy efficacy with a negative predictive value for pCR of 97%. CONCLUSION Diagnosis by intrinsic subtype adds significant prognostic and predictive information to standard parameters for patients with breast cancer. The prognostic properties of the continuous risk score will be of value for the management of node-negative breast cancers. The subtypes and risk score can also be used to assess the likelihood of efficacy from neoadjuvant chemotherapy.

Gene expression profiling of breast cancer has identified two biologically distinct estrogen receptor (ER)-positive subtypes of breast cancer: luminal A and luminal B. Luminal B tumors have higher proliferation and poorer prognosis than luminal A tumors. In this study, we developed a clinically practical immunohistochemistry assay to distinguish luminal B from luminal A tumors and investigated its ability to separate tumors according to breast cancer recurrence-free and disease-specific survival.Tumors from a cohort of 357 patients with invasive breast carcinomas were subtyped by gene expression profile. Hormone receptor status, HER2 status, and the Ki67 index (percentage of Ki67-positive cancer nuclei) were determined immunohistochemically. Receiver operating characteristic curves were used to determine the Ki67 cut point to distinguish luminal B from luminal A tumors. The prognostic value of the immunohistochemical assignment for breast cancer recurrence-free and disease-specific survival was investigated with an independent tissue microarray series of 4046 breast cancers by use of Kaplan-Meier curves and multivariable Cox regression.Gene expression profiling classified 101 (28%) of the 357 tumors as luminal A and 69 (19%) as luminal B. The best Ki67 index cut point to distinguish luminal B from luminal A tumors was 13.25%. In an independent cohort of 4046 patients with breast cancer, 2847 had hormone receptor-positive tumors. When HER2 immunohistochemistry and the Ki67 index were used to subtype these 2847 tumors, we classified 1530 (59%, 95% confidence interval [CI] = 57% to 61%) as luminal A, 846 (33%, 95% CI = 31% to 34%) as luminal B, and 222 (9%, 95% CI = 7% to 10%) as luminal-HER2 positive. Luminal B and luminal-HER2-positive breast cancers were statistically significantly associated with poor breast cancer recurrence-free and disease-specific survival in all adjuvant systemic treatment categories. Of particular relevance are women who received tamoxifen as their sole adjuvant systemic therapy, among whom the 10-year breast cancer-specific survival was 79% (95% CI = 76% to 83%) for luminal A, 64% (95% CI = 59% to 70%) for luminal B, and 57% (95% CI = 47% to 69%) for luminal-HER2 subtypes.Expression of ER, progesterone receptor, and HER2 proteins and the Ki67 index appear to distinguish luminal A from luminal B breast cancer subtypes.

Abstract Purpose: Basal-like breast cancer is associated with high grade, poor prognosis, and younger patient age. Clinically, a triple-negative phenotype definition [estrogen receptor, progesterone receptor, and human epidermal growth factor receptor (HER)-2, all negative] is commonly used to identify such cases. EGFR and cytokeratin 5/6 are readily available positive markers of basal-like breast cancer applicable to standard pathology specimens. This study directly compares the prognostic significance between three- and five-biomarker surrogate panels to define intrinsic breast cancer subtypes, using a large clinically annotated series of breast tumors. Experimental Design: Four thousand forty-six invasive breast cancers were assembled into tissue microarrays. All had staging, pathology, treatment, and outcome information; median follow-up was 12.5 years. Cox regression analyses and likelihood ratio tests compared the prognostic significance for breast cancer death-specific survival (BCSS) of the two immunohistochemical panels. Results: Among 3,744 interpretable cases, 17% were basal using the triple-negative definition (10-year BCSS, 6 7%) and 9% were basal using the five-marker method (10-year BCSS, 62%). Likelihood ratio tests of multivariable Cox models including standard clinical variables show that the five-marker panel is significantly more prognostic than the three-marker panel. The poor prognosis of triple-negative phenotype is conferred almost entirely by those tumors positive for basal markers. Among triple-negative patients treated with adjuvant anthracycline-based chemotherapy, the additional positive basal markers identified a cohort of patients with significantly worse outcome. Conclusions: The expanded surrogate immunopanel of estrogen receptor, progesterone receptor, human HER-2, EGFR, and cytokeratin 5/6 provides a more specific definition of basal-like breast cancer that better predicts breast cancer survival.

Although it has long been appreciated that ovarian carcinoma subtypes (serous, clear cell, endometrioid, and mucinous) are associated with different natural histories, most ovarian carcinoma biomarker studies and current treatment protocols for women with this disease are not subtype specific. With the emergence of high-throughput molecular techniques, distinct pathogenetic pathways have been identified in these subtypes. We examined variation in biomarker expression rates between subtypes, and how this influences correlations between biomarker expression and stage at diagnosis or prognosis.In this retrospective study we assessed the protein expression of 21 candidate tissue-based biomarkers (CA125, CRABP-II, EpCam, ER, F-Spondin, HE4, IGF2, K-Cadherin, Ki-67, KISS1, Matriptase, Mesothelin, MIF, MMP7, p21, p53, PAX8, PR, SLPI, TROP2, WT1) in a population-based cohort of 500 ovarian carcinomas that was collected over the period from 1984 to 2000. The expression of 20 of the 21 biomarkers differs significantly between subtypes, but does not vary across stage within each subtype. Survival analyses show that nine of the 21 biomarkers are prognostic indicators in the entire cohort but when analyzed by subtype only three remain prognostic indicators in the high-grade serous and none in the clear cell subtype. For example, tumor proliferation, as assessed by Ki-67 staining, varies markedly between different subtypes and is an unfavourable prognostic marker in the entire cohort (risk ratio [RR] 1.7, 95% confidence interval [CI] 1.2%-2.4%) but is not of prognostic significance within any subtype. Prognostic associations can even show an inverse correlation within the entire cohort, when compared to a specific subtype. For example, WT1 is more frequently expressed in high-grade serous carcinomas, an aggressive subtype, and is an unfavourable prognostic marker within the entire cohort of ovarian carcinomas (RR 1.7, 95% CI 1.2%-2.3%), but is a favourable prognostic marker within the high-grade serous subtype (RR 0.5, 95% CI 0.3%-0.8%).The association of biomarker expression with survival varies substantially between subtypes, and can easily be overlooked in whole cohort analyses. To avoid this effect, each subtype within a cohort should be analyzed discretely. Ovarian carcinoma subtypes are different diseases, and these differences should be reflected in clinical research study design and ultimately in the management of ovarian carcinoma.

Abstract Purpose: To compare clinical, immunohistochemical (IHC), and gene expression models of prognosis applicable to formalin-fixed, paraffin-embedded blocks in a large series of estrogen receptor (ER)–positive breast cancers from patients uniformly treated with adjuvant tamoxifen. Experimental Design: Quantitative real-time reverse transcription-PCR (qRT-PCR) assays for 50 genes identifying intrinsic breast cancer subtypes were completed on 786 specimens linked to clinical (median follow-up, 11.7 years) and IHC [ER, progesterone receptor (PR), HER2, and Ki67] data. Performance of predefined intrinsic subtype and risk-of-relapse scores was assessed using multivariable Cox models and Kaplan-Meier analysis. Harrell's C-index was used to compare fixed models trained in independent data sets, including proliferation signatures. Results: Despite clinical ER positivity, 10% of cases were assigned to nonluminal subtypes. qRT-PCR signatures for proliferation genes gave more prognostic information than clinical assays for hormone receptors or Ki67. In Cox models incorporating standard prognostic variables, hazard ratios for breast cancer disease-specific survival over the first 5 years of follow-up, relative to the most common luminal A subtype, are 1.99 [95% confidence interval (CI), 1.09-3.64] for luminal B, 3.65 (95% CI, 1.64-8.16) for HER2-enriched subtype, and 17.71 (95% CI, 1.71-183.33) for the basal-like subtype. For node-negative disease, PAM50 qRT-PCR–based risk assignment weighted for tumor size and proliferation identifies a group with >95% 10-year survival without chemotherapy. In node-positive disease, PAM50-based prognostic models were also superior. Conclusion: The PAM50 gene expression test for intrinsic biological subtype can be applied to large series of formalin-fixed, paraffin-embedded breast cancers, and gives more prognostic information than clinical factors and IHC using standard cut points. Clin Cancer Res; 16(21); 5222–32. ©2010 AACR.

Carbonic anhydrase IX (CAIX) is a hypoxia and HIF-1-inducible protein that regulates intra- and extracellular pH under hypoxic conditions and promotes tumor cell survival and invasion in hypoxic microenvironments. Interrogation of 3,630 human breast cancers provided definitive evidence of CAIX as an independent poor prognostic biomarker for distant metastases and survival. shRNA-mediated depletion of CAIX expression in 4T1 mouse metastatic breast cancer cells capable of inducing CAIX in hypoxia resulted in regression of orthotopic mammary tumors and inhibition of spontaneous lung metastasis formation. Stable depletion of CAIX in MDA-MB-231 human breast cancer xenografts also resulted in attenuation of primary tumor growth. CAIX depletion in the 4T1 cells led to caspase-independent cell death and reversal of extracellular acidosis under hypoxic conditions in vitro. Treatment of mice harboring CAIX-positive 4T1 mammary tumors with novel CAIX-specific small molecule inhibitors that mimicked the effects of CAIX depletion in vitro resulted in significant inhibition of tumor growth and metastasis formation in both spontaneous and experimental models of metastasis, without inhibitory effects on CAIX-negative tumors. Similar inhibitory effects on primary tumor growth were observed in mice harboring orthotopic tumors comprised of lung metatstatic MDA-MB-231 LM2-4(Luc+) cells. Our findings show that CAIX is vital for growth and metastasis of hypoxic breast tumors and is a specific, targetable biomarker for breast cancer metastasis.

Automated quantification of thousands of morphologic features in microscopic images of breast cancer allows the construction of a robust prognostic model.

Classification of endometrial carcinomas (ECs) by morphologic features is inconsistent, and yields limited prognostic and predictive information. A new system for classification based on the molecular categories identified in The Cancer Genome Atlas is proposed. Genomic data from the Cancer Genome Atlas (TCGA) support classification of endometrial carcinomas into four prognostically significant subgroups; we used the TCGA data set to develop surrogate assays that could replicate the TCGA classification, but without the need for the labor-intensive and cost-prohibitive genomic methodology. Combinations of the most relevant assays were carried forward and tested on a new independent cohort of 152 endometrial carcinoma cases, and molecular vs clinical risk group stratification was compared. Replication of TCGA survival curves was achieved with statistical significance using multiple different molecular classification models (16 total tested). Internal validation supported carrying forward a classifier based on the following components: mismatch repair protein immunohistochemistry, POLE mutational analysis and p53 immunohistochemistry as a surrogate for ‘copy-number’ status. The proposed molecular classifier was associated with clinical outcomes, as was stage, grade, lymph-vascular space invasion, nodal involvement and adjuvant treatment. In multivariable analysis both molecular classification and clinical risk groups were associated with outcomes, but differed greatly in composition of cases within each category, with half of POLE and mismatch repair loss subgroups residing within the clinically defined ‘high-risk’ group. Combining the molecular classifier with clinicopathologic features or risk groups provided the highest C-index for discrimination of outcome survival curves. Molecular classification of ECs can be achieved using clinically applicable methods on formalin-fixed paraffin-embedded samples, and provides independent prognostic information beyond established risk factors. This pragmatic molecular classification tool has potential to be used routinely in guiding treatment for individuals with endometrial carcinoma and in stratifying cases in future clinical trials.

BACKGROUND Classification of endometrial carcinomas (ECs) by morphologic features is irreproducible and imperfectly reflects tumor biology. The authors developed the Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE), a molecular classification system based on The Cancer Genome Atlas genomic subgroups, and sought to confirm both feasibility and prognostic ability in a new, large cohort of ECs. METHODS Immunohistochemistry (IHC) for the presence or absence of mismatch repair (MMR) proteins (to identify MMR deficiency [MMR‐D]), sequencing for polymerase‐ɛ ( POLE ) exonuclease domain mutations ( POLE EDMs), and IHC for tumor protein 53 (p53) (wild type vs null/missense mutations; p53 wt and p53 abn, respectively) were performed on 319 new EC samples. Subgroups were characterized and assessed relative to outcomes. The prognostic ability of ProMisE was compared with that of current risk‐stratification systems (European Society of Medical Oncology [ESMO]). RESULTS ProMisE decision‐tree classification achieved categorization of all cases and identified 4 prognostic subgroups with distinct overall, disease‐specific, and progression‐free survival ( P < .001). Tumors with POLE EDMs had the most favorable prognosis, and those with p53 abn the worst prognosis, and separation of the 2 middle survival curves (p53 wt and MMR‐D) was observed. There were no significant differences in survival between the ESMO low‐risk and intermediate‐risk groups. ProMisE improved the ability to discriminate outcomes compared with ESMO risk stratification. There was substantial overlap (89%) between the p53 abn and high‐risk ESMO subgroups; but, otherwise, there were no predictable associations between molecular and ESMO risk groups. CONCLUSIONS Molecular classification of ECs can be achieved using clinically applicable methods and provides independent prognostic information beyond established clinicopathologic risk factors available at diagnosis. Consistent, biologically relevant categorization enables stratification for clinical trials and/or targeted therapy, identification of women who are at increased risk of having Lynch syndrome, and may guide clinical management. Cancer 2017;123:802–13. © 2016 American Cancer Society .

In breast cancer, immunohistochemical assessment of proliferation using the marker Ki67 has potential use in both research and clinical management. However, lack of consistency across laboratories has limited Ki67's value. A working group was assembled to devise a strategy to harmonize Ki67 analysis and increase scoring concordance. Toward that goal, we conducted a Ki67 reproducibility study.Eight laboratories received 100 breast cancer cases arranged into 1-mm core tissue microarrays-one set stained by the participating laboratory and one set stained by the central laboratory, both using antibody MIB-1. Each laboratory scored Ki67 as percentage of positively stained invasive tumor cells using its own method. Six laboratories repeated scoring of 50 locally stained cases on 3 different days. Sources of variation were analyzed using random effects models with log2-transformed measurements. Reproducibility was quantified by intraclass correlation coefficient (ICC), and the approximate two-sided 95% confidence intervals (CIs) for the true intraclass correlation coefficients in these experiments were provided.Intralaboratory reproducibility was high (ICC = 0.94; 95% CI = 0.93 to 0.97). Interlaboratory reproducibility was only moderate (central staining: ICC = 0.71, 95% CI = 0.47 to 0.78; local staining: ICC = 0.59, 95% CI = 0.37 to 0.68). Geometric mean of Ki67 values for each laboratory across the 100 cases ranged 7.1% to 23.9% with central staining and 6.1% to 30.1% with local staining. Factors contributing to interlaboratory discordance included tumor region selection, counting method, and subjective assessment of staining positivity. Formal counting methods gave more consistent results than visual estimation.Substantial variability in Ki67 scoring was observed among some of the world's most experienced laboratories. Ki67 values and cutoffs for clinical decision-making cannot be transferred between laboratories without standardizing scoring methodology because analytical validity is limited.

Metabolic reprogramming is a hallmark of cellular transformation, yet little is known about metabolic changes that accompany tumor metastasis. Here we show that primary breast cancer cells display extensive metabolic heterogeneity and engage distinct metabolic programs depending on their site of metastasis. Liver-metastatic breast cancer cells exhibit a unique metabolic program compared to bone- or lung-metastatic cells, characterized by increased conversion of glucose-derived pyruvate into lactate and a concomitant reduction in mitochondrial metabolism. Liver-metastatic cells displayed increased HIF-1α activity and expression of the HIF-1α target Pyruvate dehydrogenase kinase-1 (PDK1). Silencing HIF-1α reversed the glycolytic phenotype of liver-metastatic cells, while PDK1 was specifically required for metabolic adaptation to nutrient limitation and hypoxia. Finally, we demonstrate that PDK1 is required for efficient liver metastasis, and its expression is elevated in liver metastases from breast cancer patients. Our data implicate PDK1 as a key regulator of metabolism and metastatic potential in breast cancer.

We have previously developed and confirmed a pragmatic molecular classifier for endometrial cancers; ProMisE (Proactive Molecular Risk Classifier for Endometrial Cancer). Inspired by the Cancer Genome Atlas, ProMisE identifies four prognostically distinct molecular subtypes and can be applied to diagnostic specimens (biopsy/curettings) enabling earlier informed decision-making. We have strictly adhered to the Institute of Medicine (IOM) guidelines for the development of genomic biomarkers, and herein present the final validation step of a locked-down classifier before clinical application.We assessed a retrospective cohort of women from the Tübingen University Women's Hospital treated for endometrial carcinoma between 2003 and 2013. Primary outcomes of overall, disease-specific, and progression-free survival were evaluated for clinical, pathological, and molecular features.Complete clinical and molecular data were evaluable from 452 women. Patient age ranged from 29 to 93 (median 65) years, and 87.8% cases were endometrioid histotype. Grade distribution included 282 (62.4%) G1, 75 (16.6%) G2, and 95 (21.0%) G3 tumors. 276 (61.1%) patients had stage IA disease, with the remaining stage IB [89 (19.7%)], stage II [26 (5.8%)], and stage III/IV [61 (13.5%)]. ProMisE molecular classification yielded 127 (28.1%) MMR-D, 42 (9.3%) POLE, 55 (12.2%) p53abn, and 228 (50.4%) p53wt. ProMisE was a prognostic marker for progression-free (P = 0.001) and disease-specific (P = 0.03) survival even after adjusting for known risk factors. Concordance between diagnostic and surgical specimens was highly favorable; accuracy 0.91, κ 0.88.We have developed, confirmed, and now validated a pragmatic molecular classification tool (ProMisE) that provides consistent categorization of tumors and identifies four distinct prognostic molecular subtypes. ProMisE can be applied to diagnostic samples and thus could be used to inform surgical procedure(s) and/or need for adjuvant therapy. Based on the IOM guidelines this classifier is now ready for clinical evaluation through prospective clinical trials.

Tumor infiltrating lymphocytes may indicate an immune response to cancer development, but their significance remains controversial in breast cancer. We conducted this study to assess CD8+ (cytotoxic T) lymphocyte infiltration in a large cohort of invasive early stage breast cancers, and to evaluate its prognostic effect in different breast cancer intrinsic subtypes.Immunohistochemistry for CD8 staining was performed on tissue microarrays from 3992 breast cancer patients. CD8+ tumor infiltrating lymphocytes were counted as intratumoral when in direct contact with tumor cells, and as stromal in adjacent locations. Kaplan-Meier functions and Cox proportional hazards regression models were applied to examine the associations between tumor infiltrating lymphocytes and breast cancer specific survival.Among 3403 cases for which immunohistochemical results were obtained, CD8+ tumor infiltrating lymphocytes were identified in an intratumoral pattern in 32% and stromal pattern in 61% of the cases. In the whole cohort, the presence of intratumoral tumor-infiltrating lymphocytes was significantly correlated with young age, high grade, estrogen receptor negativity, human epidermal growth factor receptor-2 positivity and core basal intrinsic subtype, and was associated with superior breast cancer specific survival. Multivariate analysis indicated that the favorable prognostic effect of CD8+ tumor infiltrating lymphocytes was significant only in the core basal intrinsic subgroup (Hazard ratio, HR = 0.35, 95% CI = 0.23-0.54). No association with improved survival was present in those triple negative breast cancers that lack expression of basal markers (HR = 0.99, 95% CI = 0.48-2.04) nor in the other intrinsic subtypes.CD8+ tumor infiltrating lymphocytes are an independent prognostic factor associated with better patient survival in basal-like breast cancer, but not in non-basal triple negative breast cancers nor in other intrinsic molecular subtypes.

The four intrinsic subtypes of breast cancer, defined by differential expression of 50 genes (PAM50), have been shown to be predictive of risk of recurrence and benefit of hormonal therapy and chemotherapy. Here we describe the development of Prosigna™, a PAM50-based subtype classifier and risk model on the NanoString nCounter Dx Analysis System intended for decentralized testing in clinical laboratories.514 formalin-fixed, paraffin-embedded (FFPE) breast cancer patient samples were used to train prototypical centroids for each of the intrinsic subtypes of breast cancer on the NanoString platform. Hierarchical cluster analysis of gene expression data was used to identify the prototypical centroids defined in previous PAM50 algorithm training exercises. 304 FFPE patient samples from a well annotated clinical cohort in the absence of adjuvant systemic therapy were then used to train a subtype-based risk model (i.e. Prosigna ROR score). 232 samples from a tamoxifen-treated patient cohort were used to verify the prognostic accuracy of the algorithm prior to initiating clinical validation studies.The gene expression profiles of each of the four Prosigna subtype centroids were consistent with those previously published using the PCR-based PAM50 method. Similar to previously published classifiers, tumor samples classified as Luminal A by Prosigna had the best prognosis compared to samples classified as one of the three higher-risk tumor subtypes. The Prosigna Risk of Recurrence (ROR) score model was verified to be significantly associated with prognosis as a continuous variable and to add significant information over both commonly available IHC markers and Adjuvant! Online.The results from the training and verification data sets show that the FDA-cleared and CE marked Prosigna test provides an accurate estimate of the risk of distant recurrence in hormone receptor positive breast cancer and is also capable of identifying a tumor's intrinsic subtype that is consistent with the previously published PCR-based PAM50 assay. Subsequent analytical and clinical validation studies confirm the clinical accuracy and technical precision of the Prosigna PAM50 assay in a decentralized setting.

Ki67 immunohistochemistry (IHC), commonly used as a proliferation marker in breast cancer, has limited value for treatment decisions due to questionable analytical validity. The International Ki67 in Breast Cancer Working Group (IKWG) consensus meeting, held in October 2019, assessed the current evidence for Ki67 IHC analytical validity and clinical utility in breast cancer, including the series of scoring studies the IKWG conducted on centrally stained tissues. Consensus observations and recommendations are: 1) as for estrogen receptor and HER2 testing, preanalytical handling considerations are critical; 2) a standardized visual scoring method has been established and is recommended for adoption; 3) participation in and evaluation of quality assurance and quality control programs is recommended to maintain analytical validity; and 4) the IKWG accepted that Ki67 IHC as a prognostic marker in breast cancer has clinical validity but concluded that clinical utility is evident only for prognosis estimation in anatomically favorable estrogen receptor-positive and HER2-negative patients to identify those who do not need adjuvant chemotherapy. In this T1-2, N0-1 patient group, the IKWG consensus is that Ki67 5% or less, or 30% or more, can be used to estimate prognosis. In conclusion, analytical validity of Ki67 IHC can be reached with careful attention to preanalytical issues and calibrated standardized visual scoring. Currently, clinical utility of Ki67 IHC in breast cancer care remains limited to prognosis assessment in stage I or II breast cancer. Further development of automated scoring might help to overcome some current limitations.

Gene expression profiling classifies breast cancer into intrinsic subtypes based on the biology of the underlying disease pathways. We have used material from a prospective randomized trial of tamoxifen versus placebo in premenopausal women with primary breast cancer (NCIC CTG MA.12) to evaluate the prognostic and predictive significance of intrinsic subtypes identified by both the PAM50 gene set and by immunohistochemistry.Total RNA from 398 of 672 (59%) patients was available for intrinsic subtyping with a quantitative reverse transcriptase PCR (qRT-PCR) 50-gene predictor (PAM50) for luminal A, luminal B, HER-2-enriched, and basal-like subtypes. A tissue microarray was also constructed from 492 of 672 (73%) of the study population to assess a panel of six immunohistochemical IHC antibodies to define the same intrinsic subtypes.Classification into intrinsic subtypes by the PAM50 assay was prognostic for both disease-free survival (DFS; P = 0.0003) and overall survival (OS; P = 0.0002), whereas classification by the IHC panel was not. Luminal subtype by PAM50 was predictive of tamoxifen benefit [DFS: HR, 0.52; 95% confidence interval (CI), 0.32-0.86 vs. HR, 0.80; 95% CI, 0.50-1.29 for nonluminal subtypes], although the interaction test was not significant (P = 0.24), whereas neither subtyping by central immunohistochemistry nor by local estrogen receptor (ER) or progesterone receptor (PR) status were predictive. Risk of relapse (ROR) modeling with the PAM50 assay produced a continuous risk score in both node-negative and node-positive disease.In the MA.12 study, intrinsic subtype classification by qRT-PCR with the PAM50 assay was superior to IHC profiling for both prognosis and prediction of benefit from adjuvant tamoxifen.

Although an important biomarker in breast cancer, Ki67 lacks scoring standardization, which has limited its clinical use. Our previous study found variability when laboratories used their own scoring methods on centrally stained tissue microarray slides. In this current study, 16 laboratories from eight countries calibrated to a specific Ki67 scoring method and then scored 50 centrally MIB-1 stained tissue microarray cases. Simple instructions prescribed scoring pattern and staining thresholds for determination of the percentage of stained tumor cells. To calibrate, laboratories scored 18 ‘training’ and ‘test’ web-based images. Software tracked object selection and scoring. Success for the calibration was prespecified as Root Mean Square Error of scores compared with reference <0.6 and Maximum Absolute Deviation from reference <1.0 (log2-transformed data). Prespecified success criteria for tissue microarray scoring required intraclass correlation significantly >0.70 but aiming for observed intraclass correlation ≥0.90. Laboratory performance showed non-significant but promising trends of improvement through the calibration exercise (mean Root Mean Square Error decreased from 0.6 to 0.4, Maximum Absolute Deviation from 1.6 to 0.9; paired t-test: P=0.07 for Root Mean Square Error, 0.06 for Maximum Absolute Deviation). For tissue microarray scoring, the intraclass correlation estimate was 0.94 (95% credible interval: 0.90–0.97), markedly and significantly >0.70, the prespecified minimum target for success. Some discrepancies persisted, including around clinically relevant cutoffs. After calibrating to a common scoring method via a web-based tool, laboratories can achieve high inter-laboratory reproducibility in Ki67 scoring on centrally stained tissue microarray slides. Although these data are potentially encouraging, suggesting that it may be possible to standardize scoring of Ki67 among pathology laboratories, clinically important discrepancies persist. Before this biomarker could be recommended for clinical use, future research will need to extend this approach to biopsies and whole sections, account for staining variability, and link to outcomes.

The infiltration of FOXP3+ regulatory T cells into invasive tumors has been reported to be associated with survival in a variety of cancers. The prognostic significance of FOXP3+ tumor-infiltrating lymphocytes (TILs) in breast cancer, however, remains controversial.FOXP3+ TILs were assessed by immunohistochemistry on tissue microarrays constructed from a well-defined cohort of 3,992 breast cancer patients linked to detailed demographic, biomarker, treatment and outcome data. Survival analyses were performed using the Kaplan-Meier function and Cox proportional hazards regression models to evaluate the association of FOXP3+ TILs with breast cancer-specific survival, stratified by intrinsic subtype and cytotoxic T-cell infiltration status (as defined by CD8 immunohistochemistry).The presence of high numbers of FOXP3+ TILs was significantly associated with young age, high grade, estrogen receptor (ER) negativity, concurrent CD8+ cytotoxic T-cell infiltration, and human epidermal growth factor receptor-2 positive (HER2+)/ER- and core basal subtypes. On multivariate survival analysis, a high level of FOXP3+ TILs was significantly associated with poor survival in ER+ breast cancers that lacked CD8+ T-cell infiltrates (hazard ratio (HR) = 1.30, 95% confidence interval (CI) = 1.02 to 1.66). However, in ER- breast cancers, FOXP3+ TILs were strongly associated with improved survival in the HER2+/ER- subgroup, particularly in those with co-existent CD8+ T-cell infiltrates (HR = 0.48, 95% CI = 0.23 to 0.98), for which the presence of high levels of FOXP3+ TILs was independent of standard clinical prognostic factors.FOXP3+ regulatory TILs are a poor prognostic indicator in ER+ breast cancer, but a favorable prognostic factor in the HER2+/ER- subtype. The prognostic value of FOXP3+ TILs in breast cancer differs depending on ER and HER2 expression status and CD8+ T-cell infiltration.

There are 5 major histotypes of ovarian carcinomas. Diagnostic typing criteria have evolved over time, and past cohorts may be misclassified by current standards. Our objective was to reclassify the recently assembled Canadian Ovarian Experimental Unified Resource and the Alberta Ovarian Tumor Type cohorts using immunohistochemical (IHC) biomarkers and to develop an IHC algorithm for ovarian carcinoma histotyping. A total of 1626 ovarian carcinoma samples from the Canadian Ovarian Experimental Unified Resource and the Alberta Ovarian Tumor Type were subjected to a reclassification by comparing the original with the predicted histotype. Histotype prediction was derived from a nominal logistic regression modeling using a previously reclassified cohort (N=784) with the binary input of 8 IHC markers. Cases with discordant original or predicted histotypes were subjected to arbitration. After reclassification, 1762 cases from all cohorts were subjected to prediction models (χ Automatic Interaction Detection, recursive partitioning, and nominal logistic regression) with a variable IHC marker input. The histologic type was confirmed in 1521/1626 (93.5%) cases of the Canadian Ovarian Experimental Unified Resource and the Alberta Ovarian Tumor Type cohorts. The highest misclassification occurred in the endometrioid type, where most of the changes involved reclassification from endometrioid to high-grade serous carcinoma, which was additionally supported by mutational data and outcome. Using the reclassified histotype as the endpoint, a 4-marker prediction model correctly classified 88%, a 6-marker 91%, and an 8-marker 93% of the 1762 cases. This study provides statistically validated, inexpensive IHC algorithms, which have versatile applications in research, clinical practice, and clinical trials.

Small cell carcinoma of the ovary, hypercalcaemic type (SCCOHT) is a lethal and sometimes familial ovarian tumour of young women and children. We and others recently discovered that over 90% of SCCOHTs harbour inactivating mutations in the chromatin remodelling gene SMARCA4 with concomitant loss of its encoded protein SMARCA4 (BRG1), one of two mutually exclusive ATPases of the SWI/SNF chromatin remodelling complex. To determine the specificity of SMARCA4 loss for SCCOHT, we examined the expression of SMARCA4 by immunohistochemistry in more than 3000 primary gynaecological tumours. Among ovarian tumours, it was only absent in clear cell carcinoma (15 of 360, 4%). In the uterus, it was absent in endometrial stromal sarcomas (4 of 52, 8%) and high-grade endometrioid carcinomas (2 of 338, 1%). Recent studies have shown that SMARCA2 (BRM), the other mutually exclusive ATPase of the SWI/SNF complex, is necessary for survival of tumour cells lacking SMARCA4. Therefore, we examined SMARCA2 expression and discovered that all SMARCA4-negative SCCOHTs also lacked SMARCA2 protein by IHC, including the SCCOHT cell lines BIN67 and SCCOHT1. Among ovarian tumours, the SMARCA4/SMARCA2 dual loss phenotype appears completely specific for SCCOHT. SMARCA2 loss was not due to mutation but rather from an absence of mRNA expression, which was restored by treatment with the histone deacetylase inhibitor trichostatin A. Re-expression of SMARCA4 or SMARCA2 inhibited the growth of BIN67 and SCCOHT1 cell lines. Our results indicate that SMARCA4 loss, either alone or with SMARCA2, is highly sensitive and specific for SCCOHT and that restoration of either SWI/SNF ATPase can inhibit the growth of SCCOHT cell lines.

Background A proportion of endometrial carcinomas (ECs) are associated with deficient DNA mismatch repair (MMR). These tumors are characterized by high levels of microsatellite instability (MSI). Identification of MSI is important in identifying women who should be tested for Lynch syndrome and identifying a phenotype that may have specific prognostic and predictive implications. Genomic characterization of ECs has shown that MSI tumors form a distinct subgroup. The two most common methodologies for MSI assessment have not been compared in EC. Methods Pentaplex mono and di-nucleotide PCR for MSI testing was compared to MMR IHC (presence/absence of MLH1, MSH2, MSH6, PMS2) in a cohort of patients with EC. Concordance, Kappa statistic, sensitivity, specificity, positive and negative predictive values were obtained on the cross-tabulation of results. Results Comparison of both MSI and MMR status was complete for 89 cases. Overall agreement between methods (concordance) was 93.3% (95% CI[85.9%–97.5%]). A one-sided test to determine whether the accuracy is better than the “no information rate,” which is taken to be the largest class percentage in the data, is significant (p < 0.00001). Unweighted Kappa was 0.84, along with the sensitivity (88.5%), specificity (95.2%), PPV (88.5%), and NPV (95.2%). The balanced accuracy (i.e. the average between sensitivity and specificity) was 92%. Discussion We show the equivalence of MSI testing and MMR IHC. We advocate the implementation of MMR IHC in future EC classification schemes, enabling stratification of cases for future clinical trials as well as assisting identification of Lynch syndrome, so that screening and risk reducing interventions can be undertaken.

Categorization and risk stratification of endometrial carcinomas is inadequate; histomorphologic assessment shows considerable interobserver variability, and risk of metastases and recurrence can only be derived after surgical staging. We have developed a Proactive Molecular Risk classification tool for Endometrial cancers (ProMisE) that identifies four distinct prognostic subgroups. Our objective was to assess whether molecular classification could be performed on diagnostic endometrial specimens obtained prior to surgical staging and its concordance with molecular classification performed on the subsequent hysterectomy specimen.Sequencing of tumors for exonuclease domain mutations (EDMs) in POLE and immunohistochemistry for mismatch repair (MMR) proteins and p53 were applied to both pre- and post-staging archival specimens from 60 individuals to identify four molecular subgroups: MMR-D, POLE EDM, p53 wild type, p53 abn (abnormal). Three gynecologic subspecialty pathologists assigned histotype and grade to a subset of samples. Concordance of molecular and clinicopathologic subgroup assignments were determined, comparing biopsy/curetting to hysterectomy specimens.Complete molecular and pathologic categorization was achieved in 57 cases. Concordance metrics for pre- vs. post-staging endometrial samples categorized by ProMisE were highly favorable; average per ProMisE class sensitivity(0.9), specificity(0.96), PPV(0.9), NPV(0.96) and kappa statistic 0.86(95%CI, 0.72-0.93), indicating excellent agreement. We observed the highest level of concordance for 'p53 abn' tumors, the group associated with the worst prognosis. In contrast, grade and histotype assignment from original pathology reports pre- vs. post-staging showed only moderate levels of agreement (kappa=0.55 and 0.44 respectively); even with subspecialty pathology review only moderate levels of agreement were observed.Molecular classification can be achieved on diagnostic endometrial samples and accurately predicts the molecular features in the final hysterectomy specimens, demonstrating concordance superior to grade and histotype. This biologically relevant information, available at initial diagnosis, has the potential to inform management (surgery, adjuvant therapy) from the earliest time point in cancer care.

Novel immune checkpoint blockade strategies are being evaluated in clinical trials and include targeting the lymphocyte activation gene 3 (LAG-3) checkpoint, alone or in combination with PD-1/PD-L1 blockade. We investigated LAG-3 expression and its prognostic value in a large series of breast cancer patients, and correlated LAG-3 expression with key biomarkers including PD-1 and PD-L1.LAG-3 expression was evaluated by immunohistochemistry on two tissue microarray series incorporating 4322 breast cancer primary excision specimens (N = 330 in the training and N= 3992 in the validation set) linked to detailed clinicopathologic, biomarker and long-term clinical outcome data. PD-1 and PD-L1 expressions were also evaluated by immunohistochemistry. Stromal or intra-epithelial tumor infiltrating lymphocytes (sTILs or iTILs) expressing LAG-3 or PD-1 were assessed by absolute count. PD-L1 expression was evaluated as the percentage of positive carcinoma cells per core. Kaplan-Meier curves and Cox proportional hazard models were used for survival analyses.After locking down interpretation cut-offs on the training set, LAG-3+ iTILs were found in 11% of cases in the validation set. In both sets, LAG-3+ iTILs were significantly associated with negative prognostic factors: young age, large tumor size, high proliferation, HER2E and basal-like breast cancer subtypes. In multivariate analyses, breast cancer patients with LAG-3+ iTILs had a significantly improved breast cancer-specific survival [hazard ratio (HR): 0.71, 95% CI 0.56-0.90], particularly among estrogen receptor-negative patients (HR: 0.50, 95% CI 0.36-0.69). Furthermore, we found that 53% of PD-L1+ and 61% of PD-1+ cases were also positive for LAG-3+ iTILs. Concurrent infiltration of LAG-3+ and CD8+ iTILs was significantly associated with increased breast cancer-specific survival (HR: 0.49, 95% CI 0.32-0.74).LAG-3+ iTILs are enriched in estrogen receptor-negative breast cancers and represent an independent favorable prognostic factor. In addition, a high proportion of PD-1/PD-L1+ tumors are co-infiltrated with LAG-3+ TILs, supporting potential immune checkpoint blockade combination strategies as a treatment option for breast cancer patients.

Abstract Purpose: The aim of this study was to confirm the prognostic significance of POLE exonuclease domain mutations (EDM) in endometrial carcinoma patients. In addition, the effect of treatment on POLE-mutated tumors was assessed. Experimental Design: A retrospective patient cohort of 496 endometrial carcinoma patients was identified for targeted sequencing of the POLE exonuclease domain, yielding 406 evaluable tumors. Univariable and multivariable analyses were performed to determine the effect of POLE mutation status on progression-free survival (PFS), disease-specific survival (DSS), and overall survival (OS). Combining results from eight studies in a meta-analysis, we computed pooled HR for PFS, DSS, and OS. Results: POLEEDMs were identified in 39 of 406 (9.6%) endometrial carcinomas. Women with POLE-mutated endometrial carcinomas were younger, with stage I (92%) tumors, grade 3 (62%), endometrioid histology (82%), and frequent (49%) lymphovascular invasion. In univariable analysis, POLE-mutated endometrial carcinomas had significantly improved outcomes compared with patients with no EDMs for PFS, DSS, and OS. In multivariable analysis, POLE EDMs were only significantly associated with improved PFS. The effect of adjuvant treatment on POLE-mutated cases could not be determined conclusively; however, both treated and untreated patients with POLE EDMs had good outcomes. Meta-analysis revealed an association between POLE EDMs and improved PFS and DSS with pooled HRs 0.34 [95% confidence interval (CI), 0.15–0.73] and 0.35 (95% CI, 0.13–0.92), respectively. Conclusions: POLE EDMs are prognostic markers associated with excellent outcomes for endometrial carcinoma patients. Further investigation is needed to conclusively determine if treatment is necessary for this group of women. Clin Cancer Res; 22(12); 2865–73. ©2016 AACR.

Rho/ROCK signaling and caveolin-1 (Cav1) are implicated in tumor cell migration and metastasis; however, the underlying molecular mechanisms remain poorly defined. Cav1 was found here to be an independent predictor of decreased survival in breast and rectal cancer and significantly associated with the presence of distant metastasis for colon cancer patients. Rho/ROCK signaling promotes tumor cell migration by regulating focal adhesion (FA) dynamics through tyrosine (Y14) phosphorylation of Cav1. Phosphorylated Cav1 is localized to protrusive domains of tumor cells and Cav1 tyrosine phosphorylation is dependent on Src kinase and Rho/ROCK signaling. Increased levels of phosphorylated Cav1 were associated with elevated GTP-RhoA levels in metastatic tumor cells of various tissue origins. Stable expression and knockdown studies of Cav1 in tumor cells showed that phosphorylated Cav1 expression stimulates Rho activation, stabilizes FAK association with FAs, and promotes cell migration and invasion in a ROCK-dependent and Src-dependent manner. Tyrosine-phosphorylated Cav1, therefore, functions as an effector of Rho/ROCK signaling in the regulation of FA turnover and, thereby, tumor cell migration and invasion. These studies define a feedback loop between Rho/ROCK, Src, and phosphorylated Cav1 in tumor cell protrusions, identifying a novel function for Cav1 in tumor metastasis that may contribute to the poor prognosis of some Cav1-expressing tumors.

The histologic subtype of non-small cell lung carcinoma is important in selecting appropriate chemotherapy for patients with advanced disease. As many of these patients are not operative candidates, they are treated medically after biopsy for diagnosis. Inherent limitations of small biopsy samples can make distinguishing poorly differentiated lung adenocarcinoma (ADC) from squamous cell carcinoma (SCC) difficult. The value of histochemical and immunohistochemical markers to help separate poorly differentiated ADC from SCC in resection specimens is well established; however, the optimal use of markers in small tissue samples has only recently been examined and the correlation of marker expression in small tissue samples with histologic subtype determined on resection specimens has not been well documented. We address this issue by examining the expression of 9 markers (p63, TTF1, CK5/6, CK7, 34βE12, Napsin A, mucicarmine, NTRK1, and NTRK2) on 200 cases of ADC and 225 cases of SCC in tissue microarray format to mimic small tissue specimens. The single best marker to separate ADC from SCC is p63 (for SCC: sensitivity 84%, specificity 85%). Logistic regression analysis identifies p63, TTF1, CK5/6, CK7, Napsin A, and mucicarmine as the optimal panel to separate ADC from SCC. Reduction of the panel to p63, TTF1, CK5/6, and CK7 is marginally less effective but may be the best compromise when tissue is limited. We present an algorithm for the stepwise application of p63, TTF1, CK5/6, CK7, Napsin A, and mucicarmine in situations in which separation of ADC from SCC in small specimens cannot be accomplished by morphology alone.

Endometrial stromal sarcoma (ESS) characterized by YWHAE-FAM22 genetic fusion is histologically higher grade and clinically more aggressive than ESS with JAZF1-SUZ12 or equivalent genetic rearrangements, hence it is clinically important to recognize this subset of ESS. To identify diagnostic immunomarkers for this biologically defined ESS subset, we compared gene expression profiles between YWHAE-FAM22 ESS and JAZF1-rearranged ESS. These studies showed consistent upregulation of cyclin D1 in YWHAE-FAM22 ESS compared with JAZF1-SUZ12 ESS. Immunohistochemically, the high-grade round cell component of all 12 YWHAE-FAM22 ESS demonstrated diffuse (≥70%) moderate to strong nuclear cyclin D1 staining, and this diffuse positivity was not seen in 34 ESSs with JAZF1 and equivalent genetic rearrangements or in 21 low-grade ESS with no demonstrable genetic rearrangements. In a series of 243 non-ESS pure uterine mesenchymal and mixed epithelial-mesenchymal tumors, only 2 of 8 undifferentiated endometrial sarcomas with nuclear uniformity and 1 of 80 uterine leiomyosarcomas demonstrate diffuse cyclin D1 immunoreactivity. Both cyclin D1-positive undifferentiated endometrial sarcomas showed diffuse strong CD10 staining, which is consistently absent in the high-grade round cell component of YWHAE-FAM22 ESS. The low-grade spindle cell component of YWHAE-FAM22 ESS showed a spatially heterogenous cyclin D1 staining pattern that was weaker and less diffuse overall. Our findings indicate that cyclin D1 is a sensitive and specific diagnostic immunomarker for YWHAE-FAM22 ESS. When evaluating high-grade uterine sarcomas, cyclin D1 can be included in the immunohistochemical panel as an indicator of YWHAE-FAM22 ESS.

Recent reports indicate that prostate cancers (CaP) frequently over-express the potential oncogenes, ERG or ETV1. Many cases have chromosomal rearrangements leading to the fusion of the 5' end of the androgen-regulated serine protease TMPRSS2 (21q22.2) to the 3' end of either ERG (21q22.3) or ETV1 (7p21.3). The consequence of these rearrangements is aberrant androgen receptor-driven expression of the potential oncogenes, ETV1 or ERG.To determine the frequency of rearrangements involving TMPRSS2, ERG, or ETV1 genes in CaP of varying Gleason grades through fluorescence in situ hybridisation (FISH) on CaP tissue microarrays (TMAs).Two independent assays, a TMPRSS2 break-apart assay and a three-colour gene fusion FISH assay were applied to TMAs. FISH positive cases were confirmed by reverse transcriptase (RT) PCR and DNA sequence analysis.A total of 106/196 (54.1%) cases were analysed by FISH. None of the five benign prostatic hyperplasia cases analysed exhibited these gene rearrangements. TMPRSS2:ERG fusion was found more frequently in moderate to poorly differentiated tumours (35/86, 40.7%) than in well differentiated tumours (1/15, 6.7%, p = 0.017). TMPRSS2:ETV1 gene fusions were not detected in any of the cases tested. TMPRSS2:ERG fusion product was verified by RT-PCR followed by DNA sequencing in 7/7 randomly selected positive cases analysed.This study indicates that TMPRSS2:ERG gene rearrangements in CaP may be used as a diagnostic tool to identify prognostically relevant sub-classifications of these cancers.

The existence of non-small cell lung carcinoma with neuroendocrine differentiation as a distinct entity and its relevance for prognostic and treatment purposes is controversial. This study assesses the frequency and biologic and prognostic significance of neuroendocrine (NE) expression of synaptophysin (SNP), chromogranin (Ch), and neural cell adhesion molecule (N-CAM) using tissue microarray (TMA) and immunohistochemistry. Six hundred nine nonsmall cell lung carcinomas (NSCLCs) were reviewed for subclassification. TMA blocks were made using duplicate 0.6-mm-diameter tissue cores and slides stained with SNP, Ch, and N-CAM. Immunoreactivity was considered if 1% or more of tumor cells were positive. Hematoxylin and eosin-stained sections were subclassified as: 243 adenocarcinoma (ACA), 272 squamous cell carcinoma (SCC), 35 large cell carcinoma, 32 non-small cell carcinoma NOS, and 6 other (carcinosarcoma, giant cell carcinoma). Positivity for either marker was identified in 13.6% of NSCLC (76/558). NSCLC showed reactivity for Ch in 0.4% of cases (2/524), for SNP in 7.5% of cases (39/521) and for N-CAM in 8.6% of cases (44/511), whereas only 0.2% of cases (1/517) showed coexpression of SNP and Ch and none of all 3 markers. The assessment of NE differentiation in NSCLC is unnecessary and expensive and is of no clinical or prognostic significance. SNP or N-CAM stains a small minority of NSCLC, whereas Ch immunoreactivity is less common. Positivity for any 2 NE markers is rare. SNP is more likely to be expressed in adenocarcinoma (P=0.01) and N-CAM in squamous-cell carcinoma (P=0.008). Otherwise there was no correlation between immunoreactivity and tumor morphology. Disease specific and overall survival is not influenced by NE differentiation and therefore non-small cell lung carcinoma with neuroendocrine differentiation should not be a subclass distinct from the other NSCLC.

Reproducible diagnosis of ovarian carcinoma cell types is critical for cell type-specific treatment. The purpose of this study was to test the reproducibility of cell type diagnosis across Canada. Analysis of the interobserver reproducibility of histologic tumor type was performed among 6 pathologists after brief training in the use of modified World Health Organization criteria to classify ovarian carcinomas into 1 of 6 categories: high-grade serous, endometrioid, clear cell, mucinous, low-grade serous, and other. These 6 pathologists independently reviewed a test set of 40 ovarian carcinomas. A validation set of 88 consecutive ovarian carcinomas drawn from 5 centers was subject to local review by 1 of the 6 study pathologists, and central review by a single observer. Interobserver agreement was assessed through calculation of concordance and kappa values for pair-wise comparison. For the test set, the paired concordance between pathologists in cell type diagnosis ranged from 85.0% to 97.5% (average 92.3%), and the kappa values were 0.80 to 0.97 (average 0.89). Inclusion of immunostaining results did not significantly improve reproducibility (P=0.69). For the validation set, the concordance between original diagnosis and local review was 84% and between local review and central review was 94%. The kappa values were 0.73 and 0.89, respectively. With a brief training exercise and the use of defined criteria for ovarian carcinoma subtyping, there is excellent interobserver reproducibility in diagnosis of cell type. This has implications for clinical trials of subtype-specific ovarian carcinoma treatments.

Background Axillary lymph node involvement remains the most important prognostic factor in early-stage breast cancer. We hypothesized that molecular classification based on breast cancer biology would predict the presence of nodal involvement at diagnosis, which might aid treatment decisions regarding the axilla. Patients and Methods From a clinically annotated tissue microarray of 4444 early-stage breast cancers, expression of estrogen receptor (ER), progesterone receptor (PgR), HER2, epidermal growth factor receptor, and cytokeratin 5/6 was determined by immunohistochemistry. Cases were classified by published criteria into molecular subtypes of luminal, luminal/HER2 positive, HER2 positive/ER negative/PgR negative, and basal. Risk of axillary nodal involvement at diagnosis was determined in 2 multivariable logistic regression models: a “core biopsy model” including molecular subtype, age, grade, and tumor size and a “lumpectomy model,” which also included lymphovascular invasion. Luminal was used as the reference group. After internal validation of findings in 2 independent sets, we conducted combined analysis of both. Results In the core biopsy model, the molecular subtypes had a predictive effect for nodal involvement (P = .000001), with the basal subtype having an odds ratio for axillary lymph node involvement of 0.53 (95% CI, 0.41-0.69). Tumor grade (P = 5.43 × 10−12) and size (P = 8.52 × 10−35) were also predictive for nodal involvement. Similar results were found in the lumpectomy model, where lymphovascular invasion was also predictive (P = 2.74 × 10−115). Conclusion These results indicate that the basal breast cancer molecular subtype predicts a lower incidence of axillary nodal involvement, and including biomarker profiles to predict nodal status at diagnosis could help stratification for decisions regarding axillary surgery and locoregional radiation.

Recent studies suggest that intrinsic breast cancer subtypes may differ in their responsiveness to specific chemotherapy regimens. We examined this hypothesis on NCIC.CTG MA.5, a clinical trial randomizing premenopausal women with node-positive breast cancer to adjuvant CMF (cyclophosphamide-methotrexate-fluorouracil) versus CEF (cyclophosphamide-epirubicin-fluorouracil) chemotherapy.Intrinsic subtype was determined for 476 tumors using the quantitative reverse transcriptase PCR PAM50 gene expression test. Luminal A, luminal B, HER2-enriched (HER2-E), and basal-like subtypes were correlated with relapse-free survival (RFS) and overall survival (OS), estimated using Kaplan-Meier plots and log-rank testing. Multivariable Cox regression analyses determined significance of interaction between treatment and intrinsic subtypes.Intrinsic subtypes were associated with RFS (P = 0.0005) and OS (P < 0.0001) on the combined cohort. The HER2-E showed the greatest benefit from CEF versus CMF, with absolute 5-year RFS and OS differences exceeding 20%, whereas there was a less than 2% difference for non-HER2-E tumors (interaction test P = 0.03 for RFS and 0.03 for OS). Within clinically defined Her2(+) tumors, 79% (72 of 91) were classified as the HER2-E subtype by gene expression and this subset was strongly associated with better response to CEF versus CMF (62% vs. 22%, P = 0.0006). There was no significant difference in benefit between CEF and CMF in basal-like tumors [n = 94; HR, 1.1; 95% confidence interval (CI), 0.6-2.1 for RFS and HR, 1.3; 95% CI, 0.7-2.5 for OS].HER2-E strongly predicted anthracycline sensitivity. The chemotherapy-sensitive basal-like tumors showed no added benefit for CEF over CMF, suggesting that nonanthracycline regimens may be adequate in this subtype although further investigation is required.

Abstract Pathological analysis of the nuclear proliferation biomarker Ki67 has multiple potential roles in breast and other cancers. However, clinical utility of the immunohistochemical (IHC) assay for Ki67 immunohistochemistry has been hampered by unacceptable between-laboratory analytical variability. The International Ki67 Working Group has conducted a series of studies aiming to decrease this variability and improve the evaluation of Ki67. This study tries to assess whether acceptable performance can be achieved on prestained core-cut biopsies using a standardized scoring method. Sections from 30 primary ER+ breast cancer core biopsies were centrally stained for Ki67 and circulated among 22 laboratories in 11 countries. Each laboratory scored Ki67 using three methods: (1) global (4 fields of 100 cells each); (2) weighted global (same as global but weighted by estimated percentages of total area); and (3) hot-spot (single field of 500 cells). The intraclass correlation coefficient (ICC), a measure of interlaboratory agreement, for the unweighted global method (0.87; 95% credible interval (CI): 0.81–0.93) met the prespecified success criterion for scoring reproducibility, whereas that for the weighted global (0.87; 95% CI: 0.7999–0.93) and hot-spot methods (0.84; 95% CI: 0.77–0.92) marginally failed to do so. The unweighted global assessment of Ki67 IHC analysis on core biopsies met the prespecified criterion of success for scoring reproducibility. A few cases still showed large scoring discrepancies. Establishment of external quality assessment schemes is likely to improve the agreement between laboratories further. Additional evaluations are needed to assess staining variability and clinical validity in appropriate cohorts of samples.

Tumors with high mutation load are thought to engender stronger immune responses, which in turn promote prolonged patient survival. To investigate this, we assessed tumor-infiltrating lymphocytes (TILs) and immunosuppressive factors across the 4 molecular subtypes of endometrial cancer, which have characteristic mutation rates ranging from low to ultra-high.A total of 460 endometrial cancers were stratified by ProMisE (Proactive Molecular Risk Classifier in Endometrial cancer) into 4 molecular subtypes: mismatch repair-deficient (MMRd), POLE mutant (POLE), p53 abnormal (p53abn), and p53 wild-type (p53wt). Immune markers (CD3, CD8, CD79a, CD138, PD-1, PD-L1, FoxP3, IDO-1) were quantified by multiplex IHC and tested for associations with ProMisE subtype, survival, and other clinicopathologic parameters.Two major TIL patterns were observed. TILhigh tumors harbored dense T- and B-lineage infiltrates and multiple immunosuppressive features and were common in molecular subtypes associated with high mutation load (MMRd and POLE); however, equally strong responses were seen in significant numbers of p53abn and p53wt tumors, which have characteristically low mutation loads. TILlow tumors were generally devoid of immunologic features and were more prevalent in p53abn and p53wt endometrial cancers, yet were also seen in MMRd and POLE subtypes. In multivariable models involving ProMisE subtype, T-cell markers, and TIL clusters, only ProMisE showed independent prognostic significance.Immune response correlates with endometrial cancer molecular subtype but does not carry independent prognostic significance. Profound variation in immune response is seen across and within endometrial cancer molecular subtypes, suggesting that assessment of immune response rather than molecular subtype may better predict response to immunotherapy.See related commentary by Mullen and Mutch, p. 2366.

Ki67 expression has been a valuable prognostic variable in breast cancer, but has not seen broad adoption due to lack of standardization between institutions. Automation could represent a solution. Here we investigate the reproducibility of Ki67 measurement between three image analysis platforms with supervised classifiers performed by the same operator, by multiple operators, and finally we compare their accuracy in prognostic potential. Two breast cancer patient cohorts were used for this study. The standardization was done with the 30 cases of ER+ breast cancer that were used in phase 3 of International Ki67 in Breast Cancer Working Group initiatives where blocks were centrally cut and stained for Ki67. The outcome cohort was from 149 breast cancer cases from the Yale Pathology archives. A tissue microarray was built from representative tissue blocks with median follow-up of 120 months. The Mib-1 antibody (Dako) was used to detect Ki67 (dilution 1:100). HALO (IndicaLab), QuantCenter (3DHistech), and QuPath (open source software) digital image analysis (DIA) platforms were used to evaluate Ki67 expression. Intraclass correlation coefficient (ICC) was used to measure reproducibility. Between-DIA platform reproducibility was excellent (ICC: 0.933, CI: 0.879-0.966). Excellent reproducibility was found between all DIA platforms and the reference standard Ki67 values of Spectrum Webscope (QuPath-Spectrum Webscope ICC: 0.970, CI: 0.936-0.986; HALO-Spectrum Webscope ICC: 0.968, CI: 0.933-0.985; QuantCenter-Spectrum Webscope ICC: 0.964, CI: 0.919-0.983). All platforms showed excellent intra-DIA reproducibility (QuPath ICC: 0.992, CI: 0.986-0.996; HALO ICC: 0.972, CI: 0.924-0.988; QuantCenter ICC: 0.978, CI: 0.932-0.991). Comparing each DIA against outcome, the hazard ratios were similar. The inter-operator reproducibility was particularly high (ICC: 0.962-0.995). Our results showed outstanding reproducibility both within and between-DIA platforms, including one freely available DIA platform (QuPath). We also found the platforms essentially indistinguishable with respect to prediction of breast cancer patient outcome. Results justify multi-institutional DIA studies to assess clinical utility.

Median overall survival (OS) for women with high-grade serous ovarian cancer (HGSOC) is ∼4 years, yet survival varies widely between patients. There are no well-established, gene expression signatures associated with prognosis. The aim of this study was to develop a robust prognostic signature for OS in patients with HGSOC.Expression of 513 genes, selected from a meta-analysis of 1455 tumours and other candidates, was measured using NanoString technology from formalin-fixed paraffin-embedded tumour tissue collected from 3769 women with HGSOC from multiple studies. Elastic net regularization for survival analysis was applied to develop a prognostic model for 5-year OS, trained on 2702 tumours from 15 studies and evaluated on an independent set of 1067 tumours from six studies.Expression levels of 276 genes were associated with OS (false discovery rate < 0.05) in covariate-adjusted single-gene analyses. The top five genes were TAP1, ZFHX4, CXCL9, FBN1 and PTGER3 (P < 0.001). The best performing prognostic signature included 101 genes enriched in pathways with treatment implications. Each gain of one standard deviation in the gene expression score conferred a greater than twofold increase in risk of death [hazard ratio (HR) 2.35, 95% confidence interval (CI) 2.02-2.71; P < 0.001]. Median survival [HR (95% CI)] by gene expression score quintile was 9.5 (8.3 to -), 5.4 (4.6-7.0), 3.8 (3.3-4.6), 3.2 (2.9-3.7) and 2.3 (2.1-2.6) years.The OTTA-SPOT (Ovarian Tumor Tissue Analysis consortium - Stratified Prognosis of Ovarian Tumours) gene expression signature may improve risk stratification in clinical trials by identifying patients who are least likely to achieve 5-year survival. The identified novel genes associated with the outcome may also yield opportunities for the development of targeted therapeutic approaches.

Abstract Here we report targeted sequencing of 83 genes using DNA from primary breast cancer samples from 625 postmenopausal (UBC-TAM series) and 328 premenopausal (MA12 trial) hormone receptor-positive (HR+) patients to determine interactions between somatic mutation and prognosis. Independent validation of prognostic interactions was achieved using data from the METABRIC study. Previously established associations between MAP3K1 and PIK3CA mutations with luminal A status/favorable prognosis and TP53 mutations with Luminal B/non-luminal tumors/poor prognosis were observed, validating the methodological approach. In UBC-TAM, NF1 frame-shift nonsense (FS/NS) mutations were also a poor outcome driver that was validated in METABRIC. For MA12, poor outcome associated with PIK3R1 mutation was also reproducible. DDR1 mutations were strongly associated with poor prognosis in UBC-TAM despite stringent false discovery correction ( q = 0.0003). In conclusion, uncommon recurrent somatic mutations should be further explored to create a more complete explanation of the highly variable outcomes that typifies ER+ breast cancer.

Aims Vulvar squamous cell carcinoma ( VSCC ) can be subdivided by human papillomavirus ( HPV ) status into two clinicopathological entities. Studies on the prognostic significance of HPV in VSCC are discordant. Methods and results We performed a retrospective analysis of overall survival ( OS ), disease‐specific survival ( DSS ) and progression‐free survival ( PFS ) in 217 patients with VSCC . Cases were extracted from an era of more aggressive en‐bloc radical dissections (1985–95) and more localized radical surgery through separate vulvar and groin excisions (1996–2005). p16 immunohistochemistry was used as a surrogate for HPV status. HPV status could be determined in 197 tumours, 118 HPV ‐independent and 79 HPV ‐associated tumours. Patients with HPV ‐associated tumours were younger (mean 58.8 versus 71.6 years for HPV ‐independent tumours, P &lt; 0.0001) and more likely to have prior abnormal cervical cytology (41.1 versus 5.6% for HPV ‐independent tumours, P &lt; 0.0001). In univariable analysis, patients with HPV ‐associated tumours had superior PFS [hazard ratio ( HR ): 0.37, 95% confidence interval ( CI ): 0.18–0.70], DSS ( HR : 0.19, 95% CI : 0.08–0.41) and OS ( HR : 0.35, 95% CI : 0.21–0.59). This was driven by worse outcomes ( PFS , DSS and OS ) for patients with HPV ‐independent tumours compared with HPV ‐associated tumours who underwent surgery after 1995. After adjusting for age and stage in multivariable analysis, patients with HPV ‐associated tumours showed superior PFS ( HR : 0.25, 95% CI : 0.07–0.77) and DSS ( HR : 0.21, 95% CI : 0.04–0.78). Conclusions VSCC can be stratified into two prognostically different diseases based on p16 immunostaining. HPV status was associated only with prognosis in the cohort that underwent surgery after 1995, suggesting that more conservative surgery may have led to worse outcomes for patients with HPV ‐independent tumours.

Molecular subclassification of endometrial carcinoma (EC) with Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE) identifies four subtypes [DNA polymerase epsilon (POLE) mutant, mismatch repair-deficient, p53 wild-type (wt), and p53 abnormal]. The aim of this study was to evaluate additional EC biomarkers in the context of these subtypes. Tissue microarrays encompassing 460 previously characterized ECs were assessed for L1-cell adhesion molecule (L1CAM), progesterone receptor (PR), estrogen receptor (ER) alpha, stathmin, and phosphatase and tensin homolog (PTEN), by immunohistochemistry (IHC). Associations with clinicopathological parameters, molecular subtype, and outcomes were determined. About 413 ECs (75% endometrioid, >15% serous) had complete data. L1CAM overexpression was found in 16%, associated with older age, lower body mass index (BMI), advanced stage, grade 3 (97%), non-endometrioid histology (84%), deep myometrial invasion, lymphovascular space invasion (LVSI), and ER-negative, PR-negative status. Tumours overexpressing L1CAM were associated with poor outcomes {hazard ratio (HR) [95% confidence interval (CI)] 3.35 [2.10-5.23] for disease-specific survival [DSS], p < 0.0001}. PR positivity was associated with younger women, higher BMI, early stage (77% stage I), low grade (61%), endometrioid histology (90%) without LVSI or nodal disease, ER positivity (90%), p53wt tumours (55%), and favourable outcomes [HR (CI) 0.39 (0.25-0.62) for DSS, p < 0.0001]. ER positive tumours were early stage (73%), low grade, endometrioid histology, with improved DSS. Stathmin and PTEN IHC were not associated with outcomes. There was minimal agreement between IHC and mutation status for PTEN. L1CAM overexpression was significantly associated with the p53 abnormal molecular subtype, which accounted for more than 70% of the tumours overexpressing L1CAM. PR expression also correlated with molecular subtype, with most PR negative tumours being p53 abnormal. Multivariable analysis demonstrated that only ProMisE subtype [overall survival (OS), DSS, and progression-free survival] and age (OS only) maintained an association with outcomes. The prognostic significance of the single biomarkers tested could be explained based on their being covariable with the ProMisE molecular subtype.

Aims The nuclear proliferation marker Ki67 assayed by immunohistochemistry has multiple potential uses in breast cancer, but an unacceptable level of interlaboratory variability has hampered its clinical utility. The International Ki67 in Breast Cancer Working Group has undertaken a systematic programme to determine whether Ki67 measurement can be analytically validated and standardised among laboratories. This study addresses whether acceptable scoring reproducibility can be achieved on excision whole sections. Methods and results Adjacent sections from 30 primary ER + breast cancers were centrally stained for Ki67 and sections were circulated among 23 pathologists in 12 countries. All pathologists scored Ki67 by two methods: (i) global: four fields of 100 tumour cells each were selected to reflect observed heterogeneity in nuclear staining; (ii) hot‐spot: the field with highest apparent Ki67 index was selected and up to 500 cells scored. The intraclass correlation coefficient (ICC) for the global method [confidence interval (CI) = 0.87; 95% CI = 0.799–0.93] marginally met the prespecified success criterion (lower 95% CI ≥ 0.8), while the ICC for the hot‐spot method (0.83; 95% CI = 0.74–0.90) did not. Visually, interobserver concordance in location of selected hot‐spots varies between cases. The median times for scoring were 9 and 6 min for global and hot‐spot methods, respectively. Conclusions The global scoring method demonstrates adequate reproducibility to warrant next steps towards evaluation for technical and clinical validity in appropriate cohorts of cases. The time taken for scoring by either method is practical using counting software we are making publicly available. Establishment of external quality assessment schemes is likely to improve the reproducibility between laboratories further.

Objective Our aim was to characterize the pathological, molecular and clinical outcomes of clear cell carcinoma of the endometrium (CCC). Methods CCC underwent ProMisE (Proactive Molecular Risk Classifier for Endometrial Cancer) classification identifying four molecular subtypes: i) ‘POLEmut’ for ECs with pathogenic POLE mutations, ii) ‘MMRd’, if there is loss of mismatch repair proteins by immunohistochemistry (IHC), iii) ‘p53wt’ or iv) ‘p53abn’ based on p53 IHC staining. Clinicopathologic parameters, immune markers (CD3, CD8, CD79a, CD138, PD-1), ER, L1CAM, and outcomes were assessed. Results 52 CCCs were classified, including 1 (2%) POLEmut, 5 (10%) MMRd, 28 (54%) p53wt and 18 (35%) p53abn. Women with p53abn and p53wt CCCs were older than women with MMRd and POLEmut subtypes. p53wt CCC were distinct from typical p53wt endometrioid carcinomas; more likely to arise in older, thinner women, with advanced stage disease, LVSI and lymph node involvement, and a higher proportion ER negative, L1CAM overexpressing tumors with markedly worse outcomes. High levels of immune infiltrates (TILhigh) were observed in 75% of the CCC cohort. L1CAM overexpression was highest within p53abn and p53wt subtypes of CCC. Conclusion CCC is a heterogeneous disease encompassing all four molecular subtypes and a wide range of clinical outcomes. Outcomes of patients with POLEmut, MMRd and p53abn CCC are not distinguishable from those of other patients with these respective subtypes of EC; p53wt CCC, however, differ from endometrioid p53wt EC in clinical, pathological, molecular features and outcomes. Thus, p53wt CCC of endometrium appear to be a distinct clinicopathological entity within the larger group of p53wt ECs.

The nuclear proliferation biomarker Ki67 has potential prognostic, predictive, and monitoring roles in breast cancer. Unacceptable between-laboratory variability has limited its clinical value. The International Ki67 in Breast Cancer Working Group investigated whether Ki67 immunohistochemistry can be analytically validated and standardized across laboratories using automated machine-based scoring. Sets of pre-stained core-cut biopsy sections of 30 breast tumors were circulated to 14 laboratories for scanning and automated assessment of the average and maximum percentage of tumor cells positive for Ki67. Seven unique scanners and 10 software platforms were involved in this study. Pre-specified analyses included evaluation of reproducibility between all laboratories (primary) as well as among those using scanners from a single vendor (secondary). The primary reproducibility metric was intraclass correlation coefficient between laboratories, with success considered to be intraclass correlation coefficient >0.80. Intraclass correlation coefficient for automated average scores across 16 operators was 0.83 (95% credible interval: 0.73-0.91) and intraclass correlation coefficient for maximum scores across 10 operators was 0.63 (95% credible interval: 0.44-0.80). For the laboratories using scanners from a single vendor (8 score sets), intraclass correlation coefficient for average automated scores was 0.89 (95% credible interval: 0.81-0.96), which was similar to the intraclass correlation coefficient of 0.87 (95% credible interval: 0.81-0.93) achieved using these same slides in a prior visual-reading reproducibility study. Automated machine assessment of average Ki67 has the potential to achieve between-laboratory reproducibility similar to that for a rigorously standardized pathologist-based visual assessment of Ki67. The observed intraclass correlation coefficient was worse for maximum compared to average scoring methods, suggesting that maximum score methods may be suboptimal for consistent measurement of proliferation. Automated average scoring methods show promise for assessment of Ki67 scoring, but requires further standardization and subsequent clinical validation.

Objective Approximately 15% of endometrial carcinomas (ECs) arise in young women who may wish to avoid surgical menopause and/or preserve fertility. Our aim was to evaluate the prognostic significance of Proactive Molecular risk classifier for Endometrial Carcinoma (ProMisE) in young (<50 yo) women with EC. Methods ProMisE was applied to a retrospective cohort of women with ECs <50 yo at diagnosis, and associations between the four ProMisE molecular subtypes (MMR deficient (MMRd), POLE mutated (POLE), p53 wild type (p53wt), and p53 abnormal (p53abn)) and clinicopathological parameters, including outcomes, were assessed. Results Of 257 ECs, there were 48 (19%) MMRd, 34 (13%) POLE, 164 (64%) p53wt and 11 (4%) p53abn. ProMisE subtypes were associated with differences in all measured clinicopathological parameters except for presence of synchronous ovarian tumours and fertility. Age at diagnosis was youngest and BMI highest in women with p53wt ECs. MMRd and p53abn tumours were more likely to be advanced stage (III/IV), high-risk (ESMO), and receive chemotherapy. ProMisE subtypes were strongly associated with outcomes (overall, disease-specific, and progression-free survival (p < 0.0001 for all)). Advanced stage, grade, LVSI, myometrial invasion and ESMO risk groups showed associations with some but not all survival parameters. ProMisE maintained a strong association with OS and DSS on multivariable analysis. Conclusions ProMisE molecular classification is prognostic in young women with EC, enabling early stratification and risk assignment to direct care. Further studies can assess response to therapy, fertility, and cancer-related outcomes within the framework of molecular subtype.

BACKGROUND Endometrial cancers (ECs) with somatic mutations in DNA polymerase epsilon ( POLE ) are characterized by unfavorable pathological features, which prompt adjuvant treatment. Paradoxically, women with POLE ‐mutated EC have outstanding clinical outcomes, and this raises concerns of overtreatment. The authors investigated whether favorable outcomes were independent of treatment. METHODS A PubMed search for POLE and endometrial was restricted to articles published between March 1, 2012, and March 1, 2018, that provided individual patient data (IPD), adjuvant treatment, and survival. Following the Preferred Reporting Items for Systematic Review and Meta‐Analysis (PRISMA) reporting guidelines for IPD, the authors used univariate and multivariate one‐stage meta‐analyses with mixed effects Cox models (random effects for study cohorts) to infer the associations of treatment, traditional prognostic factors, and outcome, which was defined as the time from first diagnosis to any adverse event (progression/recurrence or death from EC). RESULTS Three hundred fifty‐nine women with POLE ‐mutated EC were identified; 294 (82%) had pathogenic mutations. Worse outcomes were demonstrated in patients with nonpathogenic POLE mutations (hazard ratio, 3.42; 95% confidence interval, 1.47‐7.58; log‐rank P &lt; .01). Except for stage ( P &lt; .01), traditional prognosticators were not associated with progression/recurrence or death from disease. Adverse events were rare (11 progressions/recurrences and 3 disease‐specific deaths). Salvage rates in patients who experienced recurrence were high and sustained, with 8 of 11 alive without evidence of disease (range, 5.5‐14.2 years). Adjuvant treatment was not associated with outcome. CONCLUSIONS Clinical outcomes for ECs with pathogenic POLE mutations are not associated with most traditional risk parameters, and patients do not appear to benefit from adjuvant therapy. The observed low rates of recurrence/progression and the high and sustained salvage rates raise the possibility of safely de‐escalating treatment for these patients. LAY SUMMARY Ten percent of all endometrial cancers have mutations in the DNA repair gene DNA polymerase epsilon ( POLE ). Women who have endometrial cancers with true POLE mutations experience almost no recurrences or deaths from their cancer even when their tumors appear to have very unfavorable characteristics. Additional therapy (radiation and chemotherapy) does not appear to improve outcomes for women with POLE ‐mutated endometrial cancer, and this supports the move to less therapy and less associated toxicity. Diligent classification of endometrial cancers by molecular features provides valuable information to inform prognosis and to direct treatment/no treatment.

Human papillomavirus (HPV)-independent vulvar squamous cell carcinoma (VSCC) is an aggressive clinical entity. Current diagnostic guidelines for premalignant lesions are ambiguous, and their molecular profile and progression events are still unclear. We selected 75 samples, from 40 patients, including 33 VSCC, 8 verrucous carcinomas (VC), 13 differentiated-type vulvar intraepithelial neoplasia (dVIN), 11 suspicious for dVIN (?dVIN), 6 differentiated exophytic vulvar intraepithelial lesions (DE-VIL), 2 vulvar acanthosis with altered differentiation (VAAD), and 2 usual-type vulvar intraepithelial neoplasia (uVIN/HSIL). Invasive and precursor lesions were matched in 29 cases. Clinical information, p16 immunohistochemistry, and mutation analysis were performed on all lesions. All dVIN, ?dVIN, DE-VIL, and VAAD were p16 negative, all uVIN/HSIL were p16 positive. In the HPV-independent group, mutations were identified in 6 genes: TP53 (n = 40), PIK3CA (n = 20), HRAS (n = 12), MET (n = 5), PTEN (n = 4), and BRAF (n = 1). TP53 mutations occurred in 73% (22/30) VSCC, 85% (11/13) dVIN, 70% (7/10) ?dVIN and no VC (0/8), DE-VIL (0/6) nor VAAD (0/2). Basal atypia was the only reliable feature of TP53 mutations. ?dVIN lesions that were non-acanthotic and atypical but obscured by inflammation, all harbored TP53 mutations. In lesions without TP53 mutations, PIK3CA (50% VC, 33% DE-VIL, 100% VAAD, 40% VSCC) and HRAS (63% VC, 33% DE-VIL, 0% VAAD, 20% VSCC) mutations were found. Mutational progression from in situ to invasive was seen (7/26, 27%) and usually involved TP53 (4/26, 15%). Cases with TP53 and PIK3CA co-mutations had the worse clinical outcomes (p < 0.001). We recommend testing for p53 in all HPV-independent lesions suspicious for dVIN, even in the presence of marked inflammation or non-acanthotic skin, particularly when close to a margin. VC, VAAD, and DE-VIL, were almost never mutated for TP53, but instead often harbored PIK3CA and HRAS mutations. In VSCC, combined TP53 and PIK3CA mutations may inform prognosis.

The role of lymph node assessment/dissection (LND) in endometrial cancer (EC) has been debated for decades, with significant practice variation between centers. Molecular classification of EC provides prognostic information and can be accurately performed on preoperative endometrial biopsies. We assessed the association between molecular subtype and lymph node metastases (LNM) in order to determine if this tool could be used to stratify surgical decision making.All EC patients undergoing primary staging surgery with planned complete pelvic +/- para-aortic LND from a single institution in the 2015 calendar year were identified, with clinicopathological and outcome data assessed in the context of retrospectively assigned molecular classification.172 patients were included. Molecular classification of the total cohort showed 21 POLEmut (12.2%), 47 MMRd (27.3%), 74 NSMP (43.1%), and 30 p53abn (17.4%) ECs. Complete pelvic +/- para-aortic LND was performed in 171 of 172 patients, and LNM were found in 31/171 (18.1%). This included macrometastases (19/31), micrometastases (5/31), and isolated tumour cells (ITCs) (7/31). LNM were pelvic only in 83.9%, and pelvic plus para-aortic in 16.1%. There were no isolated para-aortic LNM. Molecular subtype was significantly associated with LNM (p = 0.004). There was a strong association between the presence of LNM and p53abn EC (nodal involvement in 44.8% of cases), with LNM detected in 14.2% of POLEmut, 14.9% of MMRd, and 10.8% of NSMP EC. On multivariate analysis, molecular subtype and preoperative CA 125 > 25 were significantly associated with LNM (p = 0.021 and p = 0.022 respectively) but preoperative grade and histotype were not (p = 0.24).EC molecular subtype is significantly associated with the presence of LNM. As molecular classification can be obtained on preoperative diagnostic specimens, this information can be used to guide surgical treatment planning and may reduce the cost and morbidity of unnecessary lymph node staging in EC care.

Recent data support the predictive implications of molecular subtype assignment in endometrial cancer (EC). Our objective was to retrospectively assess clinical outcomes according to adjuvant treatment received within EC molecular subtypes.Clinical outcomes (disease-specific and progression-free survival DSS/PFS) of EC patients from a single institution and population-based cohorts that had undergone molecular classification were assessed with respect to adjuvant therapy received and 2016 ESMO risk group.2472 ECs were assessed; 184 (7.4%) POLEmut, 638 (25.8%) MMRd, 1223 (49.5%) NSMP and 427 (17.3%) p53abn. N = 774 (34.6%) of the cohort were ESMO 2016 high risk and 109 (4.8%) were advanced or metastatic. In patients with MMRd EC, assessed across and within stage, there was no observed benefit in DSS or PFS with the addition of chemotherapy +/- radiation compared to radiation alone in ESMO high risk (p = 0.694) or ESMO high, advanced, metastatic risk groups combined (p = 0.852). In patients with p53abn EC, adjuvant chemotherapy given with radiation was associated with significantly longer DSS compared to radiation alone in ESMO high risk (p = 0.007) and ESMO high, advanced and metastatic risk groups combined (p = 0.015), even when restricted to stage I disease (p < 0.001) and when compared in serous vs. non-serous histotypes (p = 0.009).Adjuvant chemotherapy is associated with more favorable outcomes for patients with p53abn EC, including stage I disease and non-serous histotypes, but does not appear to add benefit within MMRd ECs for any stage of disease, consistent with PORTEC-3 molecular subanalysis. Prospective trials, assessing treatment efficacy within molecular subtype are needed, however these 'real-world' data should be considered when discussing adjuvant treatment with patients.

Gene expression analysis is used to subtype breast cancers such that the most aggressive tumors are identified, but translating this into clinical practice can be cumbersome. Our goal is to develop a universal biomarker that distinguishes patients at high risk across all breast cancer subtypes. We previously reported that Y-box binding protein-1 (YB-1), a transcription/translation factor, was a marker of poor prognosis in a cohort of 490 patients with breast cancer, but the study was not large enough to subtype the cancers. We therefore investigated whether YB-1 identifies patients at risk for either reduced relapse free survival or decreased r breast cancer specific survival (BCSS) across all tumor subtypes by evaluating 4,049 cases.Tumor tissue microarrays, representing 4,049 cases of invasive breast cancers with 20 years of follow up, were subtyped by the expression profiles of estrogen receptor, progesterone receptor, or HER-2. We then addressed whether YB-1 expression identified patients at higher risk for relapse and/or lower BCSS.We found YB-1 to be a highly predictive biomarker of relapse (P < 2.5 x 10(-20)) and poor survival (P < 7.3 x 10(-26)) in the entire cohort and across all breast cancer subtypes. Patients with node-positive or node-negative cancer were more likely to die from the disease if YB-1 was expressed. This was further substantiated using a Cox regression model, which revealed that it was significantly associated with relapse and poor survival in a subtype independent manner (relapse patients, hazard ratio = 1.28, P < 8 x 10(-3); all patients, hazard ratio = 1.45, P < 6.7 x 10(-7)). Moreover, YB-1 was superior to estrogen receptor and HER-2 as a prognostic marker for relapse and survival. For a subset of patients who were originally considered low risk and were therefore not given chemotherapy, YB-1 was indicative of poor survival (P < 7.1 x 10 (-17)). Likewise, YB-1 was predictive of decreased BCSS in tamoxifen-treated patients (P = 0.001); in this setting a Cox regression model once again demonstrated it to be an independent biomarker indicating poor survival (hazard ratio = 1.70, P = 0.022).Expression of YB-1 universally identifies patients at high risk across all breast cancer subtypes and in situations where more aggressive treatment may be needed. We therefore propose that YB-1 may re-define high-risk breast cancer and thereby create opportunities for individualized therapy.

Understanding the signaling pathways that drive aggressive breast cancers is critical to the development of effective therapeutics. The oncogene MET is associated with decreased survival in breast cancer, yet the role that MET plays in the various breast cancer subtypes is unclear. We describe a knockin mouse with mutationally activated Met (Met(mut)) that develops a high incidence of diverse mammary tumors with basal characteristics, including metaplasia, absence of progesterone receptor and ERBB2 expression, and expression of cytokeratin 5. With gene expression and tissue microarray analysis, we show that high MET expression in human breast cancers significantly correlated with estrogen receptor negative/ERBB2 negative tumors and with basal breast cancers. Few treatment options exist for breast cancers of the basal or trastuzumab-resistant ERBB2 subtypes. We conclude from these studies that MET may play a critical role in the development of the most aggressive breast cancers and may be a rational therapeutic target.

<h2>Abstract</h2> With the emerging evidence that the five major ovarian carcinoma subtypes (high-grade serous, clear cell, endometrioid, mucinous, and low-grade serous) are distinct disease entities, management of ovarian carcinoma will become subtype specific in the future. In an effort to improve diagnostic accuracy, we set out to determine if an immunohistochemical panel of molecular markers could reproduce consensus subtype assignment. Immunohistochemical expression of 22 biomarkers were examined on tissue microarrays constructed from 322 archival ovarian carcinoma samples from the British Columbia Cancer Agency archives, for the period between 1984 and 2000, and an independent set of 242 cases of ovarian carcinoma from the Gynaecologic Tissue Bank at Vancouver General Hospital from 2001 to 2008. Nominal logistic regression was used to produce a subtype prediction model for each of these sets of cases. These models were then cross-validated against the other cohort, and then both models were further validated in an independent cohort of 81 ovarian carcinoma samples from five different centers. Starting with data for 22 markers, full model fit, backwards, nominal logistic regression identified the same nine markers (CDKN2A, DKK1, HNF1B, MDM2, PGR, TFF3, TP53, VIM, WT1) as being most predictive of ovarian carcinoma subtype in both the archival and tumor bank cohorts. These models were able to predict subtype in the respective cohort in which they were developed with a high degree of sensitivity and specificity (<i>κ</i> statistics of 0.88±0.02 and 0.86±0.04, respectively). When the models were cross-validated (ie using the model developed in one case series to predict subtype in the other series), the prediction equation's performances were reduced (<i>κ</i> statistics of 0.70±0.04 and 0.61±0.04, respectively) due to differences in frequency of expression of some biomarkers in the two case series. Both models were then validated on the independent series of 81 cases, with very good to excellent ability to predict subtype (<i>κ</i>=0.85±0.06 and 0.78±0.07, respectively). A nine-marker immunohistochemical maker panel can be used to objectively support classification into one of the five major subtypes of ovarian carcinoma.

Abstract There is an urgent need to improve prognostic classifiers in breast cancer. Ki67 and B‐cell lymphoma 2 protein (BCL2) are established prognostic markers which have traditionally been assessed separately, in a dichotomous manner. This study was conducted to test the hypothesis that combinatorial assessment of these markers would provide superior prognostic information and improve their clinical utility. Tissue microarrays were used to assess the expression of Ki67 and BCL2 in 2749 cases of invasive breast cancer. We devised a Ki67/BCL2 index representing the relative expression of each protein and assessed its association with breast cancer‐specific survival (BCSS) using a Cox proportional‐hazards model. Based on our findings, an independent cohort of 3992 cases was used to validate the prognostic significance of the Ki67/BCL2 index. All survival analyses were conducted on complete data as well as data where missing values were resolved using multiple imputation. This study complied with reporting recommendations for tumour marker prognostic studies (REMARK) criteria. The Ki67/BCL2 index showed a significant association with BCSS at 10 years in estrogen receptor (ER)‐positive disease. In multivariate analysis, adjusting for major clinical and molecular markers, the Ki67/BCL2 index retained prognostic significance, robustly classifying cases into three risk groups [intermediate‐ versus low‐risk hazard ratio (HR), 1.5; 95% confidence interval (95% CI), 1.0–2.0; p = 0.031; high‐ versus low‐risk HR, 2.6; 95% CI, 1.3–5.0; p = 0.005]. This finding was validated in an independent cohort of 3992 tumours containing 2761 ER‐positive tumours (intermediate‐ versus low‐risk HR, 1.7; 95% CI, 1.3–2.1; p &lt; 0.001; high‐ versus low‐risk HR, 2.0; 95% CI, 1.4–2.9; p &lt; 0.001). Ki67 and BCL2 can be effectively combined to produce an index which is an independent predictor of BCSS in ER‐positive breast cancer, enhancing their potential prognostic utility. Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley &amp; Sons, Ltd.

Ovarian cancer is now recognized as a number of distinct diseases primarily defined by histological subtype. Both clear cell ovarian carcinomas (CCC) and ovarian endometrioid carcinomas (EC) may arise from endometriosis and frequently harbor mutations in the ARID1A tumor suppressor gene. We studied the influence of histological subtype on protein expression with reverse phase protein array (RPPA) and assessed proteomic changes associated with ARID1A mutation/BAF250a expression in EC and CCC. Immunohistochemistry (IHC) for BAF250a expression was performed on 127 chemotherapy-naive ovarian carcinomas (33 CCC, 29 EC, and 65 high-grade serous ovarian carcinomas (HGSC)). Whole tumor lysates were prepared from frozen banked tumor samples and profiled by RPPA using 116 antibodies. ARID1A mutations were identified by exome sequencing, and PIK3CA mutations were characterized by MALDI-TOF mass spectrometry. SAM (Significance Analysis of Microarrays) was performed to determine differential protein expression by histological subtype and ARID1A mutation status. Multivariate logistic regression was used to assess the impact of ARID1A mutation status/BAF250a expression on AKT phosphorylation (pAKT). PIK3CA mutation type and PTEN expression were included in the model. BAF250a knockdown was performed in 3 clear cell lines using siRNA to ARID1A. Marked differences in protein expression were observed that are driven by histotype. Compared to HGSC, SAM identified over 50 proteins that are differentially expressed in CCC and EC. These included PI3K/AKT pathway proteins, those regulating the cell cycle, apoptosis, transcription, and other signaling pathways including steroid hormone signaling. Multivariate models showed that tumors with loss of BAF250a expression showed significantly higher levels of AKT-Thr308 and AKT-Ser473 phosphorylation (p < 0.05). In 31 CCC cases, pAKT was similarly significantly increased in tumors with BAF250a loss on IHC. Knockdown of BAF250a by siRNA in three CCC cell lines wild type for ARID1A showed no increase in either pAKT-Thr308 or pAKT-S473 suggesting that pAKT in tumor tissues is indirectly regulated by BAF250a expression. Proteomic assessment of CCC and EC demonstrates remarkable differences in protein expression that are dependent on histotype, thereby further characterizing these cancers. AKT phosphorylation is associated with ARID1A/BAF250a deficient tumors, however in ovarian cancers the mechanism remains to be elucidated.

Purpose: Consensus is lacking regarding the androgen receptor (AR) as a prognostic marker in breast cancer. The objectives of this study were to comprehensively review the literature on AR prognostication and determine optimal criteria for AR as an independent predictor of breast cancer survival.Experimental Design: AR positivity was assessed by immunostaining in two clinically validated primary breast cancer cohorts [training cohort, n = 219; validation cohort, n = 418; 77% and 79% estrogen receptor alpha (ERα) positive, respectively]. The optimal AR cut-point was determined by ROC analysis in the training cohort and applied to both cohorts.Results: AR was an independent prognostic marker of breast cancer outcome in 22 of 46 (48%) previous studies that performed multivariate analyses. Most studies used cut-points of 1% or 10% nuclear positivity. Herein, neither 1% nor 10% cut-points were robustly prognostic. ROC analysis revealed that a higher AR cut-point (78% positivity) provided optimal sensitivity and specificity to predict breast cancer survival in the training (HR, 0.41; P = 0.015) and validation (HR, 0.50; P = 0.014) cohorts. Tenfold cross-validation confirmed the robustness of this AR cut-point. Patients with ERα-positive tumors and AR positivity ≥78% had the best survival in both cohorts (P < 0.0001). Among the combined ERα-positive cases, those with comparable or higher levels of AR (AR:ERα-positivity ratio >0.87) had the best outcomes (P < 0.0001).Conclusions: This study defines an optimal AR cut-point to reliably predict breast cancer survival. Testing this cut-point in prospective cohorts is warranted for implementation of AR as a prognostic factor in the clinical management of breast cancer. Clin Cancer Res; 24(10); 2328-41. ©2018 AACR.

Recent studies of the interferon-induced transcription factor STAT1 have associated its dysregulation with poor prognosis in some cancers, but its mechanistic contributions are not well defined. In this study, we report that the STAT1 pathway is constitutively upregulated in type II endometrial cancers. STAT1 pathway alteration was especially prominent in serous papillary endometrial cancers (SPEC) that are refractive to therapy. Our results defined a "SPEC signature" as a molecular definition of its malignant features and poor prognosis. Specifically, we found that STAT1 regulated MYC as well as ICAM1, PD-L1, and SMAD7, as well as the capacity for proliferation, adhesion, migration, invasion, and in vivo tumorigenecity in cells with a high SPEC signature. Together, our results define STAT1 as a driver oncogene in SPEC that modulates disease progression. We propose that STAT1 functions as a prosurvival gene in SPEC, in a manner important to tumor progression, and that STAT1 may be a novel target for molecular therapy in this disease.

Autophagy, a lysosome-mediated degradation and recycling process, functions in advanced malignancies to promote cancer cell survival and contribute to cancer progression and drug resistance.While various autophagy inhibition strategies are under investigation for cancer treatment, corresponding patient selection criteria for these autophagy inhibitors need to be developed.Due to its central roles in the autophagy process, the cysteine protease ATG4B is one of the autophagy proteins being pursued as a potential therapeutic target.In this study, we investigated the expression of ATG4B in breast cancer, a heterogeneous disease comprised of several molecular subtypes.We examined a panel of breast cancer cell lines, xenograft tumors, and breast cancer patient specimens for the protein expression of ATG4B, and found a positive association between HER2 and ATG4B protein expression.We showed that HER2-positive cells, but not HER2negative breast cancer cells, require ATG4B to survive under stress.In HER2-positive cells, cytoprotective autophagy was dependent on ATG4B under both starvation and HER2 inhibition conditions.Combined knockdown of ATG4B and HER2 by siRNA resulted in a significant decrease in cell viability, and the combination of ATG4B knockdown with trastuzumab resulted in a greater reduction in cell viability compared to trastuzumab treatment alone, in both trastuzumab-sensitive and -resistant HER2 overexpressing breast cancer cells.Together these results demonstrate a novel association of ATG4B positive expression with HER2 positive breast cancers and indicate that this subtype is suitable for emerging ATG4B inhibition strategies.

Abstract Purpose: Endometrioid ovarian carcinoma (ENOC) is generally associated with a more favorable prognosis compared with other ovarian carcinomas. Nonetheless, current patient treatment continues to follow a “one-size-fits-all” approach. Even though tumor staging offers stratification, personalized treatments remain elusive. As ENOC shares many clinical and molecular features with its endometrial counterpart, we sought to investigate The Cancer Genome Atlas–inspired endometrial carcinoma (EC) molecular subtyping in a cohort of ENOC. Experimental Design: IHC and mutation biomarkers were used to segregate 511 ENOC tumors into four EC-inspired molecular subtypes: low-risk POLE mutant (POLEmut), moderate-risk mismatch repair deficient (MMRd), high-risk p53 abnormal (p53abn), and moderate-risk with no specific molecular profile (NSMP). Survival analysis with established clinicopathologic and subtype-specific features was performed. Results: A total of 3.5% of cases were POLEmut, 13.7% MMRd, 9.6% p53abn, and 73.2% NSMP, each showing distinct outcomes (P &amp;lt; 0.001) and survival similar to observations in EC. Median OS was 18.1 years in NSMP, 12.3 years in MMRd, 4.7 years in p53abn, and not reached for POLEmut cases. Subtypes were independent of stage, grade, and residual disease in multivariate analysis. Conclusions: EC-inspired molecular classification provides independent prognostic information in ENOC. Our findings support investigating molecular subtype–specific management recommendations for patients with ENOC; for example, subtypes may provide guidance when fertility-sparing treatment is desired. Similarities between ENOC and EC suggest that patients with ENOC may benefit from management strategies applied to EC and the opportunity to study those in umbrella trials.

Article11 March 2021Open Access Transparent process Quantitative imaging of RAD51 expression as a marker of platinum resistance in ovarian cancer Michal M Hoppe orcid.org/0000-0002-0364-6080 Cancer Science Institute of Singapore, National University of Singapore, Singapore Search for more papers by this author Patrick Jaynes Cancer Science Institute of Singapore, National University of Singapore, Singapore Search for more papers by this author Joanna D Wardyn Cancer Science Institute of Singapore, National University of Singapore, Singapore Search for more papers by this author Sai Srinivas Upadhyayula Cancer Science Institute of Singapore, National University of Singapore, Singapore Search for more papers by this author Tuan Zea Tan orcid.org/0000-0001-6624-1593 Cancer Science Institute of Singapore, National University of Singapore, Singapore Search for more papers by this author Stefanus Lie Cancer Science Institute of Singapore, National University of Singapore, Singapore Search for more papers by this author Diana G Z Lim Department of Pathology, National University Hospital, Singapore Search for more papers by this author Brendan N K Pang Cancer Science Institute of Singapore, National University of Singapore, Singapore Department of Pathology, National University Hospital, Singapore Search for more papers by this author Sherlly Lim Cancer Science Institute of Singapore, National University of Singapore, Singapore Search for more papers by this author Joe P S Yeong Cancer Science Institute of Singapore, National University of Singapore, Singapore Search for more papers by this author Anthony Karnezis British Columbia Cancer Agency, Vancouver, BC, Canada Search for more papers by this author Derek S Chiu British Columbia Cancer Agency, Vancouver, BC, Canada Search for more papers by this author Samuel Leung British Columbia Cancer Agency, Vancouver, BC, Canada Search for more papers by this author David G Huntsman British Columbia Cancer Agency, Vancouver, BC, Canada Search for more papers by this author Anna S Sedukhina Department of Pharmacogenomics, St. Marianna University, Kawasaki, Japan Search for more papers by this author Ko Sato Department of Pharmacogenomics, St. Marianna University, Kawasaki, Japan Search for more papers by this author Monique D Topp The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia Search for more papers by this author Clare L Scott The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia Search for more papers by this author Hyungwon Choi orcid.org/0000-0002-6687-3088 Saw Swee Hock School of Public Health, National University of Singapore, Singapore Search for more papers by this author Naina R Patel Division of Cancer, Imperial College London, London, UK Search for more papers by this author Robert Brown orcid.org/0000-0001-7960-5755 Division of Cancer, Imperial College London, London, UK Search for more papers by this author Stan B Kaye Department of Haematology-Oncology, National University Hospital, Singapore Search for more papers by this author Jason J Pitt Cancer Science Institute of Singapore, National University of Singapore, Singapore Search for more papers by this author David S P Tan Corresponding Author [email protected] orcid.org/0000-0001-9087-5262 Cancer Science Institute of Singapore, National University of Singapore, Singapore Department of Haematology-Oncology, National University Hospital, Singapore Search for more papers by this author Anand D Jeyasekharan Corresponding Author [email protected] orcid.org/0000-0001-9816-6137 Cancer Science Institute of Singapore, National University of Singapore, Singapore Department of Haematology-Oncology, National University Hospital, Singapore Search for more papers by this author Michal M Hoppe orcid.org/0000-0002-0364-6080 Cancer Science Institute of Singapore, National University of Singapore, Singapore Search for more papers by this author Patrick Jaynes Cancer Science Institute of Singapore, National University of Singapore, Singapore Search for more papers by this author Joanna D Wardyn Cancer Science Institute of Singapore, National University of Singapore, Singapore Search for more papers by this author Sai Srinivas Upadhyayula Cancer Science Institute of Singapore, National University of Singapore, Singapore Search for more papers by this author Tuan Zea Tan orcid.org/0000-0001-6624-1593 Cancer Science Institute of Singapore, National University of Singapore, Singapore Search for more papers by this author Stefanus Lie Cancer Science Institute of Singapore, National University of Singapore, Singapore Search for more papers by this author Diana G Z Lim Department of Pathology, National University Hospital, Singapore Search for more papers by this author Brendan N K Pang Cancer Science Institute of Singapore, National University of Singapore, Singapore Department of Pathology, National University Hospital, Singapore Search for more papers by this author Sherlly Lim Cancer Science Institute of Singapore, National University of Singapore, Singapore Search for more papers by this author Joe P S Yeong Cancer Science Institute of Singapore, National University of Singapore, Singapore Search for more papers by this author Anthony Karnezis British Columbia Cancer Agency, Vancouver, BC, Canada Search for more papers by this author Derek S Chiu British Columbia Cancer Agency, Vancouver, BC, Canada Search for more papers by this author Samuel Leung British Columbia Cancer Agency, Vancouver, BC, Canada Search for more papers by this author David G Huntsman British Columbia Cancer Agency, Vancouver, BC, Canada Search for more papers by this author Anna S Sedukhina Department of Pharmacogenomics, St. Marianna University, Kawasaki, Japan Search for more papers by this author Ko Sato Department of Pharmacogenomics, St. Marianna University, Kawasaki, Japan Search for more papers by this author Monique D Topp The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia Search for more papers by this author Clare L Scott The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia Search for more papers by this author Hyungwon Choi orcid.org/0000-0002-6687-3088 Saw Swee Hock School of Public Health, National University of Singapore, Singapore Search for more papers by this author Naina R Patel Division of Cancer, Imperial College London, London, UK Search for more papers by this author Robert Brown orcid.org/0000-0001-7960-5755 Division of Cancer, Imperial College London, London, UK Search for more papers by this author Stan B Kaye Department of Haematology-Oncology, National University Hospital, Singapore Search for more papers by this author Jason J Pitt Cancer Science Institute of Singapore, National University of Singapore, Singapore Search for more papers by this author David S P Tan Corresponding Author [email protected] orcid.org/0000-0001-9087-5262 Cancer Science Institute of Singapore, National University of Singapore, Singapore Department of Haematology-Oncology, National University Hospital, Singapore Search for more papers by this author Anand D Jeyasekharan Corresponding Author [email protected] orcid.org/0000-0001-9816-6137 Cancer Science Institute of Singapore, National University of Singapore, Singapore Department of Haematology-Oncology, National University Hospital, Singapore Search for more papers by this author Author Information Michal M Hoppe1, Patrick Jaynes1, Joanna D Wardyn1, Sai Srinivas Upadhyayula1, Tuan Zea Tan1, Stefanus Lie1, Diana G Z Lim2, Brendan N K Pang1,2, Sherlly Lim1, Joe P S Yeong1, Anthony Karnezis3,†, Derek S Chiu3, Samuel Leung3, David G Huntsman3, Anna S Sedukhina4, Ko Sato4, Monique D Topp5, Clare L Scott5, Hyungwon Choi6, Naina R Patel7, Robert Brown7, Stan B Kaye8, Jason J Pitt1, David S P Tan *,1,8 and Anand D Jeyasekharan *,1,8 1Cancer Science Institute of Singapore, National University of Singapore, Singapore 2Department of Pathology, National University Hospital, Singapore 3British Columbia Cancer Agency, Vancouver, BC, Canada 4Department of Pharmacogenomics, St. Marianna University, Kawasaki, Japan 5The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia 6Saw Swee Hock School of Public Health, National University of Singapore, Singapore 7Division of Cancer, Imperial College London, London, UK 8Department of Haematology-Oncology, National University Hospital, Singapore †Present address: Pathology and Lab medicine, UC Davis Medical Centre, Sacramento, CA, USA *Corresponding author. Tel: +65 6773 7888; E-mail: [email protected] *Corresponding author. Tel: +65 6516 5094; E-mail: [email protected] EMBO Mol Med (2021)13:e13366https://doi.org/10.15252/emmm.202013366 See also: J Schwickert et al (May 2021) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Early relapse after platinum chemotherapy in epithelial ovarian cancer (EOC) portends poor survival. A-priori identification of platinum resistance is therefore crucial to improve on standard first-line carboplatin–paclitaxel treatment. The DNA repair pathway homologous recombination (HR) repairs platinum-induced damage, and the HR recombinase RAD51 is overexpressed in cancer. We therefore designed a REMARK-compliant study of pre-treatment RAD51 expression in EOC, using fluorescent quantitative immunohistochemistry (qIHC) to overcome challenges in quantitation of protein expression in situ. In a discovery cohort (n = 284), RAD51-High tumours had shorter progression-free and overall survival compared to RAD51-Low cases in univariate and multivariate analyses. The association of RAD51 with relapse/survival was validated in a carboplatin monotherapy SCOTROC4 clinical trial cohort (n = 264) and was predominantly noted in HR-proficient cancers (Myriad HRDscore < 42). Interestingly, overexpression of RAD51 modified expression of immune-regulatory pathways in vitro, while RAD51-High tumours showed exclusion of cytotoxic T cells in situ. Our findings highlight RAD51 expression as a determinant of platinum resistance and suggest possible roles for therapy to overcome immune exclusion in RAD51-High EOC. The qIHC approach is generalizable to other proteins with a continuum instead of discrete/bimodal expression. Synopsis Quantitative immunohistochemistry (qIHC) reveals that high expression of the DNA repair protein RAD51 in epithelial ovarian cancer is associated with early relapse after platinum chemotherapy, and also with decreased cytotoxic T-cell infiltration into tumors. High nuclear expression score for RAD51 (RAD51NES) was correlated with early relapse and shorter survival in two independent EOC patient cohorts (n = 264 + 284). RAD51NES was prognostically relevant primarily for EOCs that did not have homologous recombination deficiency (HRD). RAD51 expression was correlated with a unique immune phenotype in cancer, with increased chemokines but reduced cytotoxic T-cell infiltration. The paper explained Problem Platinum chemotherapy is the cornerstone of treatment for epithelial ovarian cancer (EOC). While the typical first-line chemotherapy of Carboplatin + Paclitaxel is highly effective in EOC, a subset of patients are resistant to or relapse early after treatment and have poor overall survival. It would be advantageous to identify these cases prior to initiation of treatment, to facilitate the testing of novel agents that can supplement or even supplant platinum chemotherapy. There are no molecular markers currently used in pathology labs to define possible platinum-resistance, in large part due to challenges in quantitating expression of candidate proteins in tissue sections. Results We used a state-of-the-art method for simultaneous staining of multiple proteins in tissue sections along with automated microscopy to quantitatively measure RAD51, a DNA repair protein that is important for the resolution of platinum induced damage. We show using two large independent EOC patient cohorts, cases that expressed a high amount of RAD51 relapsed sooner than those expressing a low amount of RAD51. Furthermore, this phenomenon correlates with an exclusion of anti-tumour immune cells from the microenvironment of cancers with RAD51-High. Impact Our study identifies RAD51 as a bonafide biomarker for increased likelihood for resistance to platinum chemotherapy in ovarian cancer, which can subsequently be developed into a clinical grade assay for routine diagnostic practice. Furthermore, our observation that RAD51 tumours tend to exclude important anti-cancer immune cells sets the stage for developing therapeutic approaches to increase immune infiltration in these cancers. Introduction Epithelial ovarian cancer (EOC) is the most lethal of all female genital tract cancers. Platinum chemotherapy is the cornerstone of treatment for EOC, typically combined with paclitaxel. The duration of disease control after platinum chemotherapy is a strong predictor of overall survival in EOC (Davis et al, 2014). In recurrent EOC, the platinum treatment-free interval strongly correlates with subsequent response to platinum rechallenge therapy (Markman et al, 1991). Platinum resistance (defined as relapse within six months following completion of platinum chemotherapy) occurs in 20–30% of cases. However, recent consensus guidelines highlight the predictive limitations of this “time-based” definition (Colombo et al, 2019). There remains a need to a-priori identify patients who will have platinum resistance, and there are no molecular markers of platinum resistance in current clinical use. The identification of cases for whom first-line carboplatin–paclitaxel chemotherapy is sub-optimal will facilitate trials of early incorporation of novel agents to improve overall survival. The sensitivity of ovarian cancers to platinum chemotherapy is in part due to a high prevalence of aberrations in the DNA repair pathway of homologous recombination (HR; McMullen et al, 2020). Platinum treatment leads to inter-strand cross-links, which are typically repaired by the pathways of nucleotide excision repair (NER) and HR (De Silva et al, 2000; Sarkar et al, 2006). HR deficiency (HRD) e.g., with BRCA mutations, is associated with exquisite platinum sensitivity due to the inability to repair platinum cross-links (Tan et al, 2008). Up to 50% of EOC show HRD through mutations in other HR regulatory genes of the BRCA/Fanconi Anaemia (FA) network (Bell et al, 2011). However, unlike other BRCA/FA genes, RAD51—the central recombinase of the HR pathway—is not commonly mutated in cancer. Depletion or mutation of RAD51 is lethal due to its essential role in cellular replication (Tsuzuki et al, 1996; Sonoda et al, 1998). Conversely, RAD51 is often upregulated in multiple cancer types and is associated with poor survival (Qiao et al, 2005; Mitra et al, 2009; Tennstedt et al, 2013; Alshareeda et al, 2016). As a corollary to platinum sensitivity in ovarian cancers with HRD, it is not known if the overexpression of RAD51 confers platinum resistance. However, evaluating the clinical significance of RAD51 overexpression has been hampered by the lack of quantitative tools for proteomics in situ. In this paper, we utilize quantitative immunohistochemistry (qIHC) through multispectral imaging/ automated analysis to evaluate baseline RAD51 protein levels in formalin-fixed paraffin-embedded (FFPE) tissue. For the discovery cohort, we focused on high-grade serous ovarian carcinomas (HGSOC), the most common and aggressive subtype of EOC. Platinum is typically combined with paclitaxel, the sensitivity to which is not associated with HRD, making it challenging to dissect the contribution of a biomarker to platinum-specific survival. We therefore validated our findings in samples from the SCOTROC4 clinical trial in a REMARK-compliant retrospective biomarker analysis. SCOTROC4 was a phase III trial of carboplatin monotherapy in EOC, assessing two different dosing schedules (Banerjee et al, 2013). While the trial showed no difference between the two arms, it represents a unique cohort of platinum monotherapy in ovarian cancer, with well-annotated survival data and HRD scores (Stronach et al, 2018). Results and Discussion RAD51 forms discrete nuclear foci upon activation of HR, and this is a widely used measure of recombination proficiency in vitro (Graeser et al, 2010). The RAD51 foci counting assay has been evaluated in FFPE and ex vivo samples (Graeser et al, 2010; Naipal et al, 2014; Castroviejo-Bermejo et al, 2018; Tumiati et al, 2018). However, automated quantitation of foci counts in FFPE and ex vivo samples is logistically complex and highly reliant on sample preparation/microscopy setup. Conversely, the quantitation of mean nuclear intensity (nuclear expression score) by qIHC is relatively amenable to automated quantitation and scalability in large data sets. To setup our RAD51 qIHC assay, we first validated a rabbit monoclonal antibody EPR4030(3) (Abcam)—demonstrating specific detection of RAD51 in FFPE cell blocks, ex vivo irradiated patient-derived xenografts and control human tissues (Fig EV1-EV5). We define RAD51 nuclear expression score (RAD51NES) as the average intensity of RAD51 expression measured by qIHC across all imaged tumour cells for a given sample (Fig 1A). In a training cohort of EOC cases (n = 52), RAD51NES showed strong concordance with RAD51 H-Scores obtained independently from two board-certified pathologists (Fig 1B). We evaluated RAD51 expression in a HGSOC cohort of cases treated with standard-of-care protocols at British Columbia Cancer (BCC) Vancouver. We observed that the RAD51NES in this cohort followed a normal distribution (Fig 1C). Unlike markers such as Ki67 or ER/PR which have a distinct bimodal pattern of expression (i.e. a cell is either “positive” or “negative”), many cancer-related proteins display homogenous expression within a sample and normal distribution across samples. To cater for the normal distribution of RAD51 within a clinical cohort, RAD51NES was analysed as either a continuous variable or a categorical variable dividing the cohort into RAD51-Low (RAD51NES first quartile [Q1]), RAD51-High (fourth quartile [Q4]) and RAD51-IQR (IQR [quartiles 2 + 3]). We subsequently applied our optimized protocol for staining, imaging, scoring and analysis to assess the clinical relevance of RAD51 protein expression in the BCC cohort. In a Kaplan–Meier survival analysis, high RAD51NES was associated with poorer progression-free survival (PFS) and overall survival (OS) (Fig 1D). We used the 12-month PFS rate (%) as a surrogate for early relapse after completion of platinum-based chemotherapy. RAD51-High cases showed higher likelihood of progression than RAD51-Low cases at both 12 and 24 months (Fig 1E), pointing to the potential utility of RAD51 in predicting platinum resistance. As RAD51 expression is linked to proliferation through common regulatory pathways (Fischer et al, 2016), a possible explanation for poor survival could be increased proliferation in RAD51-High tumours. We therefore measured the proliferation marker Ki67 in the BCC cohort by qIHC. RAD51NES correlated weakly with Ki67 extent (Fig EV2A). Importantly, the proliferation status of the tumour (i.e. extent of Ki67 positivity) was not associated with survival outcomes (Fig EV2B). Furthermore, in a multivariate cox proportional hazards model (Cox PH) adjusting for Ki67 extent, age and stage, RAD51NES as a continuous variable remained a statistically significant independent predictor of PFS in HGSOC. Comparable results were obtained for OS (Table 1). Click here to expand this figure. Figure EV1. Validation of a RAD51-specific antibody Immunoblot of RAD51 in HEY-T30 control and knock-down cell lines. The EPR4030(3) antibody reveals a single band of 37 kDa size in control cells (top band results from post-translational modification of RAD51), which is not present in RAD51 knock-down cells. Blot is a representation of three independent experiments. Cells from (A) were used to create a FFPE cell block used for RAD51 fluorescent IHC (left). Cells from (A) in an FFPE block stained with an IgG isotype control (right). Scale bar is 50μm. All images are representative of three independent experiments. Fluorescent IHC FFPE block of an ovarian PDX treated ex vivo with γ-radiation and stained for RAD51 and p-H2AX S139 (left). Scale bar is 50 μm. RAD51NES score for seven ovarian PDXs before and after treatment with γ-radiation (IR) (right). Paired t-test. RAD51 fluorescent IHC on normal FFPE tissues. Testis is shown as a positive control and liver a negative control. Scale bar is 50 μm. All images are representative of three independent biological samples. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Correlation of Ki67 % extent and HRD phenotype with RAD51NES Correlation of Ki67 % extent and RAD51NES in the BCC cohort. Spearman correlation (left) and one-way ANOVA with Bonferroni correction (right). Median with interquartile range. Kaplan–Meier plots for PFS (left) and OS (right) stratified according to Ki67 extent quartile in the BCC cohort. Q—quartile. Log-rank test, shading denotes 95% confidence intervals. Correlation of Ki67 extent and RAD51NES in the SCOTROC4 cohort. Spearman correlation (left) and one-way ANOVA with Bonferroni correction (right). Median with interquartile range. Kaplan–Meier plots for PFS (left) and OS (right) stratified according to Ki67 extent quartile in the SCOTROC4 cohort. Q—quartile. Log-rank test, shading denotes 95% confidence intervals. Correlation of RAD51NES with BRCA mutation status in EOC. One-way ANOVA. Median with interquartile range. Linear regression of “genomic scar” HRD score assay and RAD51NES. Vertical dashed line denotes HRD positivity score of 42. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Exogenous Flag-RAD51 is functional Immunoblot of RAD51 upon overexpression and subsequent RNAi-mediated depletion of total (siRAD51-CDS) or endogenous (siRAD51-3’UTR) RAD51 mRNA. The exogenous stably overexpressed Flag-RAD51 represents the top band. HGSOC cell utilized indicated below the blot. Blots are representative of two independent experiments. Validation of exogenous Flag-RAD51 functionality using a cell viability assay. Flag-RAD51 was stably overexpressed in three HGSOC cell lines which were treated with increasing doses of carboplatin for 96 h. Flag-RAD51 rescues carboplatin sensitivity upon depletion of endogenous RAD51 protein. Mean with standard deviation is shown of at least three biological replicates per point. Statistical comparison is performed at 1μM concentration of carboplatin, t-test. Immunofluorescence of TYK-nu cells with stable Flag-RAD51 overexpression treated with 10μM of carboplatin for 48 h. Cells were co-stained for both RAD51 and Flag. DAPI serves as a nuclear counterstaining. Scale bar is 20 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Differential gene expression analysis of immune genes between RAD51-High and -Low tumours Differential gene expression analysis of immune genes between RAD51-High (Q4) and -Low (Q1) across three independent mRNA cohorts of EOC (see also Fig 3E). t-Test; dashed line denoted threshold of significance, Bonferroni corrected for multiple testing. Download figure Download PowerPoint Click here to expand this figure. Figure EV5. Multiplexed fluorescent IHC staining for immune markers of the tumour microenvironment Multiplexed fluorescent IHC staining for immune markers of the microenvironment in an EOC patient sample. Unmixed monochrome components are shown along with a false-coloured merge image. Cytokeratin staining was used to differentiate between the tumour and stromal compartments of the sample. Scale bar is 50 μm. Quantitation of immune populations in the BCC cohort. Results for RAD51-High and -Low tumours are shown. T/S—tumour/stroma ratio. Bar is median. Mann–Whitney test. Subset analysis of CD8+ cytotoxic T-cell infiltration in the BCC cohort stratified according to BRCA mutation status. Absolute tumour CD8+ cytotoxic T-cell infiltration numbers and tumour/stroma (T/S) cytotoxic T-cell number ratio in RAD51-High and -Low cases. Bar is median. Mann–Whitney test. Download figure Download PowerPoint Figure 1. RAD51 assay development and testing on BCC cohort Range of example RAD51 nuclear expression score (RAD51NES) values with respective unmixed monochrome fluorescent IHC staining images. EpCAM is used as a tumour marker and an internal sample quality control. Scale bar is 50 μm. Correlation of RAD51NES with two independent pathologist H-scores (top left and top right) and correlation of two pathologist with each other (bottom). Pearson correlation. Distribution of RAD51 nuclear expression score (RAD51NES) in the BCC cohort. The cohort is divided into RAD51-Low expressing cases (first quartile, Q1—blue), intermediate cases (interquartile range, IQR—grey) and RAD51-High expressing cases (fourth quartile, Q4—brown). Dashed line denotes the median RAD51NES in this cohort. Survival analysis of the BCC cohort. Kaplan–Meier plots for progression-free survival (PFS) (left) and overall survival (OS) (right) stratified according to fourth quartile (Q4) and first quartile (Q1) of RAD51NES. Log-rank test, shading denotes 95% confidence intervals. Number of cases with progression at 12 and 24 months. Chi-square test. Download figure Download PowerPoint Table 1. Multivariate analysis of continuous RAD51NES and Ki67 extent as a predictor of PFS and OS in the BCC cohort of HGSOC (Cox proportional hazards model). Variable Total cases (n = 242) missing values (n = 43) Total cases (n = 278) missing values (n = 7) PFS OS HR (95% CI) P-value HR (95% CI) P-value RAD51NES (continuous) 1.4 (1.0 to 1.9) 0.025 1.3 (0.98 to 1.9) 0.066 Ki67% (continuous) 0.97 (0.54 to 1.7) 0.975 0.84 (0.49 to 1.4) 0.529 Age <65 Ref. Ref. ≥65 1.0 (0.79 to 1.4) 0.797 1.3 (1.0 to 1.7) 0.022 Stage 0.048 0.011 I Ref. Ref. II 1.8 (0.70 to 4.4) 0.230 1.4 (0.53 to 3.7) 0.500 III 2.6 (1.2 to 5.6) 0.015 2.7 (1.2 to 6.2) 0.017 IV 2.2 (0.9 to 5.3) 0.086 2.3 (0.91 to 5.7) 0.078 BCC, British Columbia Cancer; CI, Confidence interval; HGSOC, High-grade serous ovarian cancer; HR, Hazard ratio; OS, overall survival; PFS, progression-free survival; RAD51NES, RAD51 nuclear expression score; Ref., Reference sample. Platinum is typically used along with paclitaxel in frontline chemotherapy of ovarian cancer. To negate potential confounding effects of paclitaxel on survival outcomes, we utilized the unique carboplatin monotherapy SCOTROC4 trial as a validation cohort for our findings. RAD51 protein expression within this cohort also followed a normal distribution (Fig 2A). RAD51-High patients again showed poorer PFS and OS after platinum monotherapy in comparison to RAD51-Low cases (Fig 2B). In a Cox PH multivariate analysis of continuous RAD51NES and Ki67 extent controlling for clinical prognosticators (Table 2), RAD51NES was not independently associated with poor PFS, but remained an independent statistically significant predictor of OS. Ki67 extent was not significantly associated with PFS or OS in multivariate analyses (Fig EV2CandD, Table 2). We then used PFS rate (%) at 12 months (calculated from time of randomization) as a surrogate for a shorter platinum-free interval and hence platinum resistance. Similar to the BCC cohort, RAD51-High cases were more likely to relapse within both 12 months and 24 months than RAD51-Low cases (Fig 2C). Overall, in two independent cohorts, a high RAD51NES associates with early relapses after platinum treatment in ovarian cancer and implies a higher risk of primary platinum resistance in RAD51-High tumours. Figure 2. Validation of assay and findings in SCOTROC4 cohort Distribution of RAD51NES in the SCOTROC4 cohort. Dashed line denotes the median RAD51NES in this cohort. Survival analysis of the SCOTROC4 cohort. Kaplan–Meier plots for PFS (left) and OS (right) stratified according to quartiles of RAD51NES. Log-rank test, shading denotes 95% confidence intervals. Number of cases with progression at 12 and 24 months. Chi-square test. Survival analysis of HRD-positive patients according to quartile of RAD51NES. Log-rank test, shading denotes 95% confidence intervals. Survival analysis of HRD-negative patients. Log-rank test, shading denotes 95% confidence intervals. Download figure Download PowerPoint Table 2. Multivariate analysis of continuous RAD51NES and Ki67 extent as a predictor of PFS and OS in the SCOTROC4 cohort (Cox proportional hazards model). Variable Total cases (n = 175) missing values (n = 93) PFS OS HR (95% CI) P-value HR (95% CI) P-value RAD51NES (continuous) 1.2 (0.97 to 1.5) 0.104 1.4 (1.1 to 1.9) 0.007 Ki67% (continuous) 1.0 (0.98 to 1.02) 0.971 0.98 (0.96 to 1.0) 0.227 Age <65 Ref. Ref. ≥65 1.0 (0.64 to 1.6) 0.996 0.99 (0.54 to 1.8) 0.977 Stage <0.001 0.023 I Ref. Ref. II 3.8 (0.99 to 15.0) 0.052 13.7 (1.6 to 120.5) 0.018 III 9.7 (2.8 to 33.1) <0.001 11.2 (1.4 to 88.9) 0.022 IV 5.3 (1.4 to 20.2) 0.014 4.8 (0.54 to 44.0) 0.160 Histology 0.150 <0.001 Serous Ref. Ref. Mucinous 3.9 (1.3 to 12.4) 0.01

Ki67 has potential clinical importance in breast cancer but has yet to see broad acceptance due to inter-laboratory variability. Here we tested an open source and calibrated automated digital image analysis (DIA) platform to: (i) investigate the comparability of Ki67 measurement across corresponding core biopsy and resection specimen cases, and (ii) assess section to section differences in Ki67 scoring. Two sets of 60 previously stained slides containing 30 core-cut biopsy and 30 corresponding resection specimens from 30 estrogen receptor-positive breast cancer patients were sent to 17 participating labs for automated assessment of average Ki67 expression. The blocks were centrally cut and immunohistochemically (IHC) stained for Ki67 (MIB-1 antibody). The QuPath platform was used to evaluate tumoral Ki67 expression. Calibration of the DIA method was performed as in published studies. A guideline for building an automated Ki67 scoring algorithm was sent to participating labs. Very high correlation and no systematic error (p = 0.08) was found between consecutive Ki67 IHC sections. Ki67 scores were higher for core biopsy slides compared to paired whole sections from resections (p ≤ 0.001; median difference: 5.31%). The systematic discrepancy between core biopsy and corresponding whole sections was likely due to pre-analytical factors (tissue handling, fixation). Therefore, Ki67 IHC should be tested on core biopsy samples to best reflect the biological status of the tumor.

We measured the variation in practice across all aspects of endometrial cancer (EC) management and assessed the potential impact of implementation of molecular classification.Centers from across Canada provided representative tumor samples and clinical data, including preoperative workup, operative management, hereditary cancer program (HCP) referrals, adjuvant therapy, surveillance and outcomes, for all EC patients diagnosed in 2016. Tumors were classified into the four ProMisE molecular subtypes.A total of 1336 fully evaluable EC patients were identified from 10 tertiary cancer centers (TC; n = 1022) and 19 community centers (CC; n = 314). Variation of surgical practice across TCs was profound (14-100%) for lymphadenectomy (LND) (mean 57% Gr1/2, 82% Gr3) and omental sampling (20% Gr1/2, 79% Gr3). Preoperative CT scans were inconsistently obtained (mean 32% Gr1/2, 51% Gr3) and use of adjuvant chemo or chemoRT in high risk EC ranged from 0-55% and 64-100%, respectively. Molecular subtyping was performed retrospectively and identified 6% POLEmut, 28% MMRd, 48% NSMP and 18% p53abn ECs, and was significantly associated with survival. Within patients retrospectively diagnosed with MMRd EC only 22% had been referred to HCP. Of patients with p53abn EC, LND and omental sampling was not performed in 21% and 23% respectively, and 41% received no chemotherapy. Comparison of management in 2016 with current 2020 ESGO/ESTRO/ESP guidelines identified at least 26 and 95 patients that would have been directed to less or more adjuvant therapy, respectively (10% of cohort).Molecular classification has the potential to mitigate the profound variation in practice demonstrated in current EC care, enabling reproducible risk assessment, guiding treatment and reducing health care disparities.

There are conflicting data about chemoresistance and prognosis in ovarian clear cell carcinoma (CCC). This could be due to significant interobserver variation in the diagnosis of CCC and other ovarian surface epithelial tumors containing clear cells. Thirty-two cases previously diagnosed as CCC, high-grade ovarian serous carcinoma (SC), and mixed surface epithelial carcinoma (SEC) with clear cell and serous components were reviewed by 4 gynecologic pathologists blinded to the original diagnoses. Interobserver reproducibility was evaluated. Each case was also assessed using immunohistochemical markers Wilm tumor 1, estrogen receptor, and p53. The interobserver reproducibility was greatest for pure CCC (kappa of 0.82), and lowest for the mixed SEC (kappa of 0.32). Moderate agreement was seen in the pure SC category (kappa of 0.59). All pure SC and most mixed SEC presented as stage III or IV diseases. Most pure CCC presented as stage I or II diseases. Immunoreactivities of the mixed SECs were similar to those of pure SC, but significantly different from those of pure CCC for Wilm tumor 1 (P=0.0011 for both components), estrogen receptor (P=0.0003 for clear cell component, P=0.0001 for serous component), and p53 (P=0.0062 for both components). The serous and clear cell components of mixed SEC showed higher mitotic rates than pure CCC (P=0.004 and P=0.023, respectively), but the mitotic rate of pure SC was similar to the mixed SEC. We conclude that (1) pure CCC is reproducibly diagnosed. (2) The diagnosis of mixed ovarian SEC with clear cell component is not reproducible. (3) Mixed SEC with clear cell and serous components show similar stage, mitotic activities, and immunoreactivities to those of pure SC, and likely represent SC with clear cell changes.

Abstract Background In breast cancer patients, HER2 overexpression is routinely assessed by immunohistochemistry (IHC) and equivocal cases are subject to fluorescent in situ hybridization (FISH). Our study compares HER2 scoring by histopathologists with automated quantitation of staining, and determines the concordance of IHC scores with FISH results. Methods A tissue microarray was constructed from 1,212 invasive breast carcinoma cases with linked treatment and outcome information. IHC slides were semi-quantitatively scored by two independent pathologists on a range of 0 to 3+, and also analyzed with an Ariol automated system by two operators. 616 cases were scorable by both IHC and FISH. Results Using data from unequivocal positive (3+) or negative (0, 1+) results, both visual and automated scores were highly consistent: there was excellent concordance between two pathologists (kappa = 1.000, 95% CI: 1-1), between two machines (kappa = 1.000, 95% CI: 1-1), and between both visual and both machine scores (kappa = 0.898, 95% CI: 0.775–0.979). Two pathologists successfully distinguished negative, positive and equivocal cases (kappa = 0.929, 95% CI: 0.909–0.946), with excellent agreement with machine 1 scores (kappa = 0.835, 95% CI: 0.806–0.862; kappa = 0.837, 95% CI: 0.81–0.862), and good agreement with machine 2 scores (kappa = 0.698, 95% CI: 0.6723–0.723; kappa = 0.709, 95% CI: 0.684–0.732), whereas the two machines showed good agreement (kappa = 0.806, 95% CI: 0.785–0.826). When comparing categorized IHC scores and FISH results, the agreement was excellent for visual 1 (kappa = 0.814, 95% CI: 0.768–0.856), good for visual 2 (kappa = 0.763, 95% CI: 0.712–0.81) and machine 1 (kappa = 0.665, 95% CI: 0.609–0.718), and moderate for machine 2 (kappa = 0.535, 95% CI: 0.485–0.584). Conclusion A fully automated image analysis system run by an experienced operator can provide results consistent with visual HER2 scoring. Further development of such systems will likely improve the accuracy of detection and categorization of membranous staining, making this technique suitable for use in quality assurance programs and eventually in clinical practice.

<h2>Abstract</h2> P-cadherin is a calcium-dependent cell–cell adhesion glycoprotein. P-cadherin expression is restricted to the myoepithelial cells in normal breast tissue, and aberrant staining has also been described in invasive tumors. Several small studies have reported P-cadherin as a marker of poor outcome in breast cancer patients but its prognostic significance in relation to other variables has not been established in a large series of breast cancers. A tissue microarray was constructed from 3992 cases of invasive breast carcinoma, and P-cadherin expression was evaluated using immunohistochemistry. Median follow-up was 12.5 years. The immunohistochemistry-based definitions of cancer subtypes were luminal (ER+ or PR+/HER2−), luminal/HER2+ (ER+ or PR+/HER2+), HER2+ (ER−/PR−/HER2+), and basal (ER−/PR−/HER2−/CK5/6+ or EGFR+). Clinical covariate and biomarker associations were assessed using contingency tables, and Pearson's χ² or Fisher's exact test. Survival associations were assessed using Kaplan–Meier plots, logrank and Breslow tests, and Cox proportional hazards regression analysis. P-cadherin was expressed in 34.8% (1290/3710, 50% cut point) of cases. P-cadherin staining was strongly associated with HER2+ and basal carcinoma subtypes (<i>P</i><0.0005). P-cadherin-positive patients showed significantly poorer short-term (0–10 years) overall survival, disease-specific survival, distant relapse-free interval, and locoregional relapse-free interval in univariable models (<i>P</i><0.05). In multivariable Cox models containing standard clinical covariates and cancer subtypes, P-cadherin did not show independent prognostic value. P-cadherin expression was positively associated with histological grade, chemotherapy, Ki-67, EGFR, CK5/6, p53, YB-1, and HER2 expression (<i>P</i><0.002), and negatively associated with age at diagnosis, ER, PR, and Bcl-2 expression (<i>P</i><0.0005). This study shows the value of P-cadherin as a marker of poor prognosis. The large sample size of this series clarifies contradictory findings of many smaller studies. P-cadherin positivity is associated with high-grade tumor subtypes and well-established markers of poor prognosis, and may represent a promising antibody therapeutic target.

ABSTRACT Tumor-infiltrating lymphocytes (TILs) are predominantly present in breast cancer patients with estrogen receptor negative tumors, among whom increasing levels correlate with favorable outcomes. Nevertheless, currently available immune checkpoint inhibitors appear to benefit only a small number of women with breast cancer. Upregulation of additional immune checkpoint markers is one mechanism of resistance to current inhibitors that might be amenable to targeting with newer agents. T-cell Immunoglobulin and Mucin domain-containing molecule 3 (TIM-3) is an immune checkpoint receptor that is an emerging target for cancer immunotherapy. We investigated TIM-3 immunohistochemical expression in 3,992 breast cancer specimens assembled into tissue microarrays, linked to detailed outcome, clinico-pathological parameters and biomarkers including CD8, PD-1, PD-L1 and LAG-3. We scored and reported absolute counts for TIM-3+ intra-epithelial and stromal TILs (iTILs and sTILs), and find that breast cancer patients with TIM-3+ iTILs (≥ 1) represent a minority of cases (11%), with a predilection for basal-like breast cancers (among which 28% had TIM-3+ iTILs). TIM-3+ sTILs (≥ 2) represented 20% of cases and included more non-basal cases. The presence of TIM-3+ iTILs highly correlates with hematoxylin and eosin-stained stromal TILs and with other immune checkpoint markers (PD-1+ iTILs, LAG-3+ iTILs and PD-L1+ tumors). In prognostic analyses, early breast cancer patients with TIM-3+ iTILs have significantly improved breast cancer-specific survival whereas TIM-3+ sTILs did not reach statistical significance. In multivariate analyses, the presence of TIM-3+ iTILs is an independent favorable prognostic factor in the whole cohort as well as among ER negative patients. Our study supports TIM-3 as a target for breast cancer immunotherapy.

Abstract Purpose Alterations to mismatch repair (MMR) pathways are a known cause of cancer, particularly colorectal and endometrial carcinomas. Recently, checkpoint inhibitors have been approved for use in MMR-deficient cancers of any type (Prasad et al. in JAMA Oncol 4:157–158, 2018). Functional studies in breast cancer have shown associations between MMR loss, resistance to aromatase inhibitors and sensitivity to palbociclib (Haricharan et al. in Cancer Discov 7:1168–1183, 2017). Herein, we investigate the clinical meaning of MMR deficiency in breast cancer by immunohistochemical assessment of MSH2, MSH6, MLH1 and PMS2 on a large series of breast cancers linked to detailed biomarker and long-term outcome data. Methods Cases were classified as MMR intact when all four markers expressed nuclear reactivity, but MMR-deficient when at least one of the four biomarkers displayed loss of nuclear staining in the presence of positive internal stromal controls on the tissue microarray core. Results Among the 1635 cases with interpretable staining, we identified 31 (1.9%) as MMR-deficient. In our cohort, MMR deficiency was present across all major breast cancer subtypes, and was associated with high-grade, low-progesterone receptor expression and high tumor-infiltrating lymphocyte counts. MMR deficiency is significantly associated with inferior overall (HR 2.29, 95% CI 1.02–5.17, p = 0.040) and disease-specific survival (HR 2.71, 95% CI 1.00–7.35, p = 0.042) in the 431 estrogen receptor-positive patients who were uniformly treated with tamoxifen as their sole adjuvant systemic therapy. Conclusion Overall, this study supports the concept that breast cancer patients with MMR deficiency as assessed by immunohistochemistry may be good candidates for alternative treatment approaches such as immune checkpoint or CDK4 inhibitors.

Abstract Background PTEN loss is a putative driver in histotypes of ovarian cancer (high-grade serous (HGSOC), endometrioid (ENOC), clear cell (CCOC), mucinous (MOC), low-grade serous (LGSOC)). We aimed to characterise PTEN expression as a biomarker in epithelial ovarian cancer in a large population-based study. Methods Tumours from 5400 patients from a multicentre observational, prospective cohort study of the Ovarian Tumour Tissue Analysis Consortium were used to evaluate associations between immunohistochemical PTEN patterns and overall survival time, age, stage, grade, residual tumour, CD8+ tumour-infiltrating lymphocytes (TIL) counts, expression of oestrogen receptor (ER), progesterone receptor (PR) and androgen receptor (AR) by means of Cox proportional hazard models and generalised Cochran–Mantel–Haenszel tests. Results Downregulation of cytoplasmic PTEN expression was most frequent in ENOC (most frequently in younger patients; p value = 0.0001) and CCOC and was associated with longer overall survival in HGSOC (hazard ratio: 0.78, 95% CI: 0.65–0.94, p value = 0.022). PTEN expression was associated with ER, PR and AR expression ( p values: 0.0008, 0.062 and 0.0002, respectively) in HGSOC and with lower CD8 counts in CCOC ( p value &lt; 0.0001). Heterogeneous expression of PTEN was more prevalent in advanced HGSOC ( p value = 0.019) and associated with higher CD8 counts ( p value = 0.0016). Conclusions PTEN loss is a frequent driver in ovarian carcinoma associating distinctly with expression of hormonal receptors and CD8+ TIL counts in HGSOC and CCOC histotypes.

Abstract Endometrial carcinoma, the most common gynaecological cancer, develops from endometrial epithelium which is composed of secretory and ciliated cells. Pathologic classification is unreliable and there is a need for prognostic tools. We used single cell sequencing to study organoid model systems derived from normal endometrial endometrium to discover novel markers specific for endometrial ciliated or secretory cells. A marker of secretory cells (MPST) and several markers of ciliated cells (FAM92B, WDR16, and DYDC2) were validated by immunohistochemistry on organoids and tissue sections. We performed single cell sequencing on endometrial and ovarian tumours and found both secretory‐like and ciliated‐like tumour cells. We found that ciliated cell markers (DYDC2, CTH, FOXJ1, and p73) and the secretory cell marker MPST were expressed in endometrial tumours and positively correlated with disease‐specific and overall survival of endometrial cancer patients. These findings suggest that expression of differentiation markers in tumours correlates with less aggressive disease, as would be expected for tumours that retain differentiation capacity, albeit cryptic in the case of ciliated cells. These markers could be used to improve the risk stratification of endometrial cancer patients, thereby improving their management. We further assessed whether consideration of MPST expression could refine the ProMiSE molecular classification system for endometrial tumours. We found that higher expression levels of MPST could be used to refine stratification of three of the four ProMiSE molecular subgroups, and that any level of MPST expression was able to significantly refine risk stratification of the copy number high subgroup which has the worst prognosis. Taken together, this shows that single cell sequencing of putative cells of origin has the potential to uncover novel biomarkers that could be used to guide management of cancers. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley &amp; Sons, Ltd.

Abstract ARID1A (BAF250a) is a component of the SWI/SNF chromatin modifying complex, plays an important tumour suppressor role, and is considered prognostic in several malignancies. However, in ovarian carcinomas there are contradictory reports on its relationship to outcome, immune response, and correlation with clinicopathological features. We assembled a series of 1623 endometriosis‐associated ovarian carcinomas, including 1078 endometrioid (ENOC) and 545 clear cell (CCOC) ovarian carcinomas, through combining resources of the Ovarian Tumor Tissue Analysis (OTTA) Consortium, the Canadian Ovarian Unified Experimental Resource (COEUR), local, and collaborative networks. Validated immunohistochemical surrogate assays for ARID1A mutations were applied to all samples. We investigated associations between ARID1A loss/mutation, clinical features, outcome, CD8 + tumour‐infiltrating lymphocytes (CD8 + TILs), and DNA mismatch repair deficiency (MMRd). ARID1A loss was observed in 42% of CCOCs and 25% of ENOCs. We found no associations between ARID1A loss and outcomes, stage, age, or CD8 + TIL status in CCOC. Similarly, we found no association with outcome or stage in endometrioid cases. In ENOC, ARID1A loss was more prevalent in younger patients ( p = 0.012) and was associated with MMRd ( p &lt; 0.001) and the presence of CD8 + TILs ( p = 0.008). Consistent with MMRd being causative of ARID1A mutations, in a subset of ENOCs we also observed an association with ARID1A loss‐of‐function mutation as a result of small indels ( p = 0.035, versus single nucleotide variants). In ENOC, the association with ARID1A loss, CD8 + TILs, and age appears confounded by MMRd status. Although this observation does not explicitly rule out a role for ARID1A influence on CD8 + TIL infiltration in ENOC, given current knowledge regarding MMRd, it seems more likely that effects are dominated by the hypermutation phenotype. This large dataset with consistently applied biomarker assessment now provides a benchmark for the prevalence of ARID1A loss‐of‐function mutations in endometriosis‐associated ovarian cancers and brings clarity to the prognostic significance. © 2021 The Pathological Society of Great Britain and Ireland.

The association of tumor-infiltrating lymphocyte (TIL) abundance in breast cancer tissue with cancer recurrence and death in patients with early-stage triple-negative breast cancer (TNBC) who are not treated with adjuvant or neoadjuvant chemotherapy is unclear.To study the association of TIL abundance in breast cancer tissue with survival among patients with early-stage TNBC who were treated with locoregional therapy but no chemotherapy.Retrospective pooled analysis of individual patient-level data from 13 participating centers in North America (Rochester, Minnesota; Vancouver, British Columbia, Canada), Europe (Paris, Lyon, and Villejuif, France; Amsterdam and Rotterdam, the Netherlands; Milan, Padova, and Genova, Italy; Gothenburg, Sweden), and Asia (Tokyo, Japan; Seoul, Korea), including 1966 participants diagnosed with TNBC between 1979 and 2017 (with follow-up until September 27, 2021) who received treatment with surgery with or without radiotherapy but no adjuvant or neoadjuvant chemotherapy.TIL abundance in breast tissue from resected primary tumors.The primary outcome was invasive disease-free survival [iDFS]. Secondary outcomes were recurrence-free survival [RFS], survival free of distant recurrence [distant RFS, DRFS], and overall survival. Associations were assessed using a multivariable Cox model stratified by participating center.This study included 1966 patients with TNBC (median age, 56 years [IQR, 39-71]; 55% had stage I TNBC). The median TIL level was 15% (IQR, 5%-40%). Four-hundred seventeen (21%) had a TIL level of 50% or more (median age, 41 years [IQR, 36-63]), and 1300 (66%) had a TIL level of less than 30% (median age, 59 years [IQR, 41-72]). Five-year DRFS for stage I TNBC was 94% (95% CI, 91%-96%) for patients with a TIL level of 50% or more, compared with 78% (95% CI, 75%-80%) for those with a TIL level of less than 30%; 5-year overall survival was 95% (95% CI, 92%-97%) for patients with a TIL level of 50% or more, compared with 82% (95% CI, 79%-84%) for those with a TIL level of less than 30%. At a median follow-up of 18 years, and after adjusting for age, tumor size, nodal status, histological grade, and receipt of radiotherapy, each 10% higher TIL increment was associated independently with improved iDFS (hazard ratio [HR], 0.92 [0.89-0.94]), RFS (HR, 0.90 [0.87-0.92]), DRFS (HR, 0.87 [0.84-0.90]), and overall survival (0.88 [0.85-0.91]) (likelihood ratio test, P < 10e-6).In patients with early-stage TNBC who did not undergo adjuvant or neoadjuvant chemotherapy, breast cancer tissue with a higher abundance of TIL levels was associated with significantly better survival. These results suggest that breast tissue TIL abundance is a prognostic factor for patients with early-stage TNBC.

Lindsay A Brown1,2, Jeremy Hoog3, Suet-Feung Chin4,5, Yu Tao3, Abd Alnaser Zayed1,2, Koei Chin6, Andrew E Teschendorff4,5, John F Quackenbush7, John C Marioni8, Samuel Leung9,10, Charles M Perou11, Torsten O Neilsen9,10, Matthew Ellis3, Joe W Gray6, Philip S Bernard7, David G Huntsman1,2,9,10,12, and Carlos Caldas4,5,12 1Center for Translational and Applied Genomics, and the British Columbia Cancer Agency, Vancouver, British Columbia V5Z 4E6, Canada.

The loss of E-cadherin based cell-cell contacts and tumor cell migration to the vasculature and lymphatic system are hallmarks of metastasis of epithelial cancers. Type I gamma phosphatidylinositol phosphate kinase (PIPKIgamma), an enzyme that generates phosphatidylinositol 4,5-bisphosphate (PI4,5P2) a lipid messenger and precursor to many additional second messengers, was found to regulate E-cadherin cell-cell contacts and growth factor-stimulated directional cell migration, indicating that PIPKIgamma regulates key steps in metastasis. Here, we assess the expression of PIPKIgamma in breast cancers and have shown that expression correlated with disease progression and outcome.Using a tissue microarray, we analyzed 438 breast carcinomas for the levels of PIPKIgamma and investigated the correlation of PIPKIgamma expression with patient survival via Kaplan-Meier survival analysis. Moreover, via knockdown of the expression of PIPKIgamma in cultured breast cancer cells with siRNA, the roles of PIPKIgamma in breast cancer migration, invasion, and proliferation were examined.Tissue microarray data shows that approximately 18% of the cohort immunostained showed high expression of PIPKIgamma. The Kaplan-Meier survival analysis revealed a significant inverse correlation between strong PIPKIgamma expression and overall patient survival. Expression of PIPKIgamma correlated positively with epidermal growth factor receptor (EGFR) expression, which regulates breast cancer progression and metastasis. In cultured breast cancer cells, PIPKIgamma is required for growth factor stimulated migration, invasion, and proliferation of cells.The results reveal a significant correlation between PIPKIgamma expression and the progression of breast cancer. This is consistent with PIPKIgamma 's role in breast cancer cell migration, invasion, and proliferation.

Advances in molecular biology have led to the identification of potential markers of prognostic and therapeutic importance in human cancers. HER-2 testing and targeted therapy now represents a critical cornerstone in the management of breast cancer. The objectives of the current study were to determine the frequency and prognostic significance of HER-3 over-expression and HER-4 over-expression by invasive breast cancer.Tissue microarrays were constructed using clinically annotated formalin-fixed and paraffin-embedded tumor samples from 4046 patients diagnosed with invasive breast carcinoma with a median 12.5 years of follow-up. Type 1 growth factor receptor family members HER-1, HER-2, HER-3, and HER-4 expression levels were determined by immunohistochemistry, and HER-2 status was further resolved by fluorescent in-situ hybridization. The study cohort was randomly divided and analyzed as a core data set and a validation data set.HER-3 over-expression was identified in 10.0% of tumors and was a significant marker of reduced patient breast cancer-specific survival on univariate analysis (P = 1.32 x 10(-5)). Furthermore, in tumors with normal expression levels of HER-1 and HER-2, the overexpression of HER-3 had a significant negative prognostic effect on disease-specific survival (HR: 1.541, 95% CI: 1.166-2.036, P = 2.37 x 10(-3)) independent of patient age at diagnosis, Estrogen receptor status, tumor grade, tumor size, nodal status, and the presence of lymphatic or vascular invasion by cancer. HER-4 overexpression was identified in 78.2% of breast cancers and was not a significant marker of patient survival (P = 0.214). Results of all statistical tests were positively confirmed in the validation data set analysis.HER-3 status is an important prognostic marker of disease-specific survival in patients with invasive breast cancer. Accordingly, evaluation of the HER-3 expression level may identify a subset of patients with a poor disease prognosis, and who could undergo further evaluation for the efficacy of HER-3 targeted anticancer agents.

<h3>Importance</h3> Accumulating evidence indicates that tumor-infiltrating lymphocytes (TILs) are associated with clinical outcomes and may predict the efficacy of chemotherapy and human epidermal growth factor receptor 2 (HER2, encoded by the gene<i>ERBB2</i>)–targeted therapy in patients with HER2-positive breast cancer. <h3>Objective</h3> To investigate the role of TILs, particularly cytotoxic CD8⁺T cells, in the prediction of outcomes in patients with HER2-positive metastatic breast cancer randomized to an antibody-based (trastuzumab) vs a small molecule–based (lapatinib) anti-HER2 therapy. <h3>Design, Setting, and Participants</h3> The Canadian Cancer Trials Group MA.31 phase 3 clinical trial accrued patients from 21 countries and randomized 652 with HER2-positive metastatic breast cancer to receive trastuzumab or lapatinib, in combination with a taxane, from January 17, 2008, through December 1, 2011. Patients had received no prior chemotherapy or HER2-targeted therapy in the metastatic setting. The median follow-up was 21.5 months (interquartile range, 14.3-31.0). The tumor tissue collected for primary diagnosis was used in this ad hoc substudy. Sections were scored for TILs on hematoxylin-eosin (H&amp;E)–stained sections, and immunohistochemical analysis was performed to assess CD8, FOXP3, CD56, and programmed cell death protein 1 (PD-1) expression on stromal (sTILs) and intratumoral TILs. Data were analyzed from July 15, 2015, through July 27, 2016. <h3>Interventions</h3> Treatment with trastuzumab or lapatinib in combination with taxane chemotherapy (paclitaxel or docetaxel) for 24 weeks. <h3>Main Outcomes and Measures</h3> Prognostic effects of biomarkers were evaluated for progression-free survival by stratified univariate log-rank test with Kaplan-Meier curves and by multivariate Cox proportional hazards regression; predictive effects were examined with a test of interaction between treatment allocation and biomarker classification. <h3>Results</h3> Of the 647 treated women (mean [SD] age, 55.0 [10.8] years), 614 had tumor tissue samples scored for H&amp;E sTILs and 427 for CD8 biomarker assessments. Overall H&amp;E sTIL counts of greater than 5% (high) were present in 215 cases (35%) but did not show significant prognostic or predictive effects. Univariate stratified analyses detected a significant predictive effect on risk for progression with lapatinib compared with trastuzumab among patients with low CD8⁺sTIL counts (observed hazard ratio, 2.94; 95% CI, 1.40-6.17;<i>P</i> = .003) and among those with high CD8⁺sTIL counts (observed hazard ratio, 1.36; 95% CI, 1.05-1.75;<i>P</i> = .02), confirmed in stepwise multivariate analysis (interaction<i>P</i> = .04). Other immunohistochemistry biomarkers were not associated with prognostic or predictive effects. <h3>Conclusions and Relevance</h3> In this secondary analysis of a phase 3 randomized clinical trial, a low level of preexisting cytotoxic sTILs predicted the most benefit from an antibody- vs a small molecule–based drug against the same target. <h3>Trial Registration</h3> clinicaltrials.gov Identifier:NCT00667251

Introduction Vulvar squamous cell carcinoma develops through two separate pathways, associated with the presence or absence of high-risk human papilloma virus (HPV). The objective of this study was to evaluate treatment response and clinical outcomes in women with HPV-associated versus HPV-independent vulvar squamous cell carcinoma treated with primary radiation therapy, in order to determine the ability to use HPV status as a predictor of response to radiation therapy. Methods This was a retrospective cohort study combining data from British Columbia Cancer, Canada and Duke University, USA. Patients were included who had been treated with radiation therapy but excluded if they had received major surgical interventions. Immunohistochemistry for p16 (as a surrogate for high-risk HPV infection) and p53 was performed. We analyzed the univariable association between p16 status and clinico-pathological features and performed univariable survival analysis for p16. Results Forty-eight patients with vulvar squamous cell carcinoma treated with primary radiation therapy were identified: 26 p16 positive/HPV-associated patients and 22 p16 negative/HPV-independent patients. p16 positive vulvar squamous cell carcinoma demonstrated a significantly improved overall survival (HR 0.39, p=0.03) and progression-free survival (HR 0.35, p=0.02). In women treated with definitive radiation therapy, p16 positivity was associated with improved overall survival (HR 0.29, p&lt;0.01) and progression-free survival (HR 0.21, p&lt;0.01). Among patients who received sensitizing chemotherapy, a significant association was observed with p16 positive tumors and overall survival (HR 0.25, p=0.03) and progression-free survival (HR 0.09, p&lt;0.01). Conclusion This study suggests that HPV status in vulvar squamous cell carcinoma has both prognostic and predictive implications, with increased radiosensitivity demonstrated in HPV-associated vulvar squamous cell carcinoma. Implications may include radiation dose de-escalation for HPV-associated vulvar squamous cell carcinoma and increased surgical aggressiveness for HPV-independent vulvar squamous cell carcinoma.

Aims Ovarian endometrioid carcinoma ( EC ) generally has a good prognosis. Adjuvant chemotherapy can be spared in low‐stage disease, but prognostic biomarkers are needed to refine the treatment threshold. Wnt/β‐catenin signalling is commonly altered in EC . We examined immunohistochemical expression of nuclear β‐catenin and CDX 2 as prognostic biomarkers for EC ; both are mediators of the Wnt pathway. Methods and results We evaluated two ovarian EC cohorts, discovery set ( n = 183) and validation set ( n = 174), with ovarian cancer‐specific survival ( OCSS ) as the primary end‐point. In univariable analysis, nuclear β‐catenin expression was significantly associated with longer OCSS in the discovery set [hazard ratio ( HR ) = 0.36, 95% confidence interval ( CI ) = 0.16–0.74, P = 0.004] and the validation set ( HR = 0.35, 95% CI = 0.11–0.89, P = 0.006). Similar significant associations were observed with CDX 2 expression in the discovery set ( HR = 0.25, 95% CI = 0.11–0.50, P &lt; 0.001) and validation set ( HR = 0.27, 95% CI = 0.07–0.75, P = 0.020). In multivariable analysis, combined positivity of both markers was significantly associated with longer OCSS in the discovery set ( HR = 0.20, 95% CI = 0.06–0.49, P &lt; 0.001) and in the validation set ( HR = 0.33 95% CI = 0.07–0.1.06, P = 0.047). In a stratified analysis for stage IC / II EC , combined positivity identified a subset of patients with a significantly longer OCSS in the discovery cohort but only a non‐significant trend in the validation cohort. Conclusions Nuclear β‐catenin and CDX 2 expression individually or in combination are validated prognostic markers for ovarian EC . However, their full potential to stratify low risk patients at adjuvant threshold awaits further multimarker study.

Glucocorticoid-induced TNF receptor (GITR) is an emerging immunotherapy target that is expressed at high levels on regulatory T cells. Agonistic anti-GITR antibodies have anti-tumor activity in cancer mouse models, and recent phase 1 trials have demonstrated their safe pharmacological profile. However, there is limited knowledge on the relationship between GITR expression and the tumor microenvironment. GITR protein expression was assayed by immunohistochemistry on 3992 breast cancer surgical excision specimens assembled into tissue microarrays and scored visually by a pathologist for GITR expression on tumor-infiltrating lymphocytes and on carcinoma cells. GITR expression by the malignant cells was further surveyed in gastrointestinal stromal tumor (N = 713), lung carcinoma (N = 705), pancreatic cancer (N = 486), ovarian cancer (N = 445), bladder cancer (N = 88), prostate cancer (N = 88), testicular cancer (N = 76), melanoma (N = 75), renal cell carcinoma (N = 68), epithelioid sarcoma (N = 53), and neuroendocrine tumors (N = 41). In breast cancer, GITR expression on tumor-infiltrating lymphocytes (12.4%) correlated with other immune response biomarkers (PD-L1+ on tumor cells, and PD-1+, LAG-3+, TIM-3+ lymphocytes; p < 0.001), and T-cell markers (CD8+, FOXP3+; p < 0.001). GITR+ carcinoma cells were observed in 6.0% of breast cancer cases and correlated with worse relapse-free survival (p = 0.015). Among the additional tumor types examined, cancers with GITR+ malignant cells included bladder cancer (5.7%), primary (but not metastatic) melanoma (4.5%), and ovarian cancer (3.2%); no expression was identified among examined sarcomas. To our knowledge, this is the first immunohistochemistry study to report the frequency and pattern of GITR expression in a large breast cancer cohort, or to report membranous GITR expression on malignant cells. The co-infiltration of GITR with other immune biomarkers and T-cell markers supports a potential role for anti-GITR agents in combination immunotherapies. In addition, GITR expression on carcinoma cells could imply the existence of a novel cancer immune evasion strategy worthy of further investigation.

There is no consensus on the cutoff for positivity of estrogen receptor (ER) and progesterone receptor (PR) in endometrial cancer (EC). Therefore, we determined the cutoff value for ER and PR expression with the strongest prognostic impact on the outcome. Immunohistochemical expression of ER and PR was scored as a percentage of positive EC cell nuclei. Cutoff values were related to disease-specific survival (DSS) and disease-free survival (DFS) using sensitivity, specificity, and multivariable regression analysis. The results were validated in an independent cohort. The study cohort (n = 527) included 82% of grade 1–2 and 18% of grade 3 EC. Specificity for DSS and DFS was highest for the cutoff values of 1–30%. Sensitivity was highest for the cutoff values of 80–90%. ER and PR expression were independent markers for DSS at cutoff values of 10% and 80%. Consequently, three subgroups with distinct clinical outcomes were identified: 0–10% of ER/PR expression with, unfavorable outcome (5-year DSS = 75.9–83.3%); 20–80% of ER/PR expression with, intermediate outcome (5-year DSS = 93.0–93.9%); and 90–100% of ER/PR expression with, favorable outcome (5-year DSS = 97.8–100%). The association between ER/PR subgroups and outcomes was confirmed in the validation cohort (n = 265). We propose classification of ER and PR expression based on a high-risk (0–10%), intermediate-risk (20–80%), and low-risk (90–100%) group.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, characterized by substantial risks of early disease recurrence and mortality. We constructed and validated clinical calculators for predicting recurrence-free survival (RFS) and overall survival (OS) for TNBC. Data from 605 women with centrally confirmed TNBC who underwent primary breast cancer surgery at Mayo Clinic during 1985–2012 were used to train risk models. Variables included age, menopausal status, tumor size, nodal status, Nottingham grade, surgery type, adjuvant radiation therapy, adjuvant chemotherapy, Ki67, stromal tumor-infiltrating lymphocytes (sTIL) score, and neutrophil-to-lymphocyte ratio (NLR). Final models were internally validated for calibration and discrimination using ten-fold cross-validation and compared with their base-model counterparts which include only tumor size and nodal status. Independent external validation was performed using data from 478 patients diagnosed with stage II/III invasive TNBC during 1986–1992 in the British Columbia Breast Cancer Outcomes Unit database. Final RFS and OS models were well calibrated and associated with C-indices of 0.72 and 0.73, as compared with 0.64 and 0.62 of the base models (p < 0.001). In external validation, the discriminant ability of the final models was comparable to the base models (C-index: 0.59–0.61). The RFS model demonstrated greater accuracy than the base model both overall and within patient subgroups, but the advantages of the OS model were less profound. This TNBC clinical calculator can be used to predict patient outcomes and may aid physician’s communication with TNBC patients regarding their long-term disease outlook and planning treatment strategies.

Abstract Background: The prognostic value of stromal tumor-infiltrating lymphocytes (TILs) as a biomarker for triple-negative breast cancer (TNBC) has been extensively demonstrated in patients (pts) receiving (neo)adjuvant systemic therapy. In addition, several small studies suggest that a subset of pts with early-stage TNBC and high TILs have excellent long-term outcomes, even in the absence of systemic therapy [1-3]. However, data on the absolute risk of TNBC recurrence according to TIL levels in the absence of systemic therapy are limited and critical to inform the design of future systemic therapy de-escalation clinical trials. Methods: We conducted an individual patient data pooled analysis of 12 international cohorts of pts with TNBC treated with locoregional therapy but no systemic therapy. TNBC was defined as tumors with estrogen and progesterone receptor of &amp;lt; 1% and HER2 negative (IHC 0, 1+ or IHC 2+ and FISH negative) per local evaluation. TILs were locally assessed in hematoxylin &amp; eosin-stained slides according to the International Immuno-Oncology Biomarker Working Group guidelines (www.tilsinbreastcancer.org). We used the Kaplan-Meier method to assess survival outcomes according to prespecified TIL thresholds: 30% and 50%. Confidence intervals (CI) for survival probabilities were calculated using a percentile bootstrap method. The primary endpoint was invasive disease-free survival (iDFS, STEEP 2.0 definition). Key secondary outcomes included recurrence-free survival (RFS), distant disease-free survival (DDFS) and overall survival (OS). Results: 1,835 pts diagnosed with TNBC between 1982 and 2017 who did not receive systemic therapy were included. The median age at diagnosis was 56 (IQR 38-71). Menopausal status was known in 1,184 women, of whom 78% were post-menopausal. The median tumor size was 2.0 cm (IQR 1.2-2.6). Most pts (87%) had no axillary lymph node involvement (N0). Most tumors were invasive ductal carcinoma (74%) and grade 3 (70%). The median level of TILs was 15% (IQR 5-40). The median duration of follow-up was 30.4 years (95% CI 29.9, 31.1). A total of 950 (52%) iDFS, 828 (45%) RFS, 767 (42%) DDFS events, and 604 (33%) deaths were observed. In multivariable analyses, higher TILs were independently associated with improved iDFS, RFS, DDFS, and OS beyond clinicopathological factors (likelihood ratio p&amp;lt; 10e-6). Each 10% increment in stromal TILs was associated with an 8% (95% CI: 6-11), 10% (95% CI: 7-13), and 13% (95% CI: 10-15) reduction in the risk of experiencing an iDFS, RFS or DDFS event, and with a 12% (95% CI: 9-15) reduction in the risk of death. iDFS, RFS, DDFS and OS rates according to different TIL thresholds and nodal status are shown in the Table. Of note, the RFS estimates (which exclude second non-breast primaries and contralateral breast cancers) were consistently higher than the iDFS counterparts (which include both), consistent with a high rate of contralateral breast cancers and second primary tumors in this cohort. Notably, patients with node-negative—and especially stage I—TNBC with high TILs had excellent survival rates at 10-year follow-up. Conclusion: TILs are highly prognostic in pts with systemically untreated early-stage TNBC. Pts with pN0 (and especially stage I) TNBC with high TILs exhibited very favorable long-term outcomes even in the absence of systemic therapy. These data define the natural history of TIL-rich TNBC pts and are crucial to identifying the optimal patient population for future chemotherapy and immunotherapy de-escalation clinical trials. References: [1] Leon-Ferre et al, 2017, PMID: 28913760 [2] Park et al, 2019, PMID: 31566659 [3] de Jong et al, 2022, PMID: 35353548 Table 5 and 10-year survival endpoints according TIL level, nodal status, and stage Citation Format: Roberto A. Leon-Ferre, Sarah Flora Jonas, Roberto Salgado, Sherene Loi, Vincent De Jong, Jodi M. Carter, Torsten Nielson, Samuel Leung, Nazia Riaz, Giuseppe Curigliano, Carmen Criscitiello, Vincent Cockenpot, Matteo Lambertini, Vera Suman, Barbro Linderholm, John WM Martens, Carolien HM van Deurzen, Mieke Timmermans, Tatsunori Shimoi, Shu Yazaki, Masayuki Yoshida, Sung-Bae Kim, Hee Jin Lee, Maria Vittoria Dieci, Guillaume Bataillon, Anne Salomon, Fabrice Andre, Marleen Kok, Sabine Linn, Matthew P. Goetz, Stefan Michiels. Stromal tumor-infiltrating lymphocytes identify early-stage triple-negative breast cancer patients with favorable outcomes at 10-year follow-up in the absence of systemic therapy: a pooled analysis of 1835 patients [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD9-05.

Objectives Despite recommendations for integrating molecular classification of endometrial cancers (EC) into pathology reporting and clinical management, uptake is inconsistent. To assign ProMisE subtype, all molecular components must be available (POLE mutation status, mismatch repair (MMR) and p53 immunohistochemistry (IHC)) and often these are assessed at different stages of care and/or at different centres resulting in delays in treatment. We assessed a single-test DNA-based targeted next generation sequencing (NGS) molecular classifier (ProMisE NGS), comparing concordance and prognostic value to the original ProMisE classifier. Methods DNA was extracted from formalin-fixed paraffin embedded (FFPE) ECs that had previously undergone ProMisE molecular classification (POLE sequencing, IHC for p53 and MMR). DNA was sequenced using the clinically validated Imagia Canexia Health Find It™ amplicon-based NGS gene panel assay to assess for pathogenic POLE mutations (unchanged from original ProMisE), TP53 mutations (in lieu of p53 IHC), and microsatellite instability (MSI) (in lieu of MMR IHC),with the same order of segregation as original ProMisE used for subtype assignment. Molecular subtype assignment of both classifiers was compared by concordance metrics and Kaplan-Meier survival statistics. Results The new DNA-based NGS molecular classifier (ProMisE NGS) was used to determine the molecular subtype in 164 ECs previously classified with ProMisE. 159/164 cases were concordant with a kappa statistic of 0.96 and an overall accuracy of 0.97. Prognostic differences in progression-free, disease-specific and overall survival between the four molecular subtypes were observed for the new NGS classifier, recapitulating the survival curves of the original ProMisE classifier. ProMisE NGS was 100% concordant between matched biopsy and hysterectomy samples. Conclusion ProMisE NGS is feasible on standard FFPE material, demonstrates high concordance with the original ProMisE classifier and maintains prognostic value in EC. This test has the potential to facilitate implementation of molecular classification of EC at the time of first diagnosis.

Activated v-AKT murine thymoma viral oncogene homolog 1 (AKT)/protein kinase B (PKB) kinase (pAKT) is localized to the plasma membrane, cytoplasm, and/or nucleus in 50% of cancers. The clinical importance of pAKT localization and the mechanism(s) controlling this compartmentalization are unknown. In this study, we examined nuclear and cytoplasmic phospho-AKT (pAKT) expression by immunohistochemistry in a breast cancer tissue microarray (n = 377) with ≈15 years follow-up and integrated these data with the expression of estrogen receptor (ER)α, progesterone receptor (PR), and FOXA1. Nuclear localization of pAKT (nuclear-pAKT) was associated with long-term survival (P = 0.004). Within the ERα+/PR+ subgroup, patients with nuclear-pAKT positivity had better survival than nuclear-pAKT–negative patients (P ≤ 0.05). The association of nuclear-pAKT with the ERα+/PR+ subgroup was validated in an independent cohort (n = 145). TCL1 family proteins regulate nuclear transport and/or activation of AKT. TCL1B is overexpressed in ERα-positive compared with ERα-negative breast cancers and in lung metastasis–free breast cancers. Therefore, we examined the possible control of TCL1 family member(s) expression by the estrogen:ERα pathway. Estradiol increased TCL1B expression and increased nuclear-pAKT levels in breast cancer cells; short-interfering RNA against TCL1B reduced nuclear-pAKT. Overexpression of nuclear-targeted AKT1 in MCF-7 cells increased cell proliferation without compromising sensitivity to the anti-estrogen, tamoxifen. These results suggest that subcellular localization of activated AKT plays a significant role in determining its function in breast cancer, which in part is dependent on TCL1B expression. Activated v-AKT murine thymoma viral oncogene homolog 1 (AKT)/protein kinase B (PKB) kinase (pAKT) is localized to the plasma membrane, cytoplasm, and/or nucleus in 50% of cancers. The clinical importance of pAKT localization and the mechanism(s) controlling this compartmentalization are unknown. In this study, we examined nuclear and cytoplasmic phospho-AKT (pAKT) expression by immunohistochemistry in a breast cancer tissue microarray (n = 377) with ≈15 years follow-up and integrated these data with the expression of estrogen receptor (ER)α, progesterone receptor (PR), and FOXA1. Nuclear localization of pAKT (nuclear-pAKT) was associated with long-term survival (P = 0.004). Within the ERα+/PR+ subgroup, patients with nuclear-pAKT positivity had better survival than nuclear-pAKT–negative patients (P ≤ 0.05). The association of nuclear-pAKT with the ERα+/PR+ subgroup was validated in an independent cohort (n = 145). TCL1 family proteins regulate nuclear transport and/or activation of AKT. TCL1B is overexpressed in ERα-positive compared with ERα-negative breast cancers and in lung metastasis–free breast cancers. Therefore, we examined the possible control of TCL1 family member(s) expression by the estrogen:ERα pathway. Estradiol increased TCL1B expression and increased nuclear-pAKT levels in breast cancer cells; short-interfering RNA against TCL1B reduced nuclear-pAKT. Overexpression of nuclear-targeted AKT1 in MCF-7 cells increased cell proliferation without compromising sensitivity to the anti-estrogen, tamoxifen. These results suggest that subcellular localization of activated AKT plays a significant role in determining its function in breast cancer, which in part is dependent on TCL1B expression. The serine/threonine kinase AKT is a multifunctional kinase that is activated in response to a variety of extracellular signals.1Martelli AM Faenza I Billi AM Manzoli L Evangelisti C Fala F Cocco L Intranuclear 3′-phosphoinositide metabolism and Akt signaling: new mechanisms for tumorigenesis and protection against apoptosis?.Cell Signal. 2006; 18: 1101-1107Crossref PubMed Scopus (120) Google Scholar, 2Dillon RL White DE Muller WJ The phosphatidyl inositol 3-kinase signaling network: implications for human breast cancer.Oncogene. 2007; 26: 1338-1345Crossref PubMed Scopus (242) Google Scholar, 3Datta SR Brunet A Greenberg ME Cellular survival: a play in three Akts.Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3718) Google Scholar Inactive AKT from the cytosol is recruited to the plasma membrane in the presence of phosphoinositide triphosphate, where it is activated by phosphorylation of residues T308 by phosphoinositide-dependent kinase 1 (PDK1) and S473 by mTOR/rictor or unidentified kinase(s).1Martelli AM Faenza I Billi AM Manzoli L Evangelisti C Fala F Cocco L Intranuclear 3′-phosphoinositide metabolism and Akt signaling: new mechanisms for tumorigenesis and protection against apoptosis?.Cell Signal. 2006; 18: 1101-1107Crossref PubMed Scopus (120) Google Scholar, 2Dillon RL White DE Muller WJ The phosphatidyl inositol 3-kinase signaling network: implications for human breast cancer.Oncogene. 2007; 26: 1338-1345Crossref PubMed Scopus (242) Google Scholar, 4Sarbassov DD Guertin DA Ali SM Sabatini DM Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex.Science. 2005; 307: 1098-1101Crossref PubMed Scopus (5223) Google Scholar Activated AKT, as measured mostly by antibodies that recognize phosphorylated AKT at S473 (pAKT), can be found at the plasma membrane, in the cytoplasm, and in the nucleus.1Martelli AM Faenza I Billi AM Manzoli L Evangelisti C Fala F Cocco L Intranuclear 3′-phosphoinositide metabolism and Akt signaling: new mechanisms for tumorigenesis and protection against apoptosis?.Cell Signal. 2006; 18: 1101-1107Crossref PubMed Scopus (120) Google Scholar Activation of AKT, either due to a point mutation or point mutations in the upstream PI3 kinase, or loss of the upstream tumor suppressor PTEN, or growth factor overexpression, is observed in ≈50% of all cancers.2Dillon RL White DE Muller WJ The phosphatidyl inositol 3-kinase signaling network: implications for human breast cancer.Oncogene. 2007; 26: 1338-1345Crossref PubMed Scopus (242) Google Scholar, 5Carpten JD Faber AL Horn C Donoho GP Briggs SL Robbins CM Hostetter G Boguslawski S Moses TY Savage S Uhlik M Lin A Du J Qian YW Zeckner DJ Tucker-Kellogg G Touchman J Patel K Mousses S Bittner M Schevitz R Lai MH Blanchard KL Thomas JE A transforming mutation in the pleckstrin homology domain of AKT1 in cancer.Nature. 2007; 448: 439-444Crossref PubMed Scopus (999) Google Scholar, 6Manning BD Cantley LC AKT/PKB signaling: navigating downstream.Cell. 2007; 129: 1261-1274Abstract Full Text Full Text PDF PubMed Scopus (4692) Google Scholar, 7Samuels Y Wang Z Bardelli A Silliman N Ptak J Szabo S Yan H Gazdar A Powell SM Riggins GJ Willson JK Markowitz S Kinzler KW Vogelstein B Velculescu VE High frequency of mutations of the PIK3CA gene in human cancers.Science. 2004; 304: 554Crossref PubMed Scopus (2915) Google Scholar The carcinogenic action of AKT, until recently, was believed to arise exclusively from the cytoplasm, possibly through regulation of cell size, energy metabolism, and translational control.1Martelli AM Faenza I Billi AM Manzoli L Evangelisti C Fala F Cocco L Intranuclear 3′-phosphoinositide metabolism and Akt signaling: new mechanisms for tumorigenesis and protection against apoptosis?.Cell Signal. 2006; 18: 1101-1107Crossref PubMed Scopus (120) Google Scholar, 8Plas DR Thompson CB Akt-dependent transformation: there is more to growth than just surviving.Oncogene. 2005; 24: 7435-7442Crossref PubMed Scopus (339) Google Scholar However, recent studies have identified a pool of activated AKT within the nucleus (nuclear-pAKT), where it can block apoptosis.1Martelli AM Faenza I Billi AM Manzoli L Evangelisti C Fala F Cocco L Intranuclear 3′-phosphoinositide metabolism and Akt signaling: new mechanisms for tumorigenesis and protection against apoptosis?.Cell Signal. 2006; 18: 1101-1107Crossref PubMed Scopus (120) Google Scholar, 9Ahn JY Liu X Liu Z Pereira L Cheng D Peng J Wade PA Hamburger AW Ye K Nuclear Akt associates with PKC-phosphorylated Ebp1, preventing DNA fragmentation by inhibition of caspase-activated DNase.EMBO J. 2006; 25: 2083-2095Crossref PubMed Scopus (106) Google Scholar Indeed, studies in cardiomyocytes have revealed gender-specific differences in the subcellular localization of pAKT.10Camper-Kirby D Welch S Walker A Shiraishi I Setchell KD Schaefer E Kajstura J Anversa P Sussman MA Myocardial Akt activation and gender: increased nuclear activity in females versus males.Circ Res. 2001; 88: 1020-1027Crossref PubMed Scopus (248) Google Scholar Elevated nuclear-pAKT found in cardiomyocytes of premenopausal women, as compared with men or postmenopausal women, is thought to be cardioprotective.11Shiraishi I Melendez J Ahn Y Skavdahl M Murphy E Welch S Schaefer E Walsh K Rosenzweig A Torella D Nurzynska D Kajstura J Leri A Anversa P Sussman MA Nuclear targeting of Akt enhances kinase activity and survival of cardiomyocytes.Circ Res. 2004; 94: 884-891Crossref PubMed Scopus (181) Google Scholar, 12Hayward CS Kelly RP Collins P The roles of gender, the menopause and hormone replacement on cardiovascular function.Cardiovasc Res. 2000; 46: 28-49Crossref PubMed Scopus (243) Google Scholar These studies strongly suggest distinct biological actions for cytoplasmic and nuclear-pAKT, which may be controlled by sex hormones. Thus, AKT activity and/or localization may be different in estrogen receptor–positive (ERα-positive) and ERα-negative breast cancers. ERα-positive breast cancers are further subclassified into luminal type A and luminal type B.13Sorlie T Perou CM Tibshirani R Aas T Geisler S Johnsen H Hastie T Eisen MB van de Rijn M Jeffrey SS Thorsen T Quist H Matese JC Brown PO Botstein D Eystein Lonning P Borresen-Dale AL Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications.Proc Natl Acad Sci U S A. 2001; 98: 10869-10874Crossref PubMed Scopus (8513) Google Scholar Luminal type A breast cancers are associated with elevated expression of transcription factors progesterone receptor (PR), FOXA1, and GATA-3.13Sorlie T Perou CM Tibshirani R Aas T Geisler S Johnsen H Hastie T Eisen MB van de Rijn M Jeffrey SS Thorsen T Quist H Matese JC Brown PO Botstein D Eystein Lonning P Borresen-Dale AL Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications.Proc Natl Acad Sci U S A. 2001; 98: 10869-10874Crossref PubMed Scopus (8513) Google Scholar, 14Badve S Nakshatri H Oestrogen-receptor-positive breast cancer: towards bridging histopathological and molecular classifications.J Clin Pathol. 2009; 62: 6-12Crossref PubMed Scopus (65) Google Scholar, 15Badve S Turbin D Thorat MA Morimiya A Nielsen TO Perou CM Dunn S Huntsman DG Nakshatri H FOXA1 expression in breast cancer correlation with luminal subtype a and survival.Clin Cancer Res. 2007; 13: 4415-4421Crossref PubMed Scopus (197) Google Scholar ERα, FOXA1, and GATA-3 form a transcription factor network that determines hormonal response and hence anti-estrogen sensitivity of luminal type A breast cancers.16Eeckhoute J Keeton EK Lupien M Krum SA Carroll JS Brown M Positive cross-regulatory loop ties GATA-3 to estrogen receptor alpha expression in breast cancer.Cancer Res. 2007; 67: 6477-6483Crossref PubMed Scopus (286) Google Scholar In contrast, luminal type B breast cancers are associated with increased expression of proliferation-associated genes including Ki-67.17Cheang MC Chia SK Voduc D Gao D Leung S Snider J Watson M Davies S Bernard PS Parker JS Perou CM Ellis MJ Nielsen TO Ki67 index. HER2 status, and prognosis of patients with luminal B breast cancer.J Natl Cancer Inst. 2009; 101: 736-750Crossref PubMed Scopus (1594) Google Scholar These cancers can be either ERα-positive or PR-positive and include a small subfraction of ERα-positive breast cancers that overexpress HER2. The status of activated AKT and its subcellular distribution in both types of ERα-positive luminal breast cancers are unknown. To determine subcellular distribution status of activated AKT and its relationship to ERα status, we investigated the distribution pattern of pAKT in a tissue microarray (TMA) comprising samples from 377 patients with ≈15 years of clinical follow-up, and also an independent analysis of a second TMA, comprising 118 invasive breast carcinomas. We report preferential nuclear localization of pAKT in ERα-positive breast cancers and the luminal A subtype. Furthermore, we show that a TCL1B-dependent estrogen:ERα-regulated signaling pathway partly controls the activation of nuclear AKT. Patient information, tumor pathology, and the expression of a number of biomarkers in a tissue microarray (GPEC-TMA) comprising 438 patient samples have been reported previously.18Sutherland BW Kucab J Wu J Lee C Cheang MC Yorida E Turbin D Dedhar S Nelson C Pollak M Leighton Grimes H Miller K Badve S Huntsman D Blake-Gilks C Chen M Pallen CJ Dunn SE Akt phosphorylates the Y-box binding protein 1 at Ser102 located in the cold shock domain and affects the anchorage-independent growth of breast cancer cells.Oncogene. 2005; 24: 4281-4292Crossref PubMed Scopus (231) Google Scholar The histological distribution included infiltrating ductal carcinoma (n = 379), infiltrating lobular carcinoma (n = 41), and special types (n = 8). Because of the loss of cores during the staining process with one or more stains, the final analysis included data from 377 patients. Median age and median survival years were 61.48 and 11.93, respectively. The second TMA (Cedars Sinai-UCLA Medical Center) with 145 cases of invasive carcinoma that was used for validation of results has also been described previously.19Wolf I Bose S Williamson EA Miller CW Karlan BY Koeffler HP FOXA1: growth inhibitor and a favorable prognostic factor in human breast cancer.Int J Cancer. 2007; 120: 1013-1022Crossref PubMed Scopus (92) Google Scholar, 20Bose S Chandran S Mirocha JM Bose N The Akt pathway in human breast cancer: a tissue-array-based analysis.Mod Pathol. 2006; 19: 238-245Crossref PubMed Scopus (167) Google Scholar This study was performed under human institutional review board approval with strict adherence to all established guidelines. Immunohistochemistry for pAKT (S473), ERα, PR, FOXA1, and GATA-3 has been described previously.18Sutherland BW Kucab J Wu J Lee C Cheang MC Yorida E Turbin D Dedhar S Nelson C Pollak M Leighton Grimes H Miller K Badve S Huntsman D Blake-Gilks C Chen M Pallen CJ Dunn SE Akt phosphorylates the Y-box binding protein 1 at Ser102 located in the cold shock domain and affects the anchorage-independent growth of breast cancer cells.Oncogene. 2005; 24: 4281-4292Crossref PubMed Scopus (231) Google Scholar Samples that did not stain were classified as negative (score 0), whereas those showing the highest intensity staining received a score of 3. Samples showing weak intensity staining received a score of 1, whereas those showing moderate staining were scored as 2. Samples were stratified based on the nuclear versus cytoplasmic localization of pAKT. Two pathologists scored the staining intensities and nuclear versus cytoplasmic staining pattern of pAKT. Unsupervised hierarchical clustering involving four variables, ERα, PR, FOXA1, and pAKT, was performed using Genesis 1.6.0 β 1 software.21Sturn A Quackenbush J Trajanoski Z Genesis: cluster analysis of microarray data.Bioinformatics. 2002; 18: 207-208Crossref PubMed Scopus (1471) Google Scholar Clustering was based on all 438 patients. One of the groups consisted of cases with missing data for ERα, PR, FOXA1, and pAKT (including those with missing cores). This group was not further analyzed. The Kaplan–Meier method was used for survival analysis, and hazard ratios were calculated using a log-rank test. A P value of less than 0.05 was considered as significant. All survival and correlation analyses were performed using SPSS 16.0 (SPSS Inc, Chicago, IL). ERα antibodies used for chromatin immunoprecipitation (ChIP) were: ab 10 (Neomarker, Fremont, CA) and ERα sc-543 (Santa Cruz Biotechnologies, Santa Cruz, CA). poly(ADP-ribose) polymerase (PARP) (Santa Cruz Biotechnologies, Santa Cruz, CA) and α-Tubulin (Sigma, St. Louis, MO) were used to ensure separation of nuclear and cytoplasmic proteins. Antibodies against AKT, pAKT, and a Hemagglutinin (HA)-tag were purchased from Cell Signaling Technologies (Danvers, MA). TCL1B antibody was purchased from Santa Cruz Biotechnologies. HA-tagged wild-type AKT1, AKT1 with a T308D/S473D mutation (HA-AKT-CA), and a ΔNES-mutant (L277A/L280A/L282A) in pcDNA3 vector have been described.22Saji M Vasko V Kada F Allbritton EH Burman KD Ringel MD Akt1 contains a functional leucine-rich nuclear export sequence.Biochem Biophys Res Commun. 2005; 332: 167-173Crossref PubMed Scopus (65) Google Scholar Wild-type AKT1, mutant AKT1, and TCL1B expressing retroviruses were constructed using the bicistronic expression vector pcQXIN, packaged, and used for MCF-7 and/or T47-D cell infections.23Bhat-Nakshatri P Wang G Appaiah H Luktuke N Carroll JS Geistlinger TR Brown M Badve S Liu Y Nakshatri H AKT Alters genome-wide estrogen receptor alpha binding and impacts estrogen signaling in breast cancer.Mol Cell Biol. 2008; 28: 7487-7503Crossref PubMed Scopus (82) Google Scholar Infected cells were selected using G418 (600 mg/ml), and polyclonal cultures were analyzed. Cell proliferation was measured by BrdU-ELISA assay (EMD Biosciences, La Jolla, CA). All studies were done with cells maintained in phenol red–free charcoal dextran–treated fetal calf serum (CCS-5%)-containing media for at least four days before initiating experiments. Most of the cell fractionation studies were done with cells that were additionally maintained overnight in media with 1% CCS. Cells in studies involving siRNA transfections were transfected in regular media and changed to the phenol red–free 5% CCS media 24-hours after transfection to avoid transfection-associated toxicity. ChIP coupled microarray assays were performed as described previously.24Carroll JS Meyer CA Song J Li W Geistlinger TR Eeckhoute J Brodsky AS Keeton EK Fertuck KC Hall GF Wang Q Bekiranov S Sementchenko V Fox EA Silver PA Gingeras TR Liu XS Brown M Genome-wide analysis of estrogen receptor binding sites.Nat Genet. 2006; 38: 1289-1297Crossref PubMed Scopus (1093) Google Scholar RNA was prepared using Qiagen RNAeasy kits (Valencia, CA) and subjected to Northern blotting or RT-PCR as described previously.23Bhat-Nakshatri P Wang G Appaiah H Luktuke N Carroll JS Geistlinger TR Brown M Badve S Liu Y Nakshatri H AKT Alters genome-wide estrogen receptor alpha binding and impacts estrogen signaling in breast cancer.Mol Cell Biol. 2008; 28: 7487-7503Crossref PubMed Scopus (82) Google Scholar, 25Campbell RA Bhat-Nakshatri P Patel NM Constantinidou D Ali S Nakshatri H Phosphatidylinositol 3-kinase/AKT-mediated activation of estrogen receptor alpha: a new model for anti-estrogen resistance.J Biol Chem. 2001; 276: 9817-9824Crossref PubMed Scopus (813) Google Scholar TCL1B primers used for RT-PCR were: 5′-CAGCAGATATGAACCCAGCA-3′ (forward primer) and 5′-TCTTTCCTCTCCGGCTGATA-3′ (reverse primer). Real-time PCR (Q-PCR) primers were: 5′-TTGGCCCGAAATAGATCCAGTGCT-3′ (forward primer) and 5′-ATAAGCAGAAGCACAGGCCAAACC-3′ (reverse primer). Real-time PCR was done using Syber Green (Applied Biosciences, Foster City, CA). siRNA against TCL1B (on-TARGETplus Duplex J-019892-05-0005) and the luciferase control were purchased from Dharmacon (Lafayette, CA) and transfected into MCF-7 or T47-D cells using the nucleofector reagent (Amaxa, Gaithersburg, MD). Cells were harvested four or six days after transfection, and nuclear and cytoplasmic extracts were prepared as described previously and probed with the indicated antibodies.26Asada M Yamada T Ichijo H Delia D Miyazono K Fukumuro K Mizutani S Apoptosis inhibitory activity of cytoplasmic p21(Cip1/WAF1) in monocytic differentiation.EMBO J. 1999; 18: 1223-1234Crossref PubMed Scopus (535) Google Scholar Densitometric scanning was performed to determine the intensity of signals in each lane. Statistical analysis of Western blot data (n ≥ 3) was performed using GraphPad software (GraphPad Software, Inc., San Diego, CA). Breast cancer tissue microarrays were stained for pAKT (S473) and scored based on intensity and localization. Four distinct patterns of pAKT expression were observed in breast cancer samples: no pAKT, exclusively nuclear (nuclear-pAKT, 62 tumors), exclusively cytoplasmic (cytoplasmic-pAKT, 293 tumors), and distributed both in the nucleus and cytoplasm pAKT (n = 15; Figure 1A). We examined the relationship between the subcellular distribution of pAKT and intrinsic subtypes (ie, luminal subtypes A and B, HER2+/ERα-, basal-like, and normal-like).13Sorlie T Perou CM Tibshirani R Aas T Geisler S Johnsen H Hastie T Eisen MB van de Rijn M Jeffrey SS Thorsen T Quist H Matese JC Brown PO Botstein D Eystein Lonning P Borresen-Dale AL Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications.Proc Natl Acad Sci U S A. 2001; 98: 10869-10874Crossref PubMed Scopus (8513) Google Scholar, 27Perou CM Sorlie T Eisen MB van de Rijn M Jeffrey SS Rees CA Pollack JR Ross DT Johnsen H Akslen LA Fluge O Pergamenschikov A Williams C Zhu SX Lonning PE Borresen-Dale AL Brown PO Botstein D Molecular portraits of human breast tumours.Nature. 2000; 406: 747-752Crossref PubMed Scopus (11662) Google Scholar Although both the luminal subtypes are ERα-positive, subtype A expresses higher levels of ERα and FOXA1 (a cofactor for ERα) and has better prognosis than subtype B.13Sorlie T Perou CM Tibshirani R Aas T Geisler S Johnsen H Hastie T Eisen MB van de Rijn M Jeffrey SS Thorsen T Quist H Matese JC Brown PO Botstein D Eystein Lonning P Borresen-Dale AL Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications.Proc Natl Acad Sci U S A. 2001; 98: 10869-10874Crossref PubMed Scopus (8513) Google Scholar, 15Badve S Turbin D Thorat MA Morimiya A Nielsen TO Perou CM Dunn S Huntsman DG Nakshatri H FOXA1 expression in breast cancer correlation with luminal subtype a and survival.Clin Cancer Res. 2007; 13: 4415-4421Crossref PubMed Scopus (197) Google Scholar, 28Carroll JS Liu XS Brodsky AS Li W Meyer CA Szary AJ Eeckhoute J Shao W Hestermann EV Geistlinger TR Fox EA Silver PA Brown M Chromosome-wide mapping of estrogen receptor binding reveals long-range regulation requiring the forkhead protein FoxA1.Cell. 2005; 122: 33-43Abstract Full Text Full Text PDF PubMed Scopus (1081) Google Scholar Tumors with nuclear-pAKT were predominantly of luminal type A subtype as well as positive for ERα, FOXA1, and GATA-3 (which was previously linked to ERα and FOXA1 positivity13Sorlie T Perou CM Tibshirani R Aas T Geisler S Johnsen H Hastie T Eisen MB van de Rijn M Jeffrey SS Thorsen T Quist H Matese JC Brown PO Botstein D Eystein Lonning P Borresen-Dale AL Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications.Proc Natl Acad Sci U S A. 2001; 98: 10869-10874Crossref PubMed Scopus (8513) Google Scholar; Table 1). In contrast, cytoplasmic-pAKT correlated with HER2-positivity.Table 1Correlation Analysis of Phospho-AKT (Nuclear or Cytoplasmic) with Other MarkersVariablesCorrelation coefficientP value (2-tailed)Number of patientsNuclear phospho-AKT FOXA10.2530.000001377 ERα0.2540.0000007302 GATA-30.2620.000001357 Luminal subtype A0.1460.009321 Luminal subtype B0.0070.895321 HER2−0.0580.285322Cytoplasmic phospho-AKT FOXA10.1770.001364 Luminal subtype A0.0950.91315 Luminal subtype B0.0440.435315 ERα0.1070.170309 HER20.1390.012344Luminal type A is defined as ERα+ and/or PR+, HER2−, whereas luminal type B represents tumors that are ERα+ and/or PR+, HER2+.31Carey LA Perou CM Livasy CA Dressler LG Cowan D Conway K Karaca G Troester MA Tse CK Edmiston S Deming SL Geradts J Cheang MC Nielsen TO Moorman PG Earp HS Millikan RC Race, breast cancer subtypes, and survival in the Carolina Breast Cancer Study.JAMA. 2006; 295: 2492-2502Crossref PubMed Scopus (2912) Google Scholar Open table in a new tab Luminal type A is defined as ERα+ and/or PR+, HER2−, whereas luminal type B represents tumors that are ERα+ and/or PR+, HER2+.31Carey LA Perou CM Livasy CA Dressler LG Cowan D Conway K Karaca G Troester MA Tse CK Edmiston S Deming SL Geradts J Cheang MC Nielsen TO Moorman PG Earp HS Millikan RC Race, breast cancer subtypes, and survival in the Carolina Breast Cancer Study.JAMA. 2006; 295: 2492-2502Crossref PubMed Scopus (2912) Google Scholar To further explore the relationship between ERα and nuclear-pAKT, we evaluated the pAKT distribution pattern in an independent set of patients.20Bose S Chandran S Mirocha JM Bose N The Akt pathway in human breast cancer: a tissue-array-based analysis.Mod Pathol. 2006; 19: 238-245Crossref PubMed Scopus (167) Google Scholar A TMA of 118 samples from Cedars Sinai-UCLA Medical Center was scored for nuclear-pAKT; 47 cases showed nuclear-pAKT expression. Within this TMA, nuclear-pAKT also correlated with ERα+/PR+ and ERα+/FOXA1+, and luminal A phenotype and negatively with HER2 (Table 2). Thus, nuclear-pAKT expression is associated with luminal type A breast cancers as defined by ERα+ and/or PR+ phenotype.Table 2Nuclear Phospho-AKT Analysis in Second TMAVariablenCorrelation coefficientP value (2-tailed)ERα+/PR+1040.2040.024ERα+/FOXA1+850.2280.022Luminal subtype A*Luminal type A is defined as ERα+ and/or PR+, HER2−, whereas luminal B subtype represents tumors that are ERα+ and/or PR+, HER2+.31890.2570.015Luminal subtype B*Luminal type A is defined as ERα+ and/or PR+, HER2−, whereas luminal B subtype represents tumors that are ERα+ and/or PR+, HER2+.3189−0.0580.592HER289−0.2230.036Correlation of n-pAKT expression with ERα+/PR+ status, ERα+/FOXA1+ status, HER2 and luminal breast cancer subtypes was performed in a second TMA composed of 145 breast cancer samples.* Luminal type A is defined as ERα+ and/or PR+, HER2−, whereas luminal B subtype represents tumors that are ERα+ and/or PR+, HER2+.31Carey LA Perou CM Livasy CA Dressler LG Cowan D Conway K Karaca G Troester MA Tse CK Edmiston S Deming SL Geradts J Cheang MC Nielsen TO Moorman PG Earp HS Millikan RC Race, breast cancer subtypes, and survival in the Carolina Breast Cancer Study.JAMA. 2006; 295: 2492-2502Crossref PubMed Scopus (2912) Google Scholar Open table in a new tab Correlation of n-pAKT expression with ERα+/PR+ status, ERα+/FOXA1+ status, HER2 and luminal breast cancer subtypes was performed in a second TMA composed of 145 breast cancer samples. The presence of pAKT is usually considered a poor prognostic indicator, irrespective of cancer type.1Martelli AM Faenza I Billi AM Manzoli L Evangelisti C Fala F Cocco L Intranuclear 3′-phosphoinositide metabolism and Akt signaling: new mechanisms for tumorigenesis and protection against apoptosis?.Cell Signal. 2006; 18: 1101-1107Crossref PubMed Scopus (120) Google Scholar, 29Altomare DA Testa JR Perturbations of the AKT signaling pathway in human cancer.Oncogene. 2005; 24: 7455-7464Crossref PubMed Scopus (1105) Google Scholar However, none of these prior studies, with the exception of one prostate cancer study, evaluated prognostic significance of differential subcellular pAKT expression.30Van de Sande T Roskams T Lerut E Joniau S Van Poppel H Verhoeven G Swinnen JV High-level expression of fatty acid synthase in human prostate cancer tissues is linked to activation and nuclear localization of Akt/PKB.J Pathol. 2005; 206: 214-219Crossref PubMed Scopus (118) Google Scholar We used ERα, FOXA1, PR, and nuclear-pAKT expression patterns to classify patients into subgroups that were correlated with patient survival. Because of the larger sample size and availability of ≈15-year follow-up data, only the GPEC-TMA was used for survival analysis. Combining two datasets was not feasible as these two datasets originated from different institutions and contained follow-up data for different durations. Unsupervised hierarchical clustering identified five subgroups: (1) ERα+/PR+/FOXA1+/nuclear-pAKT+; (2) ERα+/PR+/FOXA1+/nuclear-pAKT-; (3) ERα+/PR+/FOXA1±(low)/nuclear-pAKT-; (4) ERα+/PR-/FOXA1+/nuclear-pAKT-; and (5) ERα-/PR-/FOXA1-/nuclear-pAKT- (Figure 1B). Survival curves of each subgroup are shown in Figure 2A. Statistical comparison among various subgroups along with 95% bootstrap confidence interval of mean biomarker scores for each cluster is shown in supplemental Table S1 and S2 (at http://ajp.amjpathol.org). As expected, the ERα-negative subgroup showed the worst survival rate. We performed survival analysis of ERα+/FOXA1+ patients (luminal type A subtype31Carey LA Perou CM Livasy CA Dressler LG Cowan D Conway K Karaca G Troester MA Tse CK Edmiston S Deming SL Geradts J Cheang MC Nielsen TO Moorman PG Earp HS Millikan RC Race, breast cancer subtypes, and survival in the Carolina Breast Cancer Study.JAMA. 2006; 295: 2492-2502Crossref PubMed Scopus (2912) Google Scholar) based on nuclear-pAKT expression. The presence of nuclear-pAKT in this patient subgroup (n = 176) was associated with better survival, emphasizing the importance of evaluating pAKT levels based on subcellular distribution patterns in clinical settings (P = 0.037, Figure 2B). Similar results were observed when ERα+/PR+ patients were subgrouped based on nuclear-pAKT expression (Figure 2C, P = 0.05). We also performed survival analysis of patients based on the nuclear-pAKT alone. Patients with no nuclear-pAKT displayed worse survival than patients with moderate and strong (2+ and 3+) levels of nuclear-pAKT (P = 0.004, Figure 2D). Thus, a clear association of nuclear-pAKT expression with better survival was observed in all of the above analyses performed with this dataset. When patients in luminal type A cluster were analyzed separately (ERα+ or PR+ and HER2-

Many ovarian tumors, including high-grade serous carcinoma (HGSC), show clear cell change. Accurate diagnosis is important, however, as ovarian clear cell carcinoma (OCCC) is known to be less responsive to traditional types of ovarian cancer chemotherapies. In a previous study, the clinical, morphologic, and immunohistochemical features of 32 ovarian carcinomas, which had been previously diagnosed as pure OCCC (n=11), pure HGSC (n=11), and mixed serous and clear cell (MSC) (n=10), were analyzed. The immunoreactivities of WT1, ER, and p53, as well as the mitotic indices and stages of presentation of the MSC, were similar to those of HGSC. It was consequently concluded that MSC represented HGSC with clear cell change. Hepatocyte nuclear factor-1β (HNF-1β) is a relatively new immunohistochemical marker that has been shown to be rather sensitive and specific for OCCC. We thus sought to evaluate this marker in this specific group of tumors. One block each of pure HGSC and pure OCCC were stained with HNF-1β. In the cases of MSC, 2 blocks were stained when the serous and clear cell components were not present on the same slide. None (0/11) of the pure HGSC showed immunoreactivity for HNF-1β, whereas all (11/11) of the pure OCCC were positive. In the cases of MSC, both the serous and clear cell components were negative for HNF-1β. HNF-1β seems to be a sensitive and specific marker for OCCC and is not expressed in HGSC with clear cell change. The pattern of immunoreactivity of HNF-1β in tumors with both serous and clear cell change supports the conclusion that MSC are HGSC with clear cells.

Survival and hospitalization are critically important outcomes considered when choosing between intensive hemodialysis (HD), conventional HD, and peritoneal dialysis (PD). However, the comparative effectiveness of these modalities is unclear.We had the following aims: (1) to compare the association of mortality and hospitalization in patients undergoing intensive HD, compared with conventional HD or PD and (2) to appraise the methodological quality of the supporting evidence.MEDLINE, Embase, ISI Web of Science, CENTRAL, and nephrology conference abstracts.We included cohort studies with comparator arm, and randomized controlled trials (RCTs) with >50% of adult patients (≥18 years) comparing any form of intensive HD (>4 sessions/wk or >5.5 h/session) with any form of chronic dialysis (PD, HD ≤4 sessions/wk or ≤5.5 h/session), that reported at least 1 predefined outcome (mortality or hospitalization).We used the GRADE approach to systematic reviews and quality appraisal. Two reviewers screened citations and full-text articles, and extracted study-level data independently, with discrepancies resolved by consensus. We pooled effect estimates of randomized and observational studies separately using generic inverse variance with random effects models, and used fixed-effects models when only 2 studies were available for pooling. Predefined subgroups for the intensive HD cohorts were classified by nocturnal versus short daily HD and home versus in-center HD.Twenty-three studies with a total of 70 506 patients were included. Of the observational studies, compared with PD, intensive HD had a significantly lower mortality risk (hazard ratio [HR]: 0.67; 95% confidence interval [CI]: 0.53-0.84; I2 = 91%). Compared with conventional HD, home nocturnal (HR: 0.46; 95% CI: 0.38-0.55; I2 = 0%), in-center nocturnal (HR: 0.73; 95% CI: 0.60-0.90; I2 = 57%) and home short daily (HR: 0.54; 95% CI: 0.31-0.95; I2 = 82%) intensive regimens had lower mortality. Of the 2 RCTs assessing mortality, in-center short daily HD had lower mortality (HR: 0.54; 95% CI: 0.31-0.93), while home nocturnal HD had higher mortality (HR: 3.88; 95% CI: 1.27-11.79) in long-term observational follow-up. Hospitalization days per patient-year (mean difference: -1.98; 95% CI: -2.37 to -1.59; I2 = 6%) were lower in nocturnal compared with conventional HD. Quality of evidence was similarly low or very low in RCTs (due to imprecision) and observational studies (due to residual confounding and selection bias).The overall quality of evidence was low or very low for critical outcomes. Outcomes such as quality of life, transplantation, and vascular access outcomes were not included in our review.Intensive HD regimens may be associated with reduced mortality and hospitalization compared with conventional HD or PD. As the quality of supporting evidence is low, patients who place a high value on survival must be adequately advised and counseled of risks and benefits when choosing intensive dialysis. Practice guidelines that promote shared decision-making are likely to be helpful.Au moment de choisir une modalité de dialyse pour le traitement des patients souffrant d’insuffisance rénale, le taux de survie et la durée des hospitalisations sont des critères décisionnels d’une importance cruciale. Pourtant, l’efficacité différentielle de l’hémodialyse (HD) intensive, de l’HD conventionnelle et de la dialyse péritonéale (DP) demeure à ce jour mal connue.Nos objectifs allaient comme suit : 1) comparer le taux de mortalité et la durée des hospitalisations associés à chacune des modalités (HD intensive, HD conventionnelle et DP), et 2) évaluer la qualité méthodologique des données venant appuyer les résultats.Les données proviennent des bases de données en ligne MEDLINE, EMBASE et ISI Web of Science, de même que de CENTRAL et de résumés de conférence en néphrologie.Ont été incluses à cette méta-analyse les études de cohorte comportant un volet comparatif et les essais contrôlés à répartition aléatoire comptant plus de 50 % de patients adultes et comparant n’importe quelle forme d’HD intensive (plus de 4 séances par semaine ou plus de 5,5 heures par séance) à n’importe quelle forme de dialyse chronique (DP ou HD à raison de 4 séances maximum par semaine ou de 5,5 heures maximum par séance). Les études retenues devaient également rapporter au moins un des deux critères décisionnels prédéfinis (mortalité et hospitalisation).Nous avons employé l’approche GRADE (Grading of Recommendations Assessment, Development and Evaluation). Cette approche s’applique aux revues systématiques et à l’évaluation de la qualité des données. Deux personnes ont passé en revue des citations et des articles complets pour en extraire les données relatives à l’étude. Les divergences ont été résolues par consensus. Nous avons regroupé les différentes mesures provenant des essais à répartition aléatoire et des études observationnelles pour ensuite les analyser, de façon isolée, à l’aide de la méthode générique de l’inverse de la variance avec modèles à effet aléatoire. Pour les données où seules deux études étaient disponibles pour le regroupement des données, nous avons plutôt employé la méthode générique de l’inverse de la variance avec modèles à effet fixe. Des sous-groupes avaient été prédéfinis dans les cohortes de patients traités par HD intensive, selon le moment (de jour ou de nuit) et le lieu (en centre de dialyse ou à domicile) du traitement.Cette méta-analyse compte 23 études totalisant 70 506 patients. Selon les études observationnelles, lorsque comparée à la DP, l’HD intensive était corrélée à un risque de mortalité significativement plus faible (HR=0,67; IC 95 0,53-0,84; I2=91 %). En comparaison avec l’HD conventionnelle, les schémas de traitement par HD intensive nocturne prodiguée à domicile (HR=0,46; IC 95 : 0,38-0,55; I2=0 %), nocturne en centre (HR=0,73; IC 95 : 0,60-0,90; I2=57 %) et de courte durée, de jour, à domicile (HR=0,54; IC 95 : 0,31-0,95; I2=82%) étaient corrélées à de plus faibles taux de mortalité. Des deux essais contrôlés à répartition aléatoire qui faisaient mention du taux de mortalité, l’HD diurne de courte durée en centre présentait le plus faible taux de mortalité (HR=0,54; IC 95 : 0,31-0,93) alors que l’HD nocturne à domicile présentait le taux de mortalité le plus élevé (HR=3,88; IC 95 : 1,27-11,79) selon les suivis observationnels faits à long terme. Le nombre de jours d’hospitalisation par année-patient (différence moyenne = -1,98 an; IC 95 : -1,59 à 2,37; I2=6 %) était plus faible chez les patients traités par HD intensive nocturne en comparaison avec ceux qui suivaient un traitement par la méthode conventionnelle. Dans tous les cas, la qualité des données recueillies s’est avérée faible ou très faible, qu’il s’agisse d’essais contrôlés à répartition aléatoire (en raison de l’imprécision) ou d’études observationnelles (en raison de facteurs de confusion et de biais de sélection).Dans l’ensemble, la qualité des données recueillies pour appuyer les critères décisionnels jugés essentiels s’est avérée faible ou très faible. De plus, des éléments tels que la qualité de vie du patient, la greffe et les enjeux liés à l’accès vasculaire n’ont pas été pris en compte dans notre revue systématique.Le traitement de l’insuffisance rénale par HD intensive pourrait être associé à un taux de mortalité réduit et à des séjours à l’hôpital écourtés en comparaison avec les traitements par HD conventionnelle ou par DP. Cependant, en raison de la piètre qualité des données appuyant ces résultats, les patients qui accordent une grande importance à la survie devraient être adéquatement informés et conseillés sur les risques et les bienfaits offerts par l’HD intensive comme modalité de traitement. L’application de lignes directrices concernant la prise de décision conjointe en pratique clinique pourrait être pertinente.

Many rare ovarian cancer subtypes, such as small-cell carcinoma of the ovary, hypercalcemic type (SCCOHT), have poor prognosis due to their aggressive nature and resistance to standard platinum- and taxane-based chemotherapy. The development of effective therapeutics has been hindered by the rarity of such tumors. We sought to identify targetable vulnerabilities in rare ovarian cancer subtypes.We compared the global proteomic landscape of six cases each of endometrioid ovarian cancer (ENOC), clear cell ovarian cancer (CCOC), and SCCOHT to the most common subtype, high-grade serous ovarian cancer (HGSC), to identify potential therapeutic targets. IHC of tissue microarrays was used as validation of arginosuccinate synthase (ASS1) deficiency. The efficacy of arginine-depriving therapeutic ADI-PEG20 was assessed in vitro using cell lines and patient-derived xenograft mouse models representing SCCOHT.Global proteomic analysis identified low ASS1 expression in ENOC, CCOC, and SCCOHT compared with HGSC. Low ASS1 levels were validated through IHC in large patient cohorts. The lowest levels of ASS1 were observed in SCCOHT, where ASS1 was absent in 12 of 31 cases, and expressed in less than 5% of the tumor cells in 9 of 31 cases. ASS1-deficient ovarian cancer cells were sensitive to ADI-PEG20 treatment regardless of subtype in vitro. Furthermore, in two cell line mouse xenograft models and one patient-derived mouse xenograft model of SCCOHT, once-a-week treatment with ADI-PEG20 (30 mg/kg and 15 mg/kg) inhibited tumor growth in vivo.Preclinical in vitro and in vivo studies identified ADI-PEG20 as a potential therapy for patients with rare ovarian cancers, including SCCOHT.

Abstract Purpose: The RET proto-oncogene has been implicated in breast cancer, and the studies herein describe the preclinical and safety assessment of an anti-RET antibody–drug conjugate (ADC) being developed for the treatment of breast cancer. Experimental Design: RET protein expression was analyzed in breast tumor samples using tissue microarrays. The fully human anti-RET antibody (Y078) was conjugated to the DM1 and DM4 derivatives of the potent cytotoxic agent maytansine using thioether and disulfide linkers, respectively. The resulting compounds, designated Y078-DM1 and Y078-DM4, were evaluated for antitumor activity using human breast cancer cell lines and established tumor xenograft models. A single-dose, 28-day, safety study of Y078-DM1 was performed in cynomolgus monkeys. Results: By immunohistochemistry, RET expression was detected in 57% of tumors (1,596 of 2,800 tumor sections) and was most common in HER2-positive and basal breast cancer subtypes. Potent in vitro cytotoxicity was achieved in human breast cancer cell lines that have expression levels comparable with those observed in breast cancer tissue samples. Dose-response studies in xenograft models demonstrated antitumor activity with both weekly and every-3-weeks dosing regimens. In cynomolgus monkeys, a single injection of Y078-DM1 demonstrated dose-dependent, reversible drug-mediated alterations in blood chemistry with evidence of on-target neuropathy. Conclusions: RET is broadly expressed in breast cancer specimens and thus represents a potential therapeutic target; Y078-DM1 and Y078-DM4 demonstrated antitumor activity in preclinical models. Optimization of the dosing schedule or an alternate cytotoxic agent with a different mechanism of action may reduce the potential risk of neuropathy. Clin Cancer Res; 21(24); 5552–62. ©2015 AACR.

Background: The study of the intrinsic molecular subtypes of breast cancer has revealed differences among them in terms of prognosis and response to chemotherapy and endocrine therapy. However, the ability of intrinsic subtypes to predict benefit from adjuvant radiotherapy has only been examined in few studies.Methods: Gene expression-based intrinsic subtyping was performed in 228 breast tumors collected from two independent post-mastectomy clinical trials (British Columbia and the Danish Breast Cancer Cooperative Group 82b trials), where pre-menopausal patients with node-positive disease were randomized to adjuvant radiotherapy or not. All patients received adjuvant chemotherapy and a subgroup of patients underwent ovarian ablation. Tumors were classified into intrinsic subtypes: Luminal A, Luminal B, HER2-enriched, Basal-like and Normal-like using the research-based PAM50 classifier.Results: In the British Columbia study, patients treated with radiation had an overall significant lower incidence of locoregional recurrence compared to the controls. For Luminal A tumors the risk of loco-regional recurrence was low and was further lowered by adjuvant radiation. These findings were validated in the DBCG 82b study. The individual data from the two cohorts were merged, the hazard ratio (HR) for loco-regional recurrence associated with giving radiation was 0.34 (0.19 to 0.61) overall and 0.12 (0.03 to 0.52) for Luminal A tumors.Conclusions: In both postmastectomy trials, patients with Luminal A tumors turned out to have a significant lower incidence of loco-regional recurrence when randomized to adjuvant radiotherapy, leaving no indication to omit postmastectomy adjuvant radiation in pre-menopausal high-risk patients with Luminal A tumors. It was not possible to evaluate the effect of radiotherapy among the other subtypes because of limited sample sizes.

Recently, the International Endocervical Adenocarcinoma Criteria and Classification (IECC) has reorganized the classification of endocervical adenocarcinomas (ECAs), separating them into human papilloma virus (HPV)-associated (HPVA) and HPVA independent (HPVI) categories. In this study, we sought to revalidate the IECC clinical findings in an independent cohort and assess the mutational differences between HPVA and HPVI ECAs using next generation sequencing. Consecutive cases of ECAs were reclassified under the IECC. Clinicopathologic information was collected and tissue was sent for targeted next-generation sequencing in 33 genes. Associations between HPV status, clinicopathologic parameters and mutation status, with survival were evaluated. The series comprised of 85/100 HPVA (63 HPVA-usual type, 4 villoglandular, 3 mucinous intestinal, 15 mucinous not otherwise specified) and 15/100 HPVI (9 gastric, 4 mesonephric, 1 clear cell, 1 not otherwise specified). HPVA ECAs presented at a lower age ( P =0.001), smaller tumor sizes ( P =0.011), less margin positivity ( P =0.027), less Silva pattern C ( P =0.002), and lower FIGO stages ( P =0.020). HPVA had superior survival compared with HPVI ECA [overall survival ( P =0.0026), disease-specific survival ( P =0.0092), and progression-free survival ( P =0.0041)]. Factors that correlated with worse prognosis irrespective of HPV status were FIGO stage, positive margins and lymphovascular invasion ( P &lt;0.05). TP53 mutations were detected in a significantly higher proportion of HPVIs than HPVAs ( P &lt;&lt;0.001). The study revalidates the IECC system by reaffirming the clinical and prognostic differences between HPVA and HPVI ECAs in an independent dataset.

Object. The purpose of this study was to confirm, by using a sequential volume mapping (SVM) technique, that gamma knife radiosurgery (GKS) induces negative growth in vestibular schwannomas (VS). Methods. Over a period of 5 years, 126 small- to medium-sized (&lt; 15 cm 3 ) VSs were treated using microradiosurgical techniques within a standard protocol. All patient data were collected prospectively. Sequential magnetic resonance imaging was performed every 6 months to assess the volume of the tumor, based on specially developed GammaPlan software. The mean follow-up duration was 22 months. At least three SVM measurements were obtained in 91 patients and at least four were obtained in 62 patients. The mean number of SVM measurements for each patient was 2.54. After GKS, the following patterns of volume change were seen: 1) 57 VSs showed transient increase in volume with a peak at 6 months, followed by shrinkage. Four VSs exhibited prolonged swelling beyond 24 months. Transient swelling and eventual shrinkage were independent of the initial VS volume; 2) 29 VSs showed direct volume shri6nkage without swelling; and 3) five VSs showed persistent volume increase. All volume changes were greater than 10%. The overall mean volume reduction was 46.8% at 30 months. Conclusions. Sequential volume mapping appears to be superior to conventional two-dimensional measurements in monitoring volume changes in VS after GKS. It confirms that transient swelling is common. Ninety-two percent of tumors responded by showing significant volume shrinkage (mean 46.8%). It would seem that GKS can induce volume reduction in VS.

Primary breast cancer involving four or more axillary lymph nodes carries a poor prognosis. We hypothesized that use of an immunohistochemical biomarker scoring system could allow for identification of variable risk subgroups.Patients with four or more positive axillary nodes were identified from a clinically annotated tissue microarray of formalin-fixed paraffin-embedded primary breast cancers and randomized into a 'test set' and a 'validation set'. A prospectively defined prognostic scoring model was developed in the test set and was further assessed in the validation set combining expression for eight biomarkers by immunohistochemistry, including estrogen receptor, human epidermal growth factor receptors 1 and 2, carbonic anhydrase IX, cytokeratin 5/6, progesterone receptor, p53 and Ki-67. Survival outcomes were analyzed by the Kaplan-Meier method, log rank tests and Cox proportional-hazards models.A total of 313 eligible patients were identified in the test set for whom 10-year relapse-free survival was 38.3% (SEM 2.9%), with complete immunohistochemical data available for 227. Tumor size, percentage of positive axillary nodes and expression status for the progesterone receptor, Ki-67 and carbonic anhydrase IX demonstrated independent prognostic significance with respect to relapse-free survival. Our combined biomarker scoring system defined three subgroups in the test set with mean 10-year relapse-free survivals of 75.4% (SEM 7.0%), 35.3% (SEM 4.1%) and 19.3% (SEM 7.0%). In the validation set, differences in relapse-free survival for these subgroups remained statistically significant but less marked.Biomarkers assessed here carry independent prognostic value for breast cancer with four or more positive axillary nodes and identified clinically relevant prognostic subgroups. This approach requires refinement and validation of methodology.

We used a large patient population to identify immunohistochemical biomarkers to enable improved prognostication in patients with non-small cell lung carcinoma (NSCLC).A tissue microarray was constructed using duplicate 0.6 mm cores of formalin-fixed paraffin-embedded tissue blocks from 609 patients with NSCLC. Immunohistochemical was used to detect 11 biomarkers including epidermal growth factor receptor, Her2, Her3, p53, p63, bcl-1, bcl-2, Thyroid transcription factor, carcinoembryonic antigen, chromogranin, and synaptophysin. A clinical database was generated prospectively at the time of tissue collection. Survival outcomes were obtained from a Provincial Cancer Registry database. Univariate and multivariate analyses were performed to look for a relationship between biomarker expression, smoking history, and survival.Survival data for 535 cases were available. As of June 2005, 429 patients (80%) had died; of these 286 (54%) died of lung cancer, 117 (22%) died of other known causes, and for 26 (5%) the cause of death was not available. Univariate analysis revealed that bcl-2 (p = 0.007) was the only biomarker prognostic for improved overall survival (OS). bcl-2 (p = 0.021) and p63 (p = 0.025) were both found to be prognostic for improved disease-specific survival (DSS). Multivariate analysis (using age and biomarker expression) revealed that bcl-2 expression is prognostic for improved OS (p = 0.005) and DSS (p = 0.021).Our results suggest that bcl-2 expression is prognostic for improved OS and DSS in NSCLC. Testing for bcl-2 expression in a prospective study will help to determine its clinical relevance in prognostication.

Objective We sought to determine whether DNA ploidy correlates with the four molecular subgroups of endometrial carcinoma (EC) as determined using ProMisE (Proactive Molecular Risk Classifier for Endometrial Cancer). Methods 90 cases of EC previously characterized by clinicopathological parameters, outcomes, and ProMisE molecular subgroup (POLE EDM, MMR-D, p53 wt or p53 abn) were assessed for DNA ploidy using image cytometry. Associations of ploidy with traditional clinicopathological parameters were also tested. Results Abnormal DNA ploidy status differed amongst the ProMisE groups (p < 0.001) and was found in 80.9% (17/21) of p53 abn, 37.0% (10/27) of p53 wt, 28.6% (4/14) of POLE EDM and 14.3% (4/28) of MMR-D. Abnormal DNA content was significantly associated with lower BMI (p = 0.034) and grade 3 tumors (p = 0.001). In the entire cohort, abnormal DNA content was significantly associated with worse progression free survival (p = 0.0094) but not disease specific survival (p = 0.249) or overall survival (p = 0.187). When examining ploidy within each of the ProMisE groups, abnormal DNA content correlated with worse overall survival (p = 0.041) and progression free survival (p = 0.011) in the MMR-D group. No statistically significant relationship was seen in the remaining 3 groups. Conclusion Abnormal DNA ploidy status did correlate with the molecular subgroups of EC; abnormal DNA content was seen in the large majority of p53 abn cases. Abnormal ploidy however was also seen in smaller numbers in the p53 wt, POLE EDM and MMR-D groups; therefore abnormal DNA content was not a specific marker for any one molecular group. The addition of ploidy to the ProMisE molecular categories conferred additional prognostic value within the MMR-D group, which merits further study.