Peter A. Prieto

Preclinical mouse models suggest that the gut microbiome modulates tumor response to checkpoint blockade immunotherapy; however, this has not been well-characterized in human cancer patients. Here we examined the oral and gut microbiome of melanoma patients undergoing anti-programmed cell death 1 protein (PD-1) immunotherapy (n = 112). Significant differences were observed in the diversity and composition of the patient gut microbiome of responders versus nonresponders. Analysis of patient fecal microbiome samples (n = 43, 30 responders, 13 nonresponders) showed significantly higher alpha diversity (P < 0.01) and relative abundance of bacteria of the Ruminococcaceae family (P < 0.01) in responding patients. Metagenomic studies revealed functional differences in gut bacteria in responders, including enrichment of anabolic pathways. Immune profiling suggested enhanced systemic and antitumor immunity in responding patients with a favorable gut microbiome as well as in germ-free mice receiving fecal transplants from responding patients. Together, these data have important implications for the treatment of melanoma patients with immune checkpoint inhibitors.

Immune checkpoint blockade represents a major breakthrough in cancer therapy; however, responses are not universal. Genomic and immune features in pretreatment tumor biopsies have been reported to correlate with response in patients with melanoma and other cancers, but robust biomarkers have not been identified. We studied a cohort of patients with metastatic melanoma initially treated with cytotoxic T-lymphocyte-associated antigen-4 (CTLA4) blockade (n = 53) followed by programmed death-1 (PD-1) blockade at progression (n = 46), and analyzed immune signatures in longitudinal tissue samples collected at multiple time points during therapy. In this study, we demonstrate that adaptive immune signatures in tumor biopsy samples obtained early during the course of treatment are highly predictive of response to immune checkpoint blockade and also demonstrate differential effects on the tumor microenvironment induced by CTLA4 and PD-1 blockade. Importantly, potential mechanisms of therapeutic resistance to immune checkpoint blockade were also identified.These studies demonstrate that adaptive immune signatures in early on-treatment tumor biopsies are predictive of response to checkpoint blockade and yield insight into mechanisms of therapeutic resistance. These concepts have far-reaching implications in this age of precision medicine and should be explored in immune checkpoint blockade treatment across cancer types. Cancer Discov; 6(8); 827-37. ©2016 AACR.See related commentary by Teng et al., p. 818This article is highlighted in the In This Issue feature, p. 803.

Immune checkpoint blockade produces clinical benefit in many patients. However, better biomarkers of response are still needed, and mechanisms of resistance remain incompletely understood. To address this, we recently studied a cohort of melanoma patients treated with sequential checkpoint blockade against cytotoxic T lymphocyte antigen-4 (CTLA-4) followed by programmed death receptor-1 (PD-1) and identified immune markers of response and resistance. Building on these studies, we performed deep molecular profiling including T cell receptor sequencing and whole-exome sequencing within the same cohort and demonstrated that a more clonal T cell repertoire was predictive of response to PD-1 but not CTLA-4 blockade. Analysis of CNAs identified a higher burden of copy number loss in nonresponders to CTLA-4 and PD-1 blockade and found that it was associated with decreased expression of genes in immune-related pathways. The effect of mutational load and burden of copy number loss on response was nonredundant, suggesting the potential utility of a combinatorial biomarker to optimize patient care with checkpoint blockade therapy.

Abstract Purpose: Treatment with ipilimumab can cause objective tumor responses in patients with metastatic melanoma. We have treated 177 evaluable patients in three clinical trials and have long-term follow-up to evaluate the durability of responses. Experimental Design: Patients with metastatic melanoma were treated in three trials from 2002 to 2005. In protocol 1, 56 patients received ipilimumab with gp100 peptides. In protocol 2, 36 patients received ipilimumab with interleukin-2. In protocol 3, 85 patients received ipilimumab with intrapatient dose-escalation and were randomized to receive gp100 peptides. We have analyzed their long-term follow-up and survival data. Results: With median follow-up for protocols 1, 2, and 3 being 92, 84, and 71 months, median survival was 14, 16, and 13 months with 5-year survival rates being 13%, 25%, and 23%, respectively. Patients in protocol 2 had a 17% complete response (CR) rate, compared with 7% in protocol 1 and 6% in protocol 3. These CR rates are higher than previously reported for the same trials because some patients who eventually became complete responders had continual tumor regression months to years after therapy. All but one of the 15 complete responders are ongoing at 54+ to 99+ months. Conclusions: This report provides the longest follow-up of patients with melanoma treated with ipilimumab and shows that ipilimumab can induce durable, potentially curative tumor regression in a small percentage of patients with metastatic melanoma. The combination of ipilimumab and interleukin-2 seems to have an increased CR rate, but this needs to be tested in a randomized trial. Clin Cancer Res; 18(7); 2039–47. ©2012 AACR.

Abstract Purpose: Tumor-infiltrating lymphocytes (TIL) and interleukin (IL)-2 administered following lymphodepletion can cause the durable complete regression of bulky metastatic melanoma in patients refractory to approved treatments. However, the generation of a unique tumor-reactive TIL culture for each patient may be prohibitively difficult. We therefore investigated the clinical and immunologic impact of unscreened, CD8+ enriched “young” TIL. Experimental Design: Methods were developed for generating TIL that minimized the time in culture and eliminated the individualized tumor-reactivity screening step. Thirty-three patients were treated with these CD8+ enriched young TIL and IL-2 following nonmyeloablative lymphodepletion (NMA). Twenty-three additional patients were treated with CD8+ enriched young TIL and IL-2 after lymphodepletion with NMA and 6 Gy of total body irradiation. Results: Young TIL cultures for therapy were successfully established from 83% of 122 consecutive melanoma patients. Nineteen of 33 patients (58%) treated with CD8+ enriched young TIL and NMA had an objective response (Response Evaluation Criteria in Solid Tumors) including 3 complete responders. Eleven of 23 patients (48%) treated with TIL and 6 Gy total body irradiation had an objective response including 2 complete responders. At 1 month after TIL infusion the absolute CD8+ cell numbers in the periphery were highly correlated with response. Conclusions: This study shows that a rapid and simplified method can be used to reliably generate CD8+ enriched young TIL for administration as an individualized therapy for advanced melanoma, and may allow this potentially effective treatment to be applied at other institutions and to reach additional patients. Clin Cancer Res; 16(24); 6122–31. ©2010 AACR.

Dual BRAF and MEK inhibition produces a response in a large number of patients with stage IV BRAF-mutant melanoma. The existing standard of care for patients with clinical stage III melanoma is upfront surgery and consideration for adjuvant therapy, which is insufficient to cure most patients. Neoadjuvant targeted therapy with BRAF and MEK inhibitors (such as dabrafenib and trametinib) might provide clinical benefit in this high-risk p opulation.We undertook this single-centre, open-label, randomised phase 2 trial at the University of Texas MD Anderson Cancer Center (Houston, TX, USA). Eligible participants were adult patients (aged ≥18 years) with histologically or cytologically confirmed surgically resectable clinical stage III or oligometastatic stage IV BRAFV600E or BRAFV600K (ie, Val600Glu or Val600Lys)-mutated melanoma. Eligible patients had to have an Eastern Cooperative Oncology Group performance status of 0 or 1, a life expectancy of more than 3 years, and no previous exposure to BRAF or MEK inhibitors. Exclusion criteria included metastases to bone, brain, or other sites where complete surgical excision was in doubt. We randomly assigned patients (1:2) to either upfront surgery and consideration for adjuvant therapy (standard of care group) or neoadjuvant plus adjuvant dabrafenib and trametinib (8 weeks of neoadjuvant oral dabrafenib 150 mg twice per day and oral trametinib 2 mg per day followed by surgery, then up to 44 weeks of adjuvant dabrafenib plus trametinib starting 1 week after surgery for a total of 52 weeks of treatment). Randomisation was not masked and was implemented by the clinical trial conduct website maintained by the trial centre. Patients were stratified by disease stage. The primary endpoint was investigator-assessed event-free survival (ie, patients who were alive without disease progression) at 12 months in the intent-to-treat population. This trial is registered at ClinicalTrials.gov, number NCT02231775.Between Oct 23, 2014, and April 13, 2016, we randomly assigned seven patients to standard of care, and 14 to neoadjuvant plus adjuvant dabrafenib and trametinib. The trial was stopped early after a prespecified interim safety analysis that occurred after a quarter of the participants had been accrued revealed significantly longer event-free survival with neoadjuvant plus adjuvant dabrafenib and trametinib than with standard of care. After a median follow-up of 18·6 months (IQR 14·6-23·1), significantly more patients receiving neoadjuvant plus adjuvant dabrafenib and trametinib were alive without disease progression than those receiving standard of care (ten [71%] of 14 patients vs none of seven in the standard of care group; median event-free survival was 19·7 months [16·2-not estimable] vs 2·9 months [95% CI 1·7-not estimable]; hazard ratio 0·016, 95% CI 0·00012-0·14, p<0·0001). Neoadjuvant plus adjuvant dabrafenib and trametinib were well tolerated with no occurrence of grade 4 adverse events or treatment-related deaths. The most common adverse events in the neoadjuvant plus adjuvant dabrafenib and trametinib group were expected grade 1-2 toxicities including chills (12 patients [92%]), headache (12 [92%]), and pyrexia (ten [77%]). The most common grade 3 adverse event was diarrhoea (two patients [15%]).Neoadjuvant plus adjuvant dabrafenib and trametinib significantly improved event-free survival versus standard of care in patients with high-risk, surgically resectable, clinical stage III-IV melanoma. Although the trial finished early, limiting generalisability of the results, the findings provide proof-of-concept and support the rationale for further investigation of neoadjuvant approaches in this disease. This trial is currently continuing accrual as a single-arm study of neoadjuvant plus adjuvant dabrafenib and trametinib.Novartis Pharmaceuticals Corporation.

Treatment with combined immune checkpoint blockade (CICB) targeting CTLA-4 and PD-1 is associated with clinical benefit across tumor types, but also a high rate of immune-related adverse events. Insights into biomarkers and mechanisms of response and toxicity to CICB are needed. To address this, we profiled the blood, tumor and gut microbiome of 77 patients with advanced melanoma treated with CICB, with a high rate of any ≥grade 3 immune-related adverse events (49%) with parallel studies in pre-clinical models. Tumor-associated immune and genomic biomarkers of response to CICB were similar to those identified for ICB monotherapy, and toxicity from CICB was associated with a more diverse peripheral T-cell repertoire. Profiling of gut microbiota demonstrated a significantly higher abundance of Bacteroides intestinalis in patients with toxicity, with upregulation of mucosal IL-1β in patient samples of colitis and in pre-clinical models. Together, these data offer potential new therapeutic angles for targeting toxicity to CICB.

Advances in the treatment of metastatic melanoma have improved responses and survival. However, many patients continue to experience resistance or toxicity to treatment, highlighting a crucial need to identify biomarkers and understand mechanisms of response and toxicity. Neoadjuvant therapy for regional metastases might improve operability and clinical outcomes over upfront surgery and adjuvant therapy, and has become an established role for drug development and biomarker discovery in other cancers (including locally advanced breast cancer, head and neck squamous cell carcinomas, gastroesophageal cancer, and anal cancer). Patients with clinically detectable stage III melanoma are ideal candidates for neoadjuvant therapy, because they represent a high-risk patient population with poor outcomes when treated with upfront surgery alone. Neoadjuvant therapy is now an active area of research for melanoma with numerous completed and ongoing trials (since 2014) with disparate designs, endpoints, and analyses under investigation. We have, therefore, established the International Neoadjuvant Melanoma Consortium with experts in medical oncology, surgical oncology, pathology, radiation oncology, radiology, and translational research to develop recommendations for investigating neoadjuvant therapy in melanoma to align future trial designs and correlative analyses. Alignment and consistency of neoadjuvant trials will facilitate optimal data organisation for future regulatory review and strengthen translational research across the melanoma disease continuum.

Treatment with therapy targeting BRAF and MEK (BRAF/MEK) has revolutionized care in melanoma and other cancers; however, therapeutic resistance is common and innovative treatment strategies are needed1,2. Here we studied a group of patients with melanoma who were treated with neoadjuvant BRAF/MEK-targeted therapy ( NCT02231775 , n = 51) and observed significantly higher rates of major pathological response (MPR; ≤10% viable tumour at resection) and improved recurrence-free survival (RFS) in female versus male patients (MPR, 66% versus 14%, P = 0.001; RFS, 64% versus 32% at 2 years, P = 0.021). The findings were validated in several additional cohorts2–4 of patients with unresectable metastatic melanoma who were treated with BRAF- and/or MEK-targeted therapy (n = 664 patients in total), demonstrating improved progression-free survival and overall survival in female versus male patients in several of these studies. Studies in preclinical models demonstrated significantly impaired anti-tumour activity in male versus female mice after BRAF/MEK-targeted therapy (P = 0.006), with significantly higher expression of the androgen receptor in tumours of male and female BRAF/MEK-treated mice versus the control (P = 0.0006 and P = 0.0025). Pharmacological inhibition of androgen receptor signalling improved responses to BRAF/MEK-targeted therapy in male and female mice (P = 0.018 and P = 0.003), whereas induction of androgen receptor signalling (through testosterone administration) was associated with a significantly impaired response to BRAF/MEK-targeted therapy in male and female patients (P = 0.021 and P < 0.0001). Together, these results have important implications for therapy. Treatment with neoadjuvant BRAF/MEK-targeted therapy results in higher rates of major pathological response in female compared with male patients with melanoma, and pharmacological inhibition of androgen receptor signalling improved the responses of male and female mice to BRAF/MEK-targeted therapy.

Appreciation for genomic and immune heterogeneity in cancer has grown though the relationship of these factors to treatment response has not been thoroughly elucidated. To better understand this, we studied a large cohort of melanoma patients treated with targeted therapy or immune checkpoint blockade (n = 60). Heterogeneity in therapeutic responses via radiologic assessment was observed in the majority of patients. Synchronous melanoma metastases were analyzed via deep genomic and immune profiling, and revealed substantial genomic and immune heterogeneity in all patients studied, with considerable diversity in T cell frequency, and few shared T cell clones (<8% on average) across the cohort. Variables related to treatment response were identified via these approaches and through novel radiomic assessment. These data yield insight into differential therapeutic responses to targeted therapy and immune checkpoint blockade in melanoma, and have key translational implications in the age of precision medicine.

Approximately 80% of patients undergoing total mastectomy in the US opt for implant-based breast reconstruction (IBBR). A two-stage reconstruction with tissue expander (TE) remains the most common technique. Since the implementation of ADMs, a prepectoral approach has gained popularity and is becoming the standard of care. Herein, we compared the surgical and postoperative outcomes of prepectoral versus subpectoral two-stage IBBR.A retrospective chart review was performed between January 2011 and December 2020. We included female patients undergoing immediate two-stage IBBR. The primary outcomes of this study were to compare the 30-day morbidity and the overall rate of complications during the first and second stages of reconstruction, and to compare the time to initiate postmastectomy radiotherapy (PMRT). Propensity score matching was implemented.After matching, 154 reconstructions were analyzed, 77 in each group. The two matched groups exhibited comparable (p > 0.05) characteristics for all analyzed demographic and intraoperative independent variables. Reconstructions in the prepectoral group had a shortened median time for drain removal (13-days vs. 15-days, p = 0.001). The intraoperative expansion volumes were higher in the prepectoral group (300 ml versus 200 ml, p = 0.025). The 30-day morbidity and first- and second-stage complication rates were not significantly different between groups. The time to start postmastectomy radiation therapy (PMRT) was not significantly different between groups (134-days versus 126.5-days, p = 0.58).Prepectoral and subpectoral TE placement had comparable complication rates during the first and second stages of IBBR. Timing for TE-to-Implant exchange and initiation of PMRT were comparable between the two approaches.

Adoptive cell therapy using tumor-infiltrating lymphocytes (TIL) can mediate objective and durable tumor regressions in patients with metastatic melanoma. CD8+ tumor-reactive TIL are well studied in humans and animals, yet the function of tumor-infiltrating CD4+ T lymphocytes in patient treatments remains controversial. We recently demonstrated that CD4+ TILs are not necessary for objective responses in patients. Coinfusion with tumor-specific CD4 TIL may enhance or increase the durability of tumor regressions, but the number of patients with tumor-reactive CD4 TIL is unknown. We screened 44 CD8+-depleted TIL for in vitro reactivity against autologous tumor. Nine (20%) showed specific reactivity by interferon-γ release assay, of which 8 were specifically blocked by an anti-HLA-DR antibody. Flow-cytometric analysis of these reactive TIL confirmed a high CD4+ composition (median 89%). Highlighting the contribution of CD4+ TIL to tumor regression, a patient with widespread metastatic disease was administered TIL containing HLA class II-restricted tumor activity with high-dose interleukin-2 therapy after lymphodepletion that mediated regression of extensive metastatic disease in the liver and spleen. These results demonstrate that at least 20% of metastatic melanomas contain CD4+ lymphocytes with specific tumor recognition and suggest a possible role for CD4+ cells in the effectiveness of adoptive cell therapy.

We have made major advances in the treatment of melanoma through the use of targeted therapy and immune checkpoint blockade; however, clinicians are posed with therapeutic dilemmas regarding timing and sequence of therapy. There is a growing appreciation of the impact of antitumor immune responses to these therapies, and we performed studies to test the hypothesis that clinical patterns and immune infiltrates differ at progression on these treatments. We observed rapid clinical progression kinetics in patients on targeted therapy compared to immune checkpoint blockade. To gain insight into possible immune mechanisms behind these differences, we performed deep immune profiling in tumors of patients on therapy. We demonstrated low CD8+ T-cell infiltrate on targeted therapy and high CD8+ T-cell infiltrate on immune checkpoint blockade at clinical progression. These data have important implications, and suggest that antitumor immune responses should be assessed when considering therapeutic options for patients with melanoma.

<h2>Summary</h2> Over 80% of pancreatic ductal adenocarcinoma (PDA) patients are diagnosed with non-resectable late-stage disease that lacks effective neoadjuvant therapies. Stereotactic body radiation therapy (SBRT) has shown promise as an emerging neoadjuvant approach for treating PDA, and here, we report that its combination with local interleukin-12 (IL-12) microsphere (MS) immunotherapy results in marked tumor reduction and cures in multiple preclinical mouse models of PDA. Our findings demonstrate an increase of intratumoral interferon gamma (IFNγ) production following SBRT/IL-12 MS administration that initiates suppressor cell reprogramming and a subsequent increase in CD8 T cell activation. Furthermore, SBRT/IL-12 MS therapy results in the generation of systemic tumor immunity that is capable of eliminating established liver metastases, providing a rationale for follow-up studies in advanced metastatic disease.

<h3>Objective</h3> Melanoma is a form of malignant skin cancer that exhibits significant inter-tumoral differences in the tumor microenvironment (TME) secondary to genetic mutations. The heterogeneity may be subtle but can complicate the treatment of metastatic melanoma, contributing to a high mortality rate. Therefore, developing an accurate and non-invasive procedure to discriminate microenvironmental heterogeneity to facilitate therapy selection is an important goal. <h3>Methods</h3> <i>In vivo</i> murine melanoma models that recapitulate human disease using synchronous implanted YUMM 1.7 (Yale University Mouse Melanoma) and YUMMER 1.7 (Yale University Mouse Melanoma Exposed to Radiation) murine melanoma lines were investigated. Mice were treated with antibodies to modulate the immune response and longitudinally scanned with ultrasound (US). US radiofrequency data were processed using the H-scan analysis, attenuation estimation and B-mode processing to extract five US features. The measures were used to compare different TMEs (YUMMER vs. YUMM) and responses to immunomodulatory therapies with CD8 depletion or programmed cell death protein 1 (PD-1) inhibition. <h3>Results</h3> Multiparametric analysis produced a combined H-scan parameter, resolving significant differences (i) between untreated YUMMER and YUMM and (ii) between untreated, PD-1-treated and CD8-treated YUMMER. However, more importantly, the B-mode and attenuation measures failed to differentiate YUMMER and YUMM and to monitor treatment responses, indicating that H-scan is required to differentiate subtle differences within the TME. <h3>Conclusion</h3> We anticipate that the H-scan analysis could discriminate heterogeneous melanoma metastases and guide diagnosis and treatment selection, potentially reducing the need for invasive biopsies or immunologic procedures.

Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) for metastatic melanoma has shown objective response rates as high as 72%. The successful application of this therapy requires the selection of unique tumor-reactive lymphocyte cultures for each patient. This is a technically and logistically difficult undertaking, and patients who do not have tumor-reactive TIL are not considered eligible for treatment. To simplify the methods of TIL generation and extend TIL-based immunotherapy to additional patients, methods were developed to use unselected, minimally cultured (“young”) TIL. Young TIL cultures contain a variable number of CD8+, CD4+, and CD3−CD56+ natural killer cells. In this study we retrospectively investigated a role for these subsets in the clinical outcome of patients treated with TIL derived from selected microcultures. This analysis demonstrated a suggestive but nonsignificant association between the number of CD8+ cells administered and tumor regression. We therefore investigated the feasibility of selecting CD8+ cells from young TIL cultures for ACT therapy. The available methods for clinical scale CD8+ enrichment proved inadequate for TIL, so an optimized CD8+ enrichment method was developed and is reported here. We observed that CD8+ enrichment of some TIL cultures revealed in vitro tumor recognition that was not evident in bulk culture, and an improved in vitro recognition of tumor in other TIL cultures. In addition, the enriched CD8+ young TIL expanded more reliably and predictably in rapid expansions than the bulk TIL. Thus, optimized CD8+ selection combines the benefits of antigen-selected TIL and young TIL for generating lymphocyte cultures for ACT, and should be evaluated in cell transfer therapy protocols.

The age of personalized medicine continues to evolve within clinical oncology with the arsenal available to clinicians in a variety of malignancies expanding at an exponential rate. The development and advancement of molecular treatment modalities, including targeted therapy and immune checkpoint blockade, continue to flourish. Treatment with targeted therapy (BRAF, MEK, and other small molecule inhibitors) can be associated with swift disease control and high response rates, but limited durability when used as monotherapy. Conversely, treatment with immune checkpoint blockade monotherapy regimens (anti–cytotoxic T-lymphocyte antigen 4 and anti–programmed cell death protein 1/programmed cell death protein 1 ligand) tends to have lower response rates than that observed with BRAF-targeted therapy, although these treatments may offer long-term durable disease control. With the advent of these forms of therapy, there was interest early on in empirically combining targeted therapy with immune checkpoint blockade with the hopes of preserving high response rates and adding durability; however, there is now strong scientific rationale for combining these forms of therapy—and early evidence of synergy in preclinical models of melanoma. Clinical trials combining these strategies are ongoing, and mature data regarding response rates and durability are not yet available. Synergy may ultimately be apparent; however, it has also become clear that complexities exist regarding toxicity when combining these therapies. Nonetheless, this increased appreciation of the complex interplay between oncogenic mutations and antitumor immunity has opened up tremendous opportunities for studying targeted agents and immunotherapy in combination, which extends far beyond melanoma to other solid tumors and also to hematologic malignancies.

Adoptive cell therapy (ACT) with tumor-infiltrating T cells (TILs) has emerged as a promising therapy for the treatment of unresectable or metastatic solid tumors. One challenge to finding a universal anticancer treatment is the heterogeneity present between different tumors as a result of genetic instability associated with tumorigenesis. As the epitome of personalized medicine, TIL-ACT bypasses the issue of intertumoral heterogeneity by utilizing the patient's existing antitumor immune response. Despite being one of the few therapies capable of inducing durable, complete tumor regression, many patients fail to respond. Recent research has focused on increasing therapeutic efficacy by refining various aspects of the TIL protocol, which includes the isolation, ex vivo expansion, and subsequent infusion of tumor specific lymphocytes. This review will explore how the therapy has evolved with time by highlighting various resistance mechanisms to TIL therapy and the novel strategies to overcome them.

Background: Approximately 50% of melanomas harbor BRAF mutations. Treatment with BRAF +/− MEK inhibition is associated with favorable changes in the tumor microenvironment thus providing the rationale for combining targeted agents with immunotherapy.Methods: Patients with unresectable Stage III or IV BRAFV600E mutant melanoma were enrolled in a single-center prospective study (n = 6). Patients were eligible to receive two courses of HD-IL-2 and vemurafenib twice daily. The primary endpoint was progression-free survival (PFS) with secondary objectives including overall survival (OS), response rates (RR), and safety of combination therapy as compared to historical controls. Immune profiling was performed in longitudinal tissue samples, when available.Results: Overall RR was 83.3% (95% CI: 36%–99%) and 66.6% at 12 weeks. All patients eventually progressed, with three progressing on treatment and three progressing after the vemurafenib continuation phase ended. Median PFS was 35.8 weeks (95% CI: 16–57 weeks). Median OS was not reached; however, the time at which 75% of patients were still alive was 104.4 weeks. Change in circulating BRAFV600E levels correlated with response. Though combination therapy was associated with enhanced CD8 T cell infiltrate, an increase in regulatory T cell frequency was seen with HD-IL-2 administration, suggesting a potential limitation in this strategy.Conclusion: Combination vemurafenib and HD-IL-2 is well tolerated and associated with treatment responses. However, the HD-IL-2 induced increase in Tregs may abrogate potential synergy. Given the efficacy of regimens targeting the PD-1 pathway, strategies combining these regimens with BRAF-targeted therapy are currently underway, and the role of combination vemurafenib and HD-IL-2 is uncertain.Trial Registration: Clinical trial information: NCT01754376; https://clinicaltrials.gov/show/NCT01754376

Background: The Wise pattern adapted to mastectomy incisions has become a valuable asset for breast reconstruction in patients with large and ptotic breasts. The authors compared the time for exchange, time to initiate postmastectomy radiotherapy, and complication rates between Wise pattern and transverse incision pattern reconstructions. Methods: Records of patients who underwent immediate, two-stage, implant-based breast reconstruction (IBBR) between January of 2011 and December of 2020 were retrospectively reviewed. Two cohorts were compared according to the incision pattern: Wise pattern versus transverse incision pattern. Complications were compared after propensity score matching. Results: The authors initially analyzed 393 two-stage immediate IBBRs in 239 patients [91 IBBRs (23.2%) in the Wise pattern group and 302 (76.8%) in the transverse pattern group]. Expansion time (53 days versus 50 days, P = 0.9), time for tissue expander-to-implant exchange (154 versus 175 days, P = 0.547), and time to initiate postmastectomy radiotherapy (144 days versus 126 days, P = 0.616) were not different between groups. Before propensity score matching, the 30-day rate of wound-related complications (32% versus 10%, P &lt; 0.001) and the 30-day rate of wound complications requiring excision/débridement and closure procedures (20% versus 7%, P &lt; 0.001) were significantly higher in the Wise pattern group. After propensity score matching, the 30-day rate of wound complications was persistently higher (25% versus 10%, P = 0.03) in the Wise pattern group. Conclusions: The Wise pattern mastectomy independently increases the incidence of wound-related complications versus only transverse patterns during two-stage IBBR, even after propensity score matching. Delayed tissue expander placement may improve the safety profile of this procedure. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, III.

The latissimus dorsi flap (LDF) has gained popularity given its versatile nature and broad applicability in breast reconstruction. Its resurgence has been attributed to its ability to be enhanced using implant or high-volume fat grafting, rendering it a primary option for selected patients. The aim of this review is to tackle current indications and subjects of controversy regarding use of complete-autologous and implant-enhanced LDF in breast reconstruction. Also, a case-series showcasing the authors' experience with this versatile reconstructive option is presented. A search across Web of Science and PubMed MEDLINE from inception through January 3, 2023, was conducted. Articles reporting postoperative outcomes of autologous breast reconstruction with LDF were included. Regarding the case series, electronic medical records of patients who underwent total mastectomy and autologous breast reconstruction with LDF from January 2011 to December 2021 were retrospectively reviewed. Data on demographic and oncologic characteristics, and surgical characteristics and outcomes were extracted. Our review suggests that LDF is suitable for patients who lack alternative donor site, have a history of abdominoplasty or no access to microsurgery, smokers or obese. Latissimus dorsi (LD) harvesting has almost complete shoulder function recovery in the long-term. Thoracodorsal nerve division does not cause volume loss or animation deformity. Multisite multilayer fat grafting, beveling the edges of the skin paddle and fat, folding the LD muscle and plicating the paddle allow adequate projection and contour achievement. Our case-series included 234 reconstructions. Almost half of the patients had immediate fat transfer during reconstruction (51.3%). The rate of recipient site hematoma was 3.0%, seroma was 7.7%, wound disruption 32.1%, wound disruption events requiring unplanned procedures was 13.7%, and surgical site infection (SSI) was 12.4%. The LDF is reliable and safe for immediate or delayed breast reconstruction or salvage after reconstruction failure. Its versatility, reliable anatomy, easy dissection, and relative low complication rate have revived this modality as valuable opportunity for breast reconstruction in this era.

Metastatic renal cell carcinoma (RCC) to the liver portrays a poor prognosis and liver directed therapy remains controversial. We aimed to determine potential selection criteria for patients who might benefit from this strategy.We evaluated 247 consecutive patients with RCC metastatic to the liver from a prospectively maintained database.Eighteen patients received liver directed therapy (18/247, 7%). Ten patients underwent liver resection (10/247, 4%) and eight patients underwent radiofrequency ablation (RFA, 8/247, 3%). All were rendered free of disease in the liver. Five had synchronous liver disease and underwent synchronous resections with their primary. Mortality was 0%. Fourteen had single (surgery 7, RFA 7) and four (surgery 3, RFA 1) had multiple liver lesions, respectively. Median size of lesions was 5cm (0.5 - 10cm) and 2.5cm (1 - 6cm) in the surgery and RFA groups, respectively. Median DFI was 10 months, and no difference was observed in those with a longer vs. shorter than median DFI (p = 0.95); liver specific progression free survival for the surgery and RFA groups were 4 and 6 months, respectively (p= 0.93). 1, 3 and 5-year actuarial survivals for the whole group were 89%, 40%, 27%. Median survival for the surgery group was 24 (3 to 254+) months, and for the RFA group 15.6 (7-56+) months (p = 0.56). Metachronous liver disease was associated with prolonged survival (p = 0.02).Liver directed therapy for RCC is safe. For highly selected patients with metachronous liver RCC metastases, liver directed therapy should be considered in a multidisciplinary manner.

Background: Limited comparability between study groups can generate significant selection and observer bias when evaluating the efficacy of the SPY system and fluorescence imaging for implant-based breast reconstruction. In this study, the authors compared the surgical outcomes and complications during the first stage of reconstruction between reconstructions evaluated intraoperatively with fluorescence imaging using the SPY system and clinical assessment using a matched analysis. Methods: The authors conducted a retrospective review of patients undergoing total mastectomy and immediate two-stage implant-based breast reconstruction with TEs from January of 2011 to December of 2020. The rate of complication, time for TE-to-implant exchange, and time to start radiotherapy were compared between groups (intraoperative fluorescence imaging versus clinical assessment) using a propensity score–matched analysis. Results: After propensity score matching, 198 reconstructions were evaluated. There were 99 reconstructions in each group. The median time for TE-to-implant exchange (140 days versus 185 days; P = 0.476) and time to initiate adjuvant radiotherapy (144 days versus 98 days; P = 0.199) were comparable between groups. The 30-day rate of wound-related complications (21% versus 9%; P = 0.017) and 30-day rate of wound-related unplanned interventions were significantly higher in reconstructions evaluated with clinical assessment when compared with the SPY system (16% versus 5%; P = 0.011). A higher 30-day rate of seroma (19% versus 14%; P = 0.041) and hematoma (8% versus 0%; P = 0.004) were found in reconstructions assessed intraoperatively with the SPY system. Conclusions: After matching, reconstructions evaluated with fluorescence imaging exhibited a lower incidence of early wound-related complications when compared with clinical evaluation alone. Nonetheless, the Wise pattern for mastectomy was found to be the only independent predictor associated with early wound-related complications. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, III.

2 Background: Tremendous advances have been made in cancer therapy through the use of immune checkpoint blockade, although responses are not always durable. There is a growing appreciation of the role of the microbiome in cancer-related outcomes and recent evidence in murine models suggests that modulation of the gut microbiome may enhance responses to immune checkpoint blockade in melanoma. However this has not been investigated in patients. Here, we demonstrate that differential bacterial “signatures” exist in the gut microbiome of responders (R) and non-responders (NR) to anti-PD1 therapy at baseline, and that insights gained could be used to derive actionable strategies to enhance responses. Methods: We collected oral (n = 222) and gut microbiome (n = 113) samples on a large cohort of patients with metastatic melanoma (n = 228), with a majority treated with anti-PD1 therapy (n = 110). Patients were classified as either R or NR based on RECIST criteria, and 16S rRNA gene sequencing was performed to characterize the diversity and composition of the microbiomes. Immune profiling (via 7-marker IHC panel of CD3, CD8, PD-1, PD-L1, Granzyme B, RORγT and FoxP3) was performed in available tumors at baseline. Results: In these studies, we observed significant differences in the diversity and composition of the gut microbiome in R versus NR to PD1 blockade at baseline, but no clear differences in oral microbiomes. Specifically, R had a significantly higher alpha diversity compared to NR (p = 0.017). Notable differences were also seen in the composition of the gut microbiome of R versus NR. Immune profiling demonstrated significantly increased immune infiltrates in baseline tumor samples of R, with a positive correlation between CD8, CD3, PD1 and FoxP3 T-cell density and abundance of specific bacteria enriched in R (e.g. Faecalibacterium). Conclusions: Differences exist in the diversity and composition of the gut microbiome in R vs NR to anti-PD1 therapy. These results have far-reaching implications and suggest that modifications to the gut microbiome could potentially enhance therapeutic responses to immune checkpoint blockade.

8544 Background: We have previously shown objective clinical responses in patients with metastatic melanoma treated with CTLA-4 blockade using ipilimumab. We have treated 179 patients in 3 separate clinical trials and now have long-term follow-up to evaluate the durability and unique features of this immunotherapy. Methods: A total of 179 patients with metastatic melanoma were treated in 3 trials: In Protocol 1, 56 patients received ipilimumab with gp100 peptide vaccines. In Protocol 2, 36 patients received ipilimumab with high-dose interleukin-2 (IL-2). In Protocol 3, 87 patients received intra-patient dose escalation of ipilimumab and were randomized to receive gp100 peptides. We have updated and analyzed the follow-up and survival data for these trials. Results: With median follow-up for Protocol 1, 2, and 3 being 80, 71, and 60 months, median survival was 15, 16, and 13 months, respectively. Objective tumor regression was 12% for Protocol 1, 25% for Protocol 2, and 21% for Protocol 3. Patients in Protocol 2 had a 17% complete response rate (6 patients: 77+, 74+, 72+, 71+, 71+, and 69+ months), as compared to 7% in Protocol 1 (4 patients: 82+, 81+, 79+, and 66+ months) and 8% in Protocol 3 (5 patients: 64+, 63+, 62+, 60+, and 55+ months); all complete responses are ongoing. Many patients who eventually became complete responders had continual tumor shrinkage after stopping therapy. Conclusions: CTLA-4 blockade with ipilimumab can achieve durable objective tumor regression in patients with metastatic melanoma. The combination of ipilimumab and IL-2 appears to have an increased complete response rate, although this needs to be tested in a prospective randomized trial. This report represents the largest single-institution experience with the longest follow-up for this agent; our results support its role as a viable treatment option for patients with metastatic melanoma. No significant financial relationships to disclose.

The aim of the study is to analyze treatment-induced gonadal damage and premature ovarian failure after adoptive cell therapy (ACT) after a cytotoxic lymphodepleting preparative regimen. Records of 66 consecutive females who received ACT at the Surgery Branch, National Cancer Institute, NIH (Bethesda, MD) were reviewed. Patients received a conditioning regimen of high-dose cyclophosphamide (60 mg/kg×2 doses) and fludarabine (25 mg/m2×5 doses). Some patients also received total body radiation at 200 or 600 cGy. Assessment of ovarian function was determined by analysis of monthly follicle stimulating hormone (FSH) levels, menstrual history, and symptoms. Among patients with serum available and normal pretreatment ovarian function, 21 had a preparative regimen with chemotherapy alone and 5 patients had received chemotherapy with total body radiation. Nine (43%) patients in the chemotherapy cohort and all 5 patients in the chemotherapy plus total body radiation cohort had persistently elevated FSH levels and were given the diagnosis of premature ovarian failure. Twelve (57%) patients had normal FSH levels at 6 months posttreatment. Median age of all patients at treatment was 34 years. Median age of women retaining normal ovarian function was 30 (range, 19–45) vs. 41 years (range, 30–49) for those who did not regain function. The conditioning regimen of 2 doses of cyclophosphamide (60 mg/kg) and 5 doses of fludarabine (25 mg/m2) may induce gonadal damage and premature ovarian failure. Younger age at treatment was associated with a higher frequency of normal ovarian function posttreatment, whereas adding total body radiation was associated with a high risk of ovarian failure.

Endometriosis is a common disease in women of childbearing age and is defined as the presence of endometrial glands and stroma in organs outside of the uterine cavity. Appendiceal endometriosis is very uncommon and accounts for a small fraction of all cases of extrapelvic endometriosis. Cases of that which occur during pregnancy are extremely rare with an incidence that ranges between 3 and 8 deliveries per 10 000. This makes the diagnosis extremely difficult and represents a challenge in the management of the patient. In this report we describe the case of a pregnant woman who underwent ileocecectomy for perforated appendicitis stemming from endometriosis and subsequent pre-term delivery of a 31-week-old fetus.

Introduction The tumor-draining lymph node (TDLN) plays a role in tumor immunity. Intratumorally administered microspheres (MS) that encapsulate immunomodulatory agents have emerged as a treatment strategy capable of causing profound changes in the tumor microenvironment (TME) and eliciting potent antitumor effects. We hypothesized that local delivery of MS to the TME may also drain to and therefore target the TDLN to initiate antitumor immune responses.Methods Fluorescent MS were injected into orthotopically implanted murine pancreatic tumors, and tissues were examined by whole-mount microscopy and imaging flow cytometry. The role of the TDLN was investigated for mice treated with intratumoral interleukin-12 (IL-12)-encapsulated MS in combination with stereotactic body radiotherapy (SBRT) by cytokine profile and TDLN ablation.Results Fluorescent AF-594 MS delivered intratumorally were detected in the tumor, peritumoral lymphatics, and the TDLN 2 h after injection. Phagocytic cells were observed with internalized fluorescent MS. SBRT + IL-12 MS-induced upregulation of Th1 and antitumor factors IL-12, IFN-γ, CXCL10, and granzyme B in the TDLN, and excision of the TDLN partially abrogated treatment efficacy.Conclusions Our results demonstrate that intratumorally administered MS not only target the TME, but also drain to the TDLN. Furthermore, MS encapsulated with a potent antitumor cytokine, IL-12, induce an antitumor cytokine profile in the TDLN, which is essential for treatment efficacy.

A 48-year-old white female patient underwent wide excision and sentinel lymph node biopsy for a T2a melanoma of the left deltoid region at Strong Memorial Hospital of the University of Rochester Medical Center (Rochester, NY, USA) on Dec 11, 2020. Final pathology revealed a negative excision margin and one of two sentinel lymph nodes with micrometastatic disease (with no extracapsular extension); T2aN1 pathological stage IIIA. Staging brain MRI was negative; however, a PET–CT scan showed substantial fluorodeoxyglucose (18F-FDG) avidity in the contralateral axilla and neck (figure, A) and postoperative changes in the ipsilateral axilla and shoulder (small seroma and biopsy clips from sentinel lymph node biopsy).

Targeted and immunotherapy regimens have revolutionized the treatment of advanced melanoma patients. Despite this, only a subset of patients respond durably. Recently, combination strategies of BRAF/MEK inhibitors with immune checkpoint inhibitor monotherapy (α-CTLA-4 or α-PD-1) have increased the rate of durable responses. Based on evidence from our group and others, these therapies appear synergistic, but at the cost of significant toxicity. We know from other treatment paradigms (e.g. hematologic malignancies) that combination strategies with multi-drug regimens (>4 drugs) are associated with more durable disease control. To better understand the mechanism of these improved outcomes, and to identify and prioritize new strategies for testing, we studied several multi-drug regimens combining BRAF/MEK targeted therapy and immunotherapy combinations in a Braf-mutant murine melanoma model (BrafV600E/Pten−/−). Short-term treatment with α-PD-1 and α-CTLA-4 monotherapies were relatively ineffective, while treatment with α-OX40 demonstrated some efficacy [17% of mice with no evidence of disease, (NED), at 60-days]. Outcomes were improved in the combined α-OX40/α-PD-1 group (42% NED). Short-term treatment with quadruplet therapy of immunotherapy doublets in combination with targeted therapy [dabrafenib and trametinib (DT)] was associated with excellent tumor control, with 100% of mice having NED after combined DT/α-CTLA-4/α-PD-1 or DT/α-OX40/α-PD-1. Notably, tumors from mice in these groups demonstrated a high proportion of effector memory T cells, and immunologic memory was maintained with tumor re-challenge. Together, these data provide important evidence regarding the potential utility of multi-drug therapy in treating advanced melanoma and suggest these models can be used to guide and prioritize combinatorial treatment strategies.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer. Most cases of HCC are secondary to either a viral hepatitis infection (hepatitis B or C) or cirrhosis secondary to chronic alcoholism (1). Etiological factors for HCC vary widely depending on geographic location. In regions where Hepatitis B (HBV) is endemic, such as the eastern hemisphere, this is uniformly the most common cause (2). It is estimated that chronic HBV and hepatitis C virus (HCV) infections account for an estimated 78% of global HCC cases (3).

Metastatic melanoma portends a poor prognosis and patients may present with multiple, simultaneous tumors. Despite recent advances in systemic immunotherapy, a majority of patients fail to respond, or exhibit lesion-specific responses wherein some metastases respond as others progress within the same patient. While intertumoral heterogeneity has been clinically associated with these mixed lesion-specific therapeutic responses, no clear mechanism has been identified, largely due to the scarcity of preclinical models. We developed a novel murine synchronous melanoma model that recapitulates this intertumoral genetic and microenvironmental heterogeneity. We show that genetic differences between tumors are sufficient to generate distinct tumor immune microenvironments (TIME) simultaneously in the same mouse. Furthermore, these TIMEs lead to the independent regulation of PD-1/PD-L1 (programmed cell death protein 1/PD-1 ligand), a popular axis targeted by immune checkpoint therapy, in response to ongoing anti-tumor immunity and the presence of interferon-gamma. Currently, therapeutic selection for metastatic melanoma patients is guided by a single biopsy, which may not represent the immune status of all tumors. As a result, patients can display heterogeneous lesion-specific responses. Further investigations into this synchronous melanoma model will provide mechanistic insight into the effects of intertumoral heterogeneity and guide therapeutic selection in this challenging patient population.

To the Editor: We read with great interest the study by Ghinolfi et al (1.Ghinolfi D Marti J De Simone Use of octogenarian donors for liver transplantation: A survival analysis.Am J Transplant. 2014; 14 (et al): 2062-2071Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) recently published in American Journal of Transplantation. In this report, 5-year patient and graft survival rates with donors ≥80 years were outstanding—78.2% and 77.1%, respectively. Hepatitis C virus (HCV) status was the most significant factor of graft survival at multivariate analysis (hazard ratio 1.749, p < 0.001). At the latest follow-up, clinically significant HCV recurrence was observed in 13.6% and 9% of the patients transplanted with donors between 70 and 79 years of age and ≥80 years, respectively; comparable to that observed with younger donors. Of note, 5-year graft survival of the HCV-positive recipients transplanted with donors ≥80 years was 62.4% which, albeit significantly lower than that of the non-HCV recipients, is surprisingly high. Unfortunately, information on late graft loss and late retransplantation is not provided by the authors. Our experience is different. In November 2009, our IRB approved a retrospective case–control study to compare a study group of 25 HCV-positive patients transplanted between January 1998 and October 2004 with donors ≥70 years with a control group of 50 patients transplanted with donors <70 years during the same period of time (2.Gastaca M Ruiz P Fernandez JR Long-term outcome of liver transplantation with donors aged 70 and above in HCV patients. A case control study.Liver Transplant. 2010; 16 (et al): S127Google Scholar). All recipients had a minimum follow-up of 5 years. An aggressive HCV recurrence, defined as cholestatic or fibrosis stage ≥2 according to Scheuer score within the first year, was assessed in 75% of patients in the study group versus 33.3% in the control group (p = 0.002). Survival at 1, 5, 7, and 10 years in the study and control groups was as follows: graft survival was 84%, 48%, 29.1%, and 29.1% versus 100%, 82%, 74%, and 57%, respectively (p < 0.008), while patient survival was 84%, 63.7%, 51.8%, and 51.8% versus 100%, 82%, 74%, and 62.8%, respectively (p = n.s.). Of note, the rate of retransplantation was 25% versus 2% in the study and control groups (p = 0.004), all due to HCV recurrence. Similar results were recently published with 50% and 40% 5-year patient and graft survival in HCV-positive recipients transplanted with donors older than 70 years (3.Cherid FM Rosen CB Nyberg SL Heimbach JK Excellent long-term patient and graft survival are possible with appropriate use of livers from deceased septuagenarian and octogenarian donors.HPB (Oxford). 2014; 16: 852-858Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The reasons for the positive results obtained by Ghinolfi et al are difficult to determine. The mean Model for End-Stage Liver Disease score of our study group was 15 ± 6.4 and our aged donors were also carefully selected with significantly less hypotension episodes, lower mean alanine aminotransferase and a mean cold ischemic time of 290 ± 65 min. Nevertheless, the fact that our patients were transplanted in an earlier period of time and the inclusion of HCV recipients with preemptive antiviral treatment in their study might account for the relative superiority of their results. For the last decade, our policy has been to avoid the use of older donors in patients with HCV owing to a prohibitively high rate of recurrence leading to graft loss and retransplantation; however, this policy will have to be revised in the near future when the new, highly effective, HCV antiviral therapies become widely available (4.Lens S Gambato M Londono MC Forns X Interferon-free regimens in the liver-transplant setting.Semin Liver Dis. 2014; 34: 58-71Crossref PubMed Scopus (28) Google Scholar). The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

Metastatic melanoma remains the deadliest form of skin cancer. Immune checkpoint inhibition (ICI) immunotherapy has defined a new age in melanoma treatment, but responses remain inconsistent and some patients develop treatment resistance. The myriad of newly developed small molecular (SM) inhibitors of specific effector targets now affords a plethora of opportunities to increase therapeutic responses, even in resistant melanoma. In this review, we will discuss the multitude of SM classes currently under investigation, current and prospective clinical combinations of ICI and SM therapies, and their potential for synergism in melanoma eradication based on established mechanisms of immunotherapy resistance.

The H-scan analysis has been developed as a sensitive measure of ultrasound scattering types. In this study, we aim to distinguish between two different melanoma tumors: YUMM and YUMMER. To characterize the tumor tissues, H-scan was applied to the melanoma samples, resulting in estimation of several parameters. Multiparametric analysis was performed by measuring the H-scan blue fraction, attenuation coefficient, H-scan signal-to-noise ratio (SNR), and B-scan SNR. Principal component analysis was applied to the parameters, forming clusters in 2D space. These were assigned as the inputs for support vector machine (SVM) classification. H-scan imaging was able to differentiate between YUMM and YUMMER; YUMMER showed as more blue (higher frequency) in color. The H-scan parameters of blue fraction and SNR show significant differences between the two types of melanoma. Combining the four parameters, principal components formed clusters of YUMM and YUMMER. The clusters were separated by the decision planes obtained using SVM analysis with 88.5% classification accuracy. We anticipate clinical use of H-scan parameters for melanoma diagnosis.

In the present work, circular Metal-Oxide-Semiconductor capacitors with 200 μm of diameter and germanium (Ge) nanoparticles (NPs) embedded in the gate oxide are studied for memory applications. Optimal process parameters are investigated for Ge NPs growing by low pressure chemical vapor deposition at different deposition times. Photoluminescence measurements showed room-temperature size-dependent green-red region bands attributed to quantum confinement effects present in the NPs. High-frequency capacitance versus voltage measurements demonstrated the memory effects on the MOS structures due to the presence of Ge NPs in the gate oxide acting as discrete floating gates. Current versus voltage measurements confirmed the Fowler-Nordheim tunneling as the programming mechanism of the devices.

Abstract Background: Melanoma therapy has benefitted greatly from immune checkpoint blockade, although responses are variable and not always durable. There is a growing appreciation of the role of the microbiome in cancer-related outcomes and recent evidence in murine models suggests that modulation of the gut microbiome may enhance responses to immune checkpoint blockade in melanoma. However this has not been investigated in patients. Here, we demonstrate that differential bacterial “signatures” exist in the gut microbiome of responders (R) and non-responders (NR) to anti-PD-1 therapy, and that insights gained could be used to derive actionable strategies to enhance responses. Methods: We collected buccal (n=105) and stool (n=53) samples from a cohort of anti-PD-1 treated metastatic melanoma patients (n=110). Patients were classified as either R or NR based on RECIST criteria, and 16S rDNA, and whole-genome shotgun sequencing was performed to characterize the diversity, composition and functional capabilities of the microbiomes. Immune profiling (via 7-marker IHC panel of CD3, CD8, PD-1, PD-L1, Granzyme B, RORγT and FoxP3) and cytokine analyses were also performed on available tumors and serum samples at baseline. Results: In these studies, we observed significant differences in the diversity and composition of the gut microbiome in R versus NR to PD-1 blockade at baseline, but no clear differences in buccal microbiomes. Specifically, R had a significantly higher alpha diversity compared to NR (p=0.017), and the Ruminococcaceae family of the Clostridiales order was enriched in R whereas Prevotellaceae family of the Bacteroidales order was enriched in NR. Immune profiling demonstrated significantly increased immune infiltrates in baseline tumor samples of R, with a positive correlation between CD8, CD3, PD-1 and FoxP3 T-cell density and abundance of specific bacteria enriched in R (e.g. Faecalibacterium). Low diversity was also associated with elevated levels of chronic inflammation markers in the serum at baseline. Lastly, we saw differentially abundant metabolic pathways in the gut microbiomes of R (pyrimidine nucleotide biosynthesis, fatty acid biosynthesis, shikimate pathway) vs NR (Tricarboxylic acid cycle, assimilatory sulphate and nitrate reduction, tryptophan biosynthesis). Conclusion: Differences exist in the diversity and composition of the gut microbiome in R vs NR to anti-PD-1 therapy and these microbiota could bridge the gap between host metabolism and anti-tumor immunity. These results have far-reaching implications and suggest that modifications to the gut microbiome could potentially enhance therapeutic responses to immune checkpoint blockade. Citation Format: Vancheswaran Gopalakrishnan, Christine Spencer, Alexandre Reuben, Peter Prieto, Diego Vicente, Tatiana V. Karpinets, Courtney W. Hudgens, Diane S. Hutchinson, Michael Tetzlaff, Alexander Lazar, Michael A. Davies, Jeffrey E. Gershenwald, Robert Jenq, Patrick Hwu, Padmanee Sharma, James Allison, Andrew Futreal, Nadim Ajami, Joseph Petrosino, Carrie Daniel-MacDougall, Jennifer A. Wargo. Response to anti-PD-1 based therapy in metastatic melanoma patients is associated with the diversity and composition of the gut microbiome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2672. doi:10.1158/1538-7445.AM2017-2672

9587 Background: Targeted and immune therapies have dramatically improved outcomes in stage IV metastatic melanoma pts. These agents are now being tested in earlier-stage disease. SOC surgery for high-risk resectable melanoma (AJCC stage IIIB/IIIC), with or without adjuvant therapy, is associated with a high risk of relapse (~70%). We hypothesized that neoadjuvant (neo) + adjuvant treatment with D+T improves RFS in these pts. Longitudinally collected biospecimens from pts receiving this treatment were analyzed to identify candidate strategies to further improve outcomes. Methods: A prospective single-institution randomized clinical trial (NCT02231775) was conducted in BRAF-mutant pts with resectable Stage IIIB/C or oligometastatic stage IV melanoma. Pts were randomized 1:2 to SOC (Arm A) versus neo + adjuvant D+T (Arm B; 8 wks neo + 44 wks adjuvant). The primary endpoint was RFS. Tumor biopsies were collected at baseline, week 3, and at surgery for molecular and immune profiling (whole exome sequencing, gene expression profiling, IHC, flow cytometry). Results: 21 of a planned 84 patients were enrolled (Arm A = 7, Arm B = 14). Arms were well balanced for standard prognostic factors, and toxicity was manageable. RECIST response rate with neo D+T was 77%, and the pathologic complete response rate (pCR) was 58%. First interim analysis revealed significantly improved RFS in the D+T arm over SOC (HR 62.5, p &lt; 0.0001), leading to early closure to enrollment. Pts with a pCR at surgery had significantly improved RFS versus pts without pCR (p = 0.04) on neo D+T. Tumor profiling revealed incomplete MAPK pathway blockade and higher levels of CD8+ T cells expressing immunomodulators Tim-3 and Lag-3 in pts who did not achieve a pCR. Conclusions: Neo + adjuvant D+T is associated with a high pCR rate and markedly improved RFS over SOC in pts with high-risk resectable BRAF-mutant metastatic melanoma. pCR at surgery is associated with improved RFS. Tumor analyses reveal candidate targets for testing in future trials to enhance responses to neo D+T. Clinical trial information: NCT02231775.

Abstract Background: The gut microbiome is increasingly being recognized as a strong modulator of anti-PD1 based cancer immunotherapy. Compelling evidence demonstrates differential bacterial enrichment and diversity in responders (R) versus non-responders (NR), mediated by profound influences on systemic and anti-tumor immune infiltrates. However, this has not been studied in the setting of treatment with combined immune checkpoint blockade (CICB), which is associated with superior response rates, but higher rates of potentially debilitating toxicities. Methods: We assembled a cohort of patients with metastatic melanoma receiving CICB (n=54). All patients were classified as R (n=31, CR + PR) or NR (n=23, SD + PD) based on RECIST v1.1, and as having grade 3 or higher (T; n=29), or less than grade 3 (NT; n=25) immune related adverse event(s) by NCI CTCAE 4.0 criteria. Baseline stool samples were characterized by 16S rRNA sequencing. Correlative analyses of peripheral immune cell populations by flow cytometry (n=12) and circulating T cell repertoire by TCR-sequencing (n=12) were done on matched pre-treatment blood samples. Results: The overall gut microbial landscape in these patients was varied with high abundance of Bacteroidales and Clostridiales. Ordination of beta-diversity distances revealed a lack of clustering by subtype of primary tumor (uveal, mucosal, cutaneous) consistent with no significant effect of the tumor histology. While no apparent response or toxicity associations were evident based on diversity, notable compositional differences were appreciated. Comparison of relative abundances by LEfSe (LDA&amp;gt;2, p&amp;lt;0.05), and pairwise Mann-Whitney tests revealed an enrichment of Bacteroides stercoris (p=0.03), and Parabacteroides distasonis (p=0.04) in R, and Lactobacillales (p=0.005) in NR. Consistent with our prior findings, the median relative abundance of the order Clostridiales was again higher in R (0.34) versus NR (0.26). On the other hand, Bacteroides intestinalis (p=0.01) and Anaerotignum lactatifermentans (p=0.006) were enriched in T and NT, respectively. Importantly, correlative analyses with circulating immune cell subsets revealed distinct associations by differential bacterial enrichment (including positive correlations between overall CD8+ T-cell abundance and R-taxa), and a clustering effect by high or low T-cell repertoire entropy. Conclusion: These findings build on our prior work and support the notion of a close link between the gut microbiome and therapeutic outcomes to checkpoint blockade therapy. Extensive studies are underway in both matched human biospecimens and in pre-clinical models to further understand mechanisms of interactions with immune markers, and to establish causality. Taken together, these data support a critical role for the gut microbiome as both a predictive tool and therapeutic target. Citation Format: Vancheswaran Gopalakrishnan, Miles Andrews, Wei-Shen Chen, Christine Spencer, Luis Vence, Alexandre Reuben, Zachary A. Cooper, Peter A. Prieto, Michael T. Tetzlaff, MA Abdul Wadud Khan, Alexander Lazar, Courtney W. Hudgens, Lauren E. Haydu, Hussein A. Tawbi, Patrick Hwu, Wen-Jen Hwu, Rodabe N. Amaria, Elizabeth M. Burton, Scottt E. Woodman, Adi Diab, Sapna P. Patel, Isabella C. Glitza, Jianhua Zhang, Joseph Petrosino, Robert R. Jenq, Michael A. Davies, Jeffrey E. Gershenwald, Padmanee Sharma, James P. Allison, Andrew Futreal, Jennifer A. Wargo. Therapeutic efficacy and tolerability of combined immune checkpoint blockade in metastatic melanoma patients is influenced by the gut microbiome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1493.

Abstract Treatment with combined immune checkpoint blockade (CICB) targeting CTLA-4 and PD-1 is associated with clinical benefit across tumor types, but a high rate of immune-related adverse events (irAE). Insights into biomarkers and mechanisms of response and toxicity to CICB are needed. To address this, we profiled the blood, tumor and gut microbiome of 77 advanced melanoma patients treated with CICB, with a high rate of any ≥Grade 3 irAEs (49%) with parallel studies in pre-clinical models. Tumor-associated immune and genomic biomarkers of response to CICB were similar to those identified for ICB monotherapy, and toxicity from CICB was associated with a more diverse peripheral T cell repertoire. Profiling of gut microbiota demonstrated a significantly higher abundance of Bacteroides intestinalis in patients with toxicity, with upregulation of mucosal IL-1b in patient samples of colitis and in pre-clinical models. Together, these data offer potential new therapeutic angles for targeting toxicity to CICB.

Airway compromise from postoperative neck hematoma remains the most feared complication after cervical endocrine operative procedures. Events are rare and potentially lethal, and clear multidisciplinary guidelines for management of these patients are lacking. The aim of our study was to review the experience of a tertiary cancer center in this scenario.Data prospectively collected over a 10-year period, between 2005 and 2014, were retrospectively analyzed. We included all adult patients who had had a neck operation and needed reoperation for postoperative neck hematoma after an endocrine procedure. We excluded pediatric patients and cases with incomplete records.The inclusion criteria were met for 21 patients (21/2,930; 0.7%). The median age at operation was 56.2 years (SD: 16.7). The M:F ratio was 1:2. All 21 patients presented with a neck swelling at the time of reoperation. Eight of 21 patients (38%) underwent emergency bedside clot evacuation. Presentation was within 6 hours for two thirds (14/21) of the patients; the remaining one third of the patients had the hematoma develop during the evening/night (from 1700-0500). The mean estimated hematoma size was 98 cc (SD: 58). A source of bleeding was identified in 12 of 21 cases (57%). A total of 15.8% of patients had an airway classified as difficult/awkward under the American Society of Anesthesiologists classification for their wound re-exploration.Postoperation, increased vigilance is needed for the first 6 hours to detect patients with neck swelling. Emergency drainage by the bedside was performed in 38% of patients. A difficult airway was uncommon in our series.

&lt;p&gt;Supplementary Figure S1; Supplementary Table S1.&lt;/p&gt;

CCR Translation for This Article from CTLA-4 Blockade with Ipilimumab: Long-term Follow-up of 177 Patients with Metastatic Melanoma

&lt;div&gt;Abstract&lt;p&gt;&lt;b&gt;Purpose:&lt;/b&gt; Tumor‐infiltrating lymphocytes (TIL) and interleukin (IL)-2 administered following lymphodepletion can cause the durable complete regression of bulky metastatic melanoma in patients refractory to approved treatments. However, the generation of a unique tumor-reactive TIL culture for each patient may be prohibitively difficult. We therefore investigated the clinical and immunologic impact of unscreened, CD8+ enriched “young” TIL.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Experimental Design:&lt;/b&gt; Methods were developed for generating TIL that minimized the time in culture and eliminated the individualized tumor-reactivity screening step. Thirty-three patients were treated with these CD8+ enriched young TIL and IL-2 following nonmyeloablative lymphodepletion (NMA). Twenty-three additional patients were treated with CD8+ enriched young TIL and IL-2 after lymphodepletion with NMA and 6 Gy of total body irradiation.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Results:&lt;/b&gt; Young TIL cultures for therapy were successfully established from 83% of 122 consecutive melanoma patients. Nineteen of 33 patients (58%) treated with CD8+ enriched young TIL and NMA had an objective response (Response Evaluation Criteria in Solid Tumors) including 3 complete responders. Eleven of 23 patients (48%) treated with TIL and 6 Gy total body irradiation had an objective response including 2 complete responders. At 1 month after TIL infusion the absolute CD8+ cell numbers in the periphery were highly correlated with response.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; This study shows that a rapid and simplified method can be used to reliably generate CD8+ enriched young TIL for administration as an individualized therapy for advanced melanoma, and may allow this potentially effective treatment to be applied at other institutions and to reach additional patients. &lt;i&gt;Clin Cancer Res; 16(24); 6122–31. ©2010 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;&lt;b&gt;Purpose:&lt;/b&gt; Treatment with ipilimumab can cause objective tumor responses in patients with metastatic melanoma. We have treated 177 evaluable patients in three clinical trials and have long-term follow-up to evaluate the durability of responses.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Experimental Design:&lt;/b&gt; Patients with metastatic melanoma were treated in three trials from 2002 to 2005. In protocol 1, 56 patients received ipilimumab with gp100 peptides. In protocol 2, 36 patients received ipilimumab with interleukin-2. In protocol 3, 85 patients received ipilimumab with intrapatient dose-escalation and were randomized to receive gp100 peptides. We have analyzed their long-term follow-up and survival data.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Results:&lt;/b&gt; With median follow-up for protocols 1, 2, and 3 being 92, 84, and 71 months, median survival was 14, 16, and 13 months with 5-year survival rates being 13%, 25%, and 23%, respectively. Patients in protocol 2 had a 17% complete response (CR) rate, compared with 7% in protocol 1 and 6% in protocol 3. These CR rates are higher than previously reported for the same trials because some patients who eventually became complete responders had continual tumor regression months to years after therapy. All but one of the 15 complete responders are ongoing at 54+ to 99+ months.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; This report provides the longest follow-up of patients with melanoma treated with ipilimumab and shows that ipilimumab can induce durable, potentially curative tumor regression in a small percentage of patients with metastatic melanoma. The combination of ipilimumab and interleukin-2 seems to have an increased CR rate, but this needs to be tested in a randomized trial. &lt;i&gt;Clin Cancer Res; 18(7); 2039–47. ©2012 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;&lt;b&gt;Purpose:&lt;/b&gt; Tumor‐infiltrating lymphocytes (TIL) and interleukin (IL)-2 administered following lymphodepletion can cause the durable complete regression of bulky metastatic melanoma in patients refractory to approved treatments. However, the generation of a unique tumor-reactive TIL culture for each patient may be prohibitively difficult. We therefore investigated the clinical and immunologic impact of unscreened, CD8+ enriched “young” TIL.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Experimental Design:&lt;/b&gt; Methods were developed for generating TIL that minimized the time in culture and eliminated the individualized tumor-reactivity screening step. Thirty-three patients were treated with these CD8+ enriched young TIL and IL-2 following nonmyeloablative lymphodepletion (NMA). Twenty-three additional patients were treated with CD8+ enriched young TIL and IL-2 after lymphodepletion with NMA and 6 Gy of total body irradiation.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Results:&lt;/b&gt; Young TIL cultures for therapy were successfully established from 83% of 122 consecutive melanoma patients. Nineteen of 33 patients (58%) treated with CD8+ enriched young TIL and NMA had an objective response (Response Evaluation Criteria in Solid Tumors) including 3 complete responders. Eleven of 23 patients (48%) treated with TIL and 6 Gy total body irradiation had an objective response including 2 complete responders. At 1 month after TIL infusion the absolute CD8+ cell numbers in the periphery were highly correlated with response.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; This study shows that a rapid and simplified method can be used to reliably generate CD8+ enriched young TIL for administration as an individualized therapy for advanced melanoma, and may allow this potentially effective treatment to be applied at other institutions and to reach additional patients. &lt;i&gt;Clin Cancer Res; 16(24); 6122–31. ©2010 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;&lt;b&gt;Purpose:&lt;/b&gt; Treatment with ipilimumab can cause objective tumor responses in patients with metastatic melanoma. We have treated 177 evaluable patients in three clinical trials and have long-term follow-up to evaluate the durability of responses.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Experimental Design:&lt;/b&gt; Patients with metastatic melanoma were treated in three trials from 2002 to 2005. In protocol 1, 56 patients received ipilimumab with gp100 peptides. In protocol 2, 36 patients received ipilimumab with interleukin-2. In protocol 3, 85 patients received ipilimumab with intrapatient dose-escalation and were randomized to receive gp100 peptides. We have analyzed their long-term follow-up and survival data.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Results:&lt;/b&gt; With median follow-up for protocols 1, 2, and 3 being 92, 84, and 71 months, median survival was 14, 16, and 13 months with 5-year survival rates being 13%, 25%, and 23%, respectively. Patients in protocol 2 had a 17% complete response (CR) rate, compared with 7% in protocol 1 and 6% in protocol 3. These CR rates are higher than previously reported for the same trials because some patients who eventually became complete responders had continual tumor regression months to years after therapy. All but one of the 15 complete responders are ongoing at 54+ to 99+ months.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; This report provides the longest follow-up of patients with melanoma treated with ipilimumab and shows that ipilimumab can induce durable, potentially curative tumor regression in a small percentage of patients with metastatic melanoma. The combination of ipilimumab and interleukin-2 seems to have an increased CR rate, but this needs to be tested in a randomized trial. &lt;i&gt;Clin Cancer Res; 18(7); 2039–47. ©2012 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

CCR Translation for This Article from CTLA-4 Blockade with Ipilimumab: Long-term Follow-up of 177 Patients with Metastatic Melanoma

&lt;p&gt;Supplementary Figure S1; Supplementary Table S1.&lt;/p&gt;

&lt;p&gt;Supplementary Table S1a. Patient Cohort Clinical Summary. Supplementary Table S1b. Patient clinical characteristics. Supplementary Table S1c. Immune profiling by IHC sample log. Supplementary Table S1d. Nanostring 54 sample log. Supplementary Table S2. Summary of immune profiling by immunohistochemistry. Supplementary Table S3. Summary of immune profiling by 4 additional myeloid markers. Supplementary Table S4. Summary of immune profiling by IHC of additional CTLA-4 blockade-naïve samples. Supplementary Table S5. Nanostring Gene List. Supplementary Table S6a. Nanostring summary 54 samples. Supplementary Table S6b. Differentially upregulated and downregulated genes in pre-treatment anti-CTLA-4 samples. Supplementary Table S6c. Differentially upregulated and downregulated genes in on-treatment anti-CTLA-4 samples. Supplementary Table S6d. Differentially upregulated and downregulated genes in pre-treatment anti-PD-1 samples. Supplementary Table S6e. Differentially upregulated and downregulated genes in on-treatment anti-PD-1 samples. Supplementary Table S7. Differentially upregulated and downregulated genes from pre- to on-treatment anti-CTLA-4. Supplementary Table S8. Differentially upregulated and downregulated genes from pre- to on-treatment anti-PD-1. Supplementary Table S9a. Nanostring gene list - chip used for comparison of CTLA-4 blockade-experienced vs -naive anti-PD1 samples (28 samples). Supplementary Table S9b. NanoString additional 28 samples to compare CTLA-4 blockade-experienced vs -naive anti-PD1 samples. Supplementary Table S9c. NanoString normalized data of additional 28 samples to compare CTLA-4 blockade-experienced vs -naive anti-PD1 samples. Supplementary Table S10. Commonly differentially regulated genes between pre- to on-treatment CTLA-4 blockade and PD-1 blockade. Supplementary Table S11a. Fold changes of significant change by anti-PD-1 therapy for paired samples. Supplementary Table S11b. Frequency of significant change by anti-PD1 therapy for paired samples.&lt;/p&gt;

&lt;p&gt;Supplementary Figure Legends&lt;/p&gt;

&lt;p&gt;Supplementary Figure 1. Immune profiling of pre-treatment, on-treatment and progression CTLA-4 blockade samples by immunohistochemistry. Supplementary Figure 2. Myeloid cell profiling of pre-treatment, on-treatment and progression CTLA-4 blockade samples by immunohistochemistry. Supplementary Figure 3. Increased contact between CD8 T cells and CD68 myeloid cells in non-responding patients to anti-CTLA-4 and anti-PD-1 therapy at pre-treatment CTLA-4 blockade, pre-treatment PD-1 blockade, and on-treatment PD-1 blockade time points. Supplementary Figure 4. Immune profiling of pre anti-PD-1, on-treatment anti-PD-1 and progression anti-PD-1 samples by immunohistochemistry. Supplementary Figure 5. Longitudinal increase in CD8, PD-1, and PD-L1 expression in responders to anti-PD-1 therapy. Supplementary Figure 6. Relative increase in CD8 T cell infiltrate at tumor center in responders to anti-PD-1 on treatment. Supplementary Figure 7. Significant increase in immune infiltrate between responders and non-responders to PD-1 blockade in absence of prior anti-CTLA-4 therapy. Supplementary Figure 8. Immune profiling of myeloid cells atpre-treatment and on-treatment PD-1 blockade time pointsby immunohistochemistry. Supplementary Figure 9. Heatmap of 54 NanoString samples. Supplementary Figure 10. Gene-specific NanoString concordance with immune profiling by IHC in pre-treatment, on-treatment and progression CTLA-4 blockade samples. Supplementary Figure 11. Gene-specific NanoString concordance with immune profiling by IHC in pre-treatment, on-treatment and progression PD-1 blockade samples. Supplementary Figure 12. Prior CTLA-4 blockade is not required for PD-1 early on-treatment profile. Supplementary Figure 13. Hierarchical clustering of gene expression across 54 samples confirms lack of batch effect.&lt;/p&gt;

&lt;p&gt;Supplementary Figure 1. Immune profiling of pre-treatment, on-treatment and progression CTLA-4 blockade samples by immunohistochemistry. Supplementary Figure 2. Myeloid cell profiling of pre-treatment, on-treatment and progression CTLA-4 blockade samples by immunohistochemistry. Supplementary Figure 3. Increased contact between CD8 T cells and CD68 myeloid cells in non-responding patients to anti-CTLA-4 and anti-PD-1 therapy at pre-treatment CTLA-4 blockade, pre-treatment PD-1 blockade, and on-treatment PD-1 blockade time points. Supplementary Figure 4. Immune profiling of pre anti-PD-1, on-treatment anti-PD-1 and progression anti-PD-1 samples by immunohistochemistry. Supplementary Figure 5. Longitudinal increase in CD8, PD-1, and PD-L1 expression in responders to anti-PD-1 therapy. Supplementary Figure 6. Relative increase in CD8 T cell infiltrate at tumor center in responders to anti-PD-1 on treatment. Supplementary Figure 7. Significant increase in immune infiltrate between responders and non-responders to PD-1 blockade in absence of prior anti-CTLA-4 therapy. Supplementary Figure 8. Immune profiling of myeloid cells atpre-treatment and on-treatment PD-1 blockade time pointsby immunohistochemistry. Supplementary Figure 9. Heatmap of 54 NanoString samples. Supplementary Figure 10. Gene-specific NanoString concordance with immune profiling by IHC in pre-treatment, on-treatment and progression CTLA-4 blockade samples. Supplementary Figure 11. Gene-specific NanoString concordance with immune profiling by IHC in pre-treatment, on-treatment and progression PD-1 blockade samples. Supplementary Figure 12. Prior CTLA-4 blockade is not required for PD-1 early on-treatment profile. Supplementary Figure 13. Hierarchical clustering of gene expression across 54 samples confirms lack of batch effect.&lt;/p&gt;

&lt;p&gt;Supplementary Figure Legends&lt;/p&gt;

&lt;p&gt;Supplementary Table S1a. Patient Cohort Clinical Summary. Supplementary Table S1b. Patient clinical characteristics. Supplementary Table S1c. Immune profiling by IHC sample log. Supplementary Table S1d. Nanostring 54 sample log. Supplementary Table S2. Summary of immune profiling by immunohistochemistry. Supplementary Table S3. Summary of immune profiling by 4 additional myeloid markers. Supplementary Table S4. Summary of immune profiling by IHC of additional CTLA-4 blockade-naïve samples. Supplementary Table S5. Nanostring Gene List. Supplementary Table S6a. Nanostring summary 54 samples. Supplementary Table S6b. Differentially upregulated and downregulated genes in pre-treatment anti-CTLA-4 samples. Supplementary Table S6c. Differentially upregulated and downregulated genes in on-treatment anti-CTLA-4 samples. Supplementary Table S6d. Differentially upregulated and downregulated genes in pre-treatment anti-PD-1 samples. Supplementary Table S6e. Differentially upregulated and downregulated genes in on-treatment anti-PD-1 samples. Supplementary Table S7. Differentially upregulated and downregulated genes from pre- to on-treatment anti-CTLA-4. Supplementary Table S8. Differentially upregulated and downregulated genes from pre- to on-treatment anti-PD-1. Supplementary Table S9a. Nanostring gene list - chip used for comparison of CTLA-4 blockade-experienced vs -naive anti-PD1 samples (28 samples). Supplementary Table S9b. NanoString additional 28 samples to compare CTLA-4 blockade-experienced vs -naive anti-PD1 samples. Supplementary Table S9c. NanoString normalized data of additional 28 samples to compare CTLA-4 blockade-experienced vs -naive anti-PD1 samples. Supplementary Table S10. Commonly differentially regulated genes between pre- to on-treatment CTLA-4 blockade and PD-1 blockade. Supplementary Table S11a. Fold changes of significant change by anti-PD-1 therapy for paired samples. Supplementary Table S11b. Frequency of significant change by anti-PD1 therapy for paired samples.&lt;/p&gt;

Background and Objective: With the incorporation of autologous fat grafting, acellular dermal matrix (ADM) products, and nipple-sparing mastectomy, prepectoral device placement has become more popular in selected patients when compared to partial submuscular (dual plane) or complete submuscular device placement. In this article, we aimed to present a review of the current state-of-the-art for implant-based breast reconstruction (IBBR) using expanders. Additionally, we present a case series of our experience with IBBR evaluating perioperative outcomes, complications, and patient-reported outcomes (PRO). Methods: For our series, we retrospectively evaluated adult female patients undergoing 2-stage immediate IBBR after total mastectomy between 2011 and 2021. We performed a systematic search across PubMed MEDLINE for articles evaluating outcomes of prepectoral versus subpectoral two-stage IBBR with expanders published from database inception through February 28th, 2023. Key Content and Findings: Both prepectoral and subpectoral are safe alternatives for two-stage IBBR. Due to current advancements in the field of breast reconstruction, prepectoral IBBR has gained popularity and has a comparable rate of complications compared to a subpectoral approach in selected patients according to high-quality articles. In patients with several comorbidities, current tobacco use, history of preoperative radiation, and limited perfusion of the mastectomy flaps, subpectoral device placement should be given special consideration as a layer of vascularized tissue can decrease the risk of major complications or unplanned procedures. As prepectoral device placement does not require dissection of the pectoral muscles, faster recovery, better implant position, decreased pain, and a shorter time to complete expansion is expected. The plane of reconstruction does not seem to significantly affect the time for expander-to-implant exchange or PRO for quality-of-life (QOL) according to most studies. Conclusions: Prepectoral and subpectoral IBBR demonstrated a comparable rate of complications in selected patients. Nonetheless, perioperative outcomes seem to be improved using a prepectoral approach in terms of reduced pain, reduced time to conclude outpatient expansions, and less animation deformity.

&lt;div&gt;Abstract&lt;p&gt;Immune checkpoint blockade represents a major breakthrough in cancer therapy; however, responses are not universal. Genomic and immune features in pretreatment tumor biopsies have been reported to correlate with response in patients with melanoma and other cancers, but robust biomarkers have not been identified. We studied a cohort of patients with metastatic melanoma initially treated with cytotoxic T-lymphocyte–associated antigen-4 (CTLA4) blockade (&lt;i&gt;n&lt;/i&gt; = 53) followed by programmed death-1 (PD-1) blockade at progression (&lt;i&gt;n&lt;/i&gt; = 46), and analyzed immune signatures in longitudinal tissue samples collected at multiple time points during therapy. In this study, we demonstrate that adaptive immune signatures in tumor biopsy samples obtained early during the course of treatment are highly predictive of response to immune checkpoint blockade and also demonstrate differential effects on the tumor microenvironment induced by CTLA4 and PD-1 blockade. Importantly, potential mechanisms of therapeutic resistance to immune checkpoint blockade were also identified.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Significance:&lt;/b&gt; These studies demonstrate that adaptive immune signatures in early on-treatment tumor biopsies are predictive of response to checkpoint blockade and yield insight into mechanisms of therapeutic resistance. These concepts have far-reaching implications in this age of precision medicine and should be explored in immune checkpoint blockade treatment across cancer types. &lt;i&gt;Cancer Discov; 6(8); 827–37. ©2016 AACR.&lt;/i&gt;&lt;/p&gt;&lt;p&gt;&lt;i&gt;See related commentary by Teng et al., p. 818&lt;/i&gt;.&lt;/p&gt;&lt;p&gt;This article is highlighted in the In This Issue feature, p. 803&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;Immune checkpoint blockade represents a major breakthrough in cancer therapy; however, responses are not universal. Genomic and immune features in pretreatment tumor biopsies have been reported to correlate with response in patients with melanoma and other cancers, but robust biomarkers have not been identified. We studied a cohort of patients with metastatic melanoma initially treated with cytotoxic T-lymphocyte–associated antigen-4 (CTLA4) blockade (&lt;i&gt;n&lt;/i&gt; = 53) followed by programmed death-1 (PD-1) blockade at progression (&lt;i&gt;n&lt;/i&gt; = 46), and analyzed immune signatures in longitudinal tissue samples collected at multiple time points during therapy. In this study, we demonstrate that adaptive immune signatures in tumor biopsy samples obtained early during the course of treatment are highly predictive of response to immune checkpoint blockade and also demonstrate differential effects on the tumor microenvironment induced by CTLA4 and PD-1 blockade. Importantly, potential mechanisms of therapeutic resistance to immune checkpoint blockade were also identified.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Significance:&lt;/b&gt; These studies demonstrate that adaptive immune signatures in early on-treatment tumor biopsies are predictive of response to checkpoint blockade and yield insight into mechanisms of therapeutic resistance. These concepts have far-reaching implications in this age of precision medicine and should be explored in immune checkpoint blockade treatment across cancer types. &lt;i&gt;Cancer Discov; 6(8); 827–37. ©2016 AACR.&lt;/i&gt;&lt;/p&gt;&lt;p&gt;&lt;i&gt;See related commentary by Teng et al., p. 818&lt;/i&gt;.&lt;/p&gt;&lt;p&gt;This article is highlighted in the In This Issue feature, p. 803&lt;/p&gt;&lt;/div&gt;

Several reconstructive methods have been reported to restore the continuity of the aerodigestive tract following resection of pharyngeal and hypopharyngeal cancers. However, high complication rates have been reported after voice prosthesis insertion. In this setting, the ileocolon free flap (ICFF) offers a tubularized flap for reconstruction of the hypopharynx while providing a natural phonation tube. Herein, we systematically reviewed the current evidence on the use of the ICFF for reconstruction of the aerodigestive tract. A systematic literature search was conducted across PubMed MEDLINE, Web of Science, ScienceDirect, Scopus, and Ovid MEDLINE(R). Data on the technical considerations and surgical and functional outcomes were extracted. Twenty-one studies were included. The mean age and follow-up were 54.65 years and 24.72 months, respectively. An isoperistaltic or antiperistaltic standard ICFF, patch flap, or chimeric seromuscular-ICFF can be used depending on the patients' needs. The seromuscular chimeric flap is useful to augment the closure of the distal anastomotic site. The maximum phonation time, frequency, and sound pressure level (dB) were higher with ileal segments of 7 to 15 cm. The incidence of postoperative leakage ranged from 0 to 13.3%, and the majority was occurring at the coloesophageal junction. The revision rate of the microanastomosis ranged from 0 to 16.6%. The ICFF provides a reliable and versatile alternative for reconstruction of middle-size defects of the aerodigestive tract. Its three-dimensional configuration and functional anatomy encourage early speech and deglutition without a prosthetic valve and minimal donor-site morbidity.

Abstract Appreciation of the immune effects of targeted agents has grown, and efforts to combine these agents with immunotherapy are underway to enhance therapeutic responses. Multiple ongoing trials are examining this concept; however, nuances exist with regard to timing, sequence, and combinatorial strategies. Clin Cancer Res; 23(2); 327–9. ©2016 AACR. See related article by Deniger et al., p. 351

Abstract Background: Major advances have been made in the treatment of metastatic melanoma through the use of molecularly targeted therapy and immunotherapy, and trials incorporating these agents are now being extended to patients with earlier-stage disease. The current standard of care (SOC) therapy for high-risk resectable melanoma (stage IIIB/IIIC) is upfront surgery and SOC adjuvant therapy; however, relapse rates are high (~70%). We hypothesized that treatment with neoadjuvant + adjuvant targeted therapy (dabrafenib + trametinib) in this patient population would result in lower relapse rates and prolonged survival over SOC therapy. Methods: To test this hypothesis, we designed a randomized clinical trial (NCT02231775) in patients with resectable Stage IIIB/C or oligometastatic stage IV BRAF-mutant melanoma. Patients were randomized 1:2 to SOC (Arm A) versus neoadjuvant + adjuvant D+T (Arm B, 8 wks neoadjuvant + 44 wks adjuvant). The primary endpoint was relapse-free survival (RFS) with additional secondary endpoints. Importantly longitudinal sampling of tumor tissue was obtained (at baseline, week 3, and surgery) and molecular profiling was performed to gain insights into mechanisms of therapeutic response. Results: 21 of 84 patients were enrolled (arm A=7, arm B=14). Arms were well matched, and toxicity to targeted therapy was limited. RECIST response rate to 8 wks D+T was 77%, with a pathologic complete response rate (pCR) of 58%. Interim analysis revealed a significantly higher RFS in the D+T arm over SOC (p&amp;lt;0.0001), substantiating early stoppage of the trial. Notably, achievement of a pCR correlated with durable clinical benefit. Tumor mutational load was similar in those achieving a pCR versus those who did not, however known recurrent gene alterations in melanoma were noted to be more abundant in those who failed to achieve a pCR. Transcriptomic profiling in longitudinal tumor samples revealed differentially expressed genes (DEGs) that were highly correlated with achieving a pCR. Initial functional analysis of DEGs implicate multiple cancer cell-intrinsic (differentiation, MAPK pathway and metabolic) features and immune microenvironmental factors to be associated with response to neoadjuvant targeted therapy. Conclusion: Neoadjuvant + adjuvant D+T is associated with a high pCR rate and improved RFS over SOC in patients with high-risk resectable metastatic melanoma. Pathological and molecular correlative analysis revealed both pre- and on-treatment tumor features to be highly associated with the high pCR rate. Citation Format: Scott E. Woodman, Peter Prieto, Miles C. Andrews, Rodabe N. Amaria, Michael Tetzlaff, Adi Diab, Sapna P. Patel, Hwu Wen-Jen, Isabella Glitza, Hussein Tawbi, Patrick Hwu, Janice Cormier, Anthony Lucci, Richard Royal, Jeffrey Lee, Roland Bassett, Lauren Simpson, Elizabeth Burton, Li Zhao, Elizabeth Grimm, Alexandre Reuben, Christine Spencer, Junna Oba, Merrick Ross, Jeffrey Gershenwald, Michael Davies, Jennifer A. Wargo. Novel neoadjuvant targeted therapy trial yields insight into molecular mechanisms of response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT156. doi:10.1158/1538-7445.AM2017-CT156

9575 Background: Although both targeted and immune therapies have significantly improved outcomes for mm pts, only a minority of pts experience durable responses with many pts with multiple SMM demonstrating differential responses to therapy. We performed multidimensional spatial characterization of immune markers in SMM from mm patients treated with targeted and immune therapies to improve our understanding of correlations and determinants of response. Methods: NanoString's Digital Spatial Profiling research platform was used on 6 SMM from 3 pts (treatment-naïve; BRAF + MEK targeted therapy treated; anti-PD-1 immunotherapy treated) for 30 immune and signaling proteins. For analysis, we selected and compared immune-rich (CD45+) and tumor-rich (S100B+) regions across SMM. Results were compared to lesion-specific clinical responses. Results: Striking differences in patterns of expression across SMM from individual pts were detected, including in Ki67, CD68 myeloid cells, and the potent immunosuppressor B7-H3. SMM progressing after targeted therapy demonstrated higher pAKT and PD-L1 expression, consistent with described resistance mechanisms. Large differences in expression of PD-L1 were noted following anti-PD-1 therapy, which could contribute to heterogeneous responses. Differential expression patterns in the TME associated with response were also detected, including in increases in CD4 and CD14 cells in progressing lesions. Conclusions: Striking differences in responding and non-responding SMM were observed, providing potential explanations for the heterogeneous clinical responses frequently observed in mm pts. Studies are ongoing to further characterize interactions and spatial distribution of cell types, as well as integrate these findings with previous molecular and immune profiling data (whole exome sequencing, gene expression profiling, flow cytometry, IHC, TCR sequencing) in these and additional SMM to identify actionable strategies to homogenize responses across metastases in mm pts.

53 Background: Cholangiocarcinoma (CCA) is the most common liver malignancy within the biliary tree with increasing incidence and poor survival. Although surgical resection can be curative, prognosis remains abysmal with high rates of unresectability and recurrence, and new systemic therapies are needed. CCA tumors are characterized by an abundant inflammatory immune cell infiltrate, yet little is known about the immune dynamics underlying the disease. Here, we present a preclinical murine model of CCA for identifying potential targets susceptible to immune-based therapies. Methods: Mice with targeted activation of Kras G12D and loss of p53 (Kras-p53 -/- ) in the liver were exposed to 5-Diethoxycarbonyl-1.4-dihydrocollidine for 4 weeks to induce spontaneous CCA tumorigenesis. Immunohistochemistry staining was performed for mouse and human CCA. Immune compartments (bone marrow, spleen, peripheral blood, tumor draining and non-draining lymph nodes, and CCA tumor) of mice were processed for CyTOF analysis. In vitro assays of isolated myeloid leukocytes were performed to demonstrate function. Results: Tumor associated neutrophils (TANs) and tumor associated macrophages (TAMs) were present in high levels in human CCA tumors and the murine model. CyTOF analysis demonstrated mobilization of TANs, TAMs, and CCR2 + inflammatory monocytes into the tumor. The checkpoint marker PD-1 was upregulated on CD8 + and CD4 + T cells and associated with PD-L1 + TANs, TAMs, monocytes, and dendritic cells. The immunosuppressive tumor microenvironment was also characterized by TAMs expressing IL-10, TGFβ, and arginase 1; regulatory B cells expressing IL-10 and TGFβ; and Gata3 + T H 2 cells. Isolated myeloid leukocytes suppressed T cell proliferation and induced invasive migration. Conclusions: Kras-p53 -/- mice developed CCA tumors histologically similar to human disease: the data portray immunosuppressive cells in the tumor microenvironment, a possible obstacle for an effective anti-tumor response. Understanding the immune dynamics of immunosuppressive populations within the tumor microenvironment will be key to mount a potent therapeutic response for durable remission and improved survival.

Toxic epidermal necrolysis (TEN) and Stevens-Johnson syndrome (SJS) are life-threatening, cutaneous reactions often associated with culprit drugs. A growing body of knowledge has deepened our understanding of the pathophysiology and clarified mechanisms such as drug-specific cytotoxicity mediated by T-cells, genetic linkage with HLA and non-HLA genes, TCR restriction, and cytotoxicity mechanisms. Physicians should broadly consider the etiology of SJS/TEN in order to better understand treatment strategies as well as identify which patients may be at risk for developing this condition. Mechanisms for how radiotherapy and rare malignancies may contribute to the development of TEN and SJS have been proposed.

Abstract Background: Resistance to BRAF V600E inhibitors (dabrafenib) and MEK inhibitors (trametinib) occurs in 6-8 months in the majority of melanoma patients with the target V600E mutation. For this reason, combination targeted therapy (TT) offers reliable but often non-durable responses. A translatable model exhibiting de novo resistance to TT with response to checkpoint inhibition (ICB) may offer an avenue for exploring mechanisms of TT resistance, responses in combined TT/ICB, and modes of treatment salvage. Methods: C57/BL6J mice flanks were injected with Yumm 1.7 melanoma. After 14 days, mice were subjected to TT with dabrafenib/trametinib chow. A select few tumors (3/40) exhibited resistance to TT. Tumors from resistant mice were harvested, morcellized, and cultured to isolate a Yumm 1.7 cell line (Y1.7 BrafR) with a resistant phenotype and passaged to ensure uniform clonality. Y1.7 BrafR was assessed for growth characteristics, morphology, and response to dabrafenib/trametinib in vitro. In a synchronous mouse melanoma model, Yumm 1.7 and Y1.7 BrafR were injected into opposing flanks, allowed 14 days growth, and mice were subjected to either TT, ICB (aPD-1/aCTLA-4 combined therapy), TT + ICB, or vehicle for 5 days. Results: Y1.7 BrafR exhibited in vitro resistance to dabrafenib at 0.1, 1, and 10uM after 72 hr exposure (12%, 32%, and 47% growth depletion vs untreated cells, Yumm 1.7 with 67%, 61%, and 84%). This effect was not noted with trametinib except at 10uM (84% vs 92% growth depletion). Both lines succumbed to combination TT of varying doses by 72 hours. Y1.7 Braf R exhibits a doubling time of 18.14 hr vs. 12.35 hr for Yumm 1.7, with no appreciable differences in morphology on 40x light microscopy, as both lines extend dendritic processes while forming dense nests. Despite slower in vitro growth, Y1.7 BrafR exhibits markedly greater growth in vivo after 14 days incubation and 5 days treatment (1.16 v 0.44 cm^3 in vehicle mice), with less response to TT chow (51% tumor volume depletion from vehicle vs. 90% in Yumm1 .7) and TT/ICB (data not shown) and greater response to ICB (15% volume depletion vs 24% volume increase in Yumm 1.7). All data are statistically significant (2-tail t-test, p&amp;lt;0.05). Conclusions: Our results show the development of a TT-resistant mouse melanoma model. This model is clinically relevant for patients who have initially potent, but non-durable responses to TT. Future studies will focus on mechanisms of dabrafenib resistance, tumor nutrition and metabolism, modes of salvage, and efficacy of T-cell recruitment to the tumor microenvironment in this de novo TT-resistant line. Citation Format: Alexander Chacon, Katherine Jackson, Shuyang Qin, Alyssa Williams, Rachel Jewell, Peter Prieto. Development of a translatable targeted therapy-resistant melanoma model [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO029.

<h2>Abstract</h2><h3>Background</h3> Lymph node involvement is a significant prognostic factor for melanoma. Both number of positive nodes and disease burden within a lymph node affects survival. However, the significance of few tumor cells within a single node and subsequent optimal management remains without consensus. We investigated the implications of minimal nodal disease on clinical outcomes. <h3>Methods</h3> We reviewed 752 patients who underwent lymph node sampling at time of primary melanoma resection at our institution over 15 years. We deemed patients who had 1 node with 1 to 4 atypical cells staining positive for either Melan-A or Sox-10 as having "picomets." We examined the initial clinicopathological features, subsequent management, and outcomes. <h3>Results</h3> Thirty-three patients (4%) met criteria for having picomets. The most common number of positively staining atypical cells was 1 (n = 13). Nodal staging at initial pathology review varied, and overall stage ranged from IA to IIIC. Four patients underwent further therapy, none of whom had recurrent disease. Of the 29 patients undergoing observation/surveillance only, 5 had disease recurrence (17%). <h3>Conclusion</h3> Although patients with picomets had better outcomes than historical stage matched cohorts, a small subset had recurrent disease. Staging patients with picomets as "N0" may not reflect the true negative prognostic significance of picomets. A larger population of patients meeting picomets criteria is needed to draw further conclusions.

Background Only 30–40% of metastatic melanoma patients experience objective responses to first line anti-PD-1 immune checkpoint inhibition (αPD-1 ICI). Cyclooxygenase (COX-1/2) inhibition with aspirin (ASA) and other non-steroidal anti-inflammatory drugs has been associated with prolonged time to recurrence and improved responsiveness to ICI in human melanoma, 1 with inhibition of myeloid-induced immunosuppression in the tumor microenvironment (TME) a purported mechanism. 2 Similarly, dietary omega-3 fatty acids metabolized by COX-2 elicit downstream effects on T-cell differentiation akin to ASA administration, abrogating murine melanoma and human breast cancer progression. Mechanisms of ICI resistance remain unclear, and adjunct therapies look to bridge the gap from current response rates to cure. Methods YUMM 1.7 melanoma cells were injected into flanks of C57-BL6/J mice. Mice were fed control diets or supplemented with omega-3 rich fish oil (FO) chow (10% weight/weight, 30%kcal/kcal), ASA in drinking water (ASA, LO – 300, MED – 600, HI - 1000 ug/mL), or the combination of these agents (COMBO, with ASA-MED) starting at day 7 post tumor implantation. Intraperitoneal αPD1 was administered every 3–4 days starting at day 12. Tumors were assessed for growth, harvested at day 32 (day 26 for ASA LO/HI), and characterized with flow cytometry. All significant results (p&lt;0.05) assessed by 2-way ANOVA or t-test as appropriate. Results FO resulted in lesser tumor volume at day 32 in αPD-1 treated mice, while ASA-HI resulted in lesser tumor volume in mice not treated with αPD-1 but did not synergize with αPD-1. ASA-MED and COMBO groups trended towards decreased tumor size (p = 0.07 and 0.07 respectively) by day 32 in αPD-1 treated mice. FO and COMBO increased total CD3+ T-cells and monocytes (CD45+, CD19-, CD11b+, Ly6C+, Ly6G -) in the TME. FO increased PD-L1 + CD4+ T-cells, while COMBO increased total CD8+ T-cells and PD1+ CD8+ T-cells. ASA-HI increased monocytes and the proportion of PD-1+, CD8+ T-cells in the TME. Conclusions Myeloid-induced suppression of T-cell function in tumors may contribute to immune checkpoint inhibition resistance. In the present study, both fish oil and aspirin altered melanoma tumor growth, with only fish oil synergizing with anti-PD-1 at the doses assessed. Both fish oil and aspirin augmented monocyte populations in the tumor microenvironment, with differential effects on T-cell populations. The partially synergistic mechanism between substrate-limited (FO) and pharmacologic (ASA) inhibition of cyclooxygenase-2 may provide a cost-effective avenue to combat immune escape in melanoma patients treated with anti-PD-1 immune checkpoint inhibition, requiring further investigation in humans. References Wang SJ, et al . Effect of cyclo-oxygenase inhibitor use during checkpoint blockade immunotherapy in patients with metastatic melanoma and non-small cell lung cancer. J Immunother Cancer 2020; 8 (2). Zelenay S, et al . Cyclooxygenase-dependent tumor growth through evasion of immunity. Cell 2015; 162 (6):1257–70.

Twenty girls were diagnosed as having Rett's Syndrome (RS) based on criteria decided upon in 1988. Fifteen suffered epileptic fits, four of which were possible to record by EEG. In this work we report on the clinical EEG semiology of these girls. All underwent clinical, biochemical, electroneurophysical, neuroimaging and cytogenetic studies. Periodic EEG were carried out while the girls were awake and the recorded fits were so obtained. All had night-time EEG. Four girls had their first fits between 5 years 4 months and 6 years 5 months (average 6 years). The ages at which their attacks were recorded varied from seven to eleven years. Two girls presented tonic-axial attacks expressed graphically by desynchronisation in the EEG in one case and rhythmically in the other, one having had atypical simple absences expressed on EEG as point-wave complexes at a rate of two per second, the other presenting two types of attack: initially tonic-axial fits expressed as a low amplitude rhythm and a year later generalised clonal fits expressed as slow waves with sharp waves in between. Outstanding is the fact that despite the diversity of epileptic fits described in RS all such attacks recorded were of a generalised type.

Abstract There have been significant advances in the treatment of metastatic melanoma through targeted and immunotherapy, however a significant proportion of patients still progress on these regimens with many experiencing mixed responses. Intense research efforts to better understand resistance are underway, and multiple molecular resistance mechanisms to targeted therapy have been identified. The appreciation of genetic heterogeneity as a contributor to resistance to therapy has grown, though immune heterogeneity has been poorly characterized. The goal of the present study is to better understand the molecular and immune heterogeneity in synchronous melanoma metastases at the time of disease progression. In this study, we prospectively evaluated 32 tumors from 15 patients who were treatment-naïve (n = 4), or had received prior targeted (n = 4) or immunotherapy (n = 7). Whole exome sequencing demonstrated between 4-41% of non-synonymous exonic mutations (NSEM) were restricted to individual tumors within a patient. Deep profiling of infiltrating immune cell subsets by flow cytometry and immunohistochemistry analyses confirmed the immune infiltrate between synchronous metastases to be highly heterogeneous, specifically in regards to CD4 and CD8 T lymphocytes. In aggregate, 92% of these T cell clones were unique to distinct tumors within the same patient, with limited overlap with clones detected in the blood. NetMHC3.4 neoantigen prediction demonstrated a large fraction of predicted neoantigens were restricted to individual tumors, with over 10% of these presenting extremely high predicted affinity. Importantly, analysis of RECIST measurements of individual lesions within the same patient suggested this molecular and immune heterogeneity could contribute to differential tumor growth and response to therapy within the same patient. This has important clinical implications, and suggests a single tumor biopsy may not be sufficiently representative of the molecular and immune landscape of multiple tumors within the same individual. Citation Format: Alexandre Reuben, Christine N. Spencer, Peter A. Prieto, John P. Miller, Xizeng Mao, Wei-Shen Chen, Hannah Cheung, Hong Jiang, Cara Haymaker, Mariana Petaccia De Macedo, Haven R. Garber, Pei-Ling Chen, Vancheswaran Gopalakrishnan, Jacob Austin-Breneman, Courtney W. Hudgens, Jason Roszik, Patrick Hwu, Scott E. Woodman, Lynda Chin, Michael A. Davies, Rodabe N. Amaria, Sapna P. Patel, Alexander J. Lazar, Michael T. Tetzlaff, Karen C. Dwyer, Ignacio I. Wistuba, Padmanee Sharma, James P. Allison, Jianhua Zhang, Andrew Futreal, Zachary A. Cooper, Jennifer A. Wargo. Genomic and immune heterogeneity in synchronous melanoma metastases is associated with differential tumor growth and response to therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2392.

Proficiency with common endocrine operations is expected of graduating, general surgery residents. However, no expert consensus guidelines exist about these expectations.Members of the American Association of Endocrine Surgeons were surveyed about their opinions on resident proficiency with common endocrine operations.Overall response rate was 38%. A total of 92% of the respondents operate with residents. On average, they believed that the steps of a total thyroidectomy for benign disease and a well-localized parathyroidectomy could be performed by a postgraduate year 4 surgery resident. Specific steps that they thought might require more training included decisions to divide the strap muscles or leaving a drain. Approximately 66% of respondents thought that a postgraduate year 5 surgery resident could independently perform a total thyroidectomy for benign disease, but only 45% felt similarly for malignant thyroid disease; 79% thought that a postgraduate year 5 surgery resident could independently perform a parathyroidectomy. Respondents’ years of experience correlated with their opinions about resident autonomy for total thyroidectomy (benign r = 0.38, P < .001; malignant r = 0.29, P = .001) but not parathyroidectomy. On multivariate analysis, sex and years of experience of the respondents were independently associated with opinions on autonomy but only for total thyroidectomy for benign disease (P = .001). Annual endocrine volume of the respondents did not correlate with beliefs in autonomy.There was general agreement among responding members of the AAES about resident proficiency and autonomy with common endocrine operations. As postgraduate year 5 residents may not be proficient in advanced endocrine operations, opportunities exist to improve training prior to the transition to independent practice for graduates that anticipate performing endocrine operations routinely.

Abstract Introduction: First line anti-PD-1 immune checkpoint inhibition (αPD-1 ICI) induces objective responses in less than 50% of metastatic melanoma patients. Aspirin (ASA) use is associated with improved responsiveness to ICI, with inhibition of myeloid-induced immunosuppression in the tumor microenvironment (TME) a purported mechanism. Cyclooxygenase modulators ASA (pharmacologic inhibitor) and dietary fish oil (FO, substrate) shift myeloid and T cell populations to anti-inflammatory, pro-resolution phenotypes in immunity and cancer. Methods: C57-BL6/J mice were injected with YUMM 1.7 melanoma in the flank. Mice were fed control chow and water (CTL) or omega-3 rich (FO) chow (10% w/w, 30%kcal/kcal), given ASA in drinking water (600 ug/mL), or FO/ASA combined (COMBO) starting 12 days post tumor implantation, the same day as starting intraperitoneal αPD1 or IgG2a vehicle (q3-4 days). An alternative cohort received pre-αPD-1 treatment ASA and FO starting day 7. Tumors were assessed for growth, harvested, and characterized via flow cytometry on day 32. Results: When diets and water were started concurrent with αPD-1/IgG2a, FO decreased tumor volume vs. CTL for both vehicle (28%) and αPD-1 (27%) treatments; ASA and COMBO (p = 0.0501) did not. Compared to CTL, FO and COMBO increased monocytes (CD45+, CD11b+, Ly6C+, Ly6G-) and PD-L1+ monocytes in the TME with no effect on tumor associated macrophages (TAMS); ASA decreased the percent of Arginase 1+, CD206+ TAMs. ASA increased PD1+ CD8 T cells, while COMBO increased CD4 and CD8 T cells including LAG3+ and PD1+ CD8 T cells. When diets and water were started on day 7, FO + αPD-1 decreased tumor volume (21%) vs. αPD-1 controls; ASA and COMBO did not. Compared to CTL, FO and COMBO similarly altered immune populations as above, with FO additionally increasing total CD3+ T-cells, PD-L1+ CD4 T cells, and PD-1+ CD8 T cells; COMBO increased dendritic cells (CD11c+, MHCII+, F4/80-). TAMS were not assessed. Results significant (p&amp;lt; 0.05) by ANOVA or t-test unless specified. Food eaten, water imbibed, and weight did not vary between groups. Conclusions: Dietary fish oil impaired in vivo melanoma tumor growth with and without αPD-1 ICI despite increases in classically deleterious monocyte populations, while aspirin decreased M2-like macrophages in the TME without alterations in tumor growth. Modulation of the cyclooxygenase axis may be a cost-effective method to skew TME immune populations to favor αPD-1 immunotherapy responses in metastatic melanoma. Citation Format: Alexander C. Chacon, Shuyang S. Qin, Alexa D. Melucci, Katherine M. Jackson, Paul R. Burchard, Yatee A. Dave, Rachel Jewell, Brian A. Belt, William Tabayoyong, David C. Linehan, Peter A. Prieto. Dietary fish oil and aspirin to augment anti-PD-1 immunotherapy in metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 254.

Abstract PD-1 (programmed cell death protein-1) blockade, an immunotherapy that re-stimulates antitumor CD8 T cell response, has revolutionized the treatment of metastatic melanoma. However, the majority of melanoma patients eventually acquire therapy resistance. The emergence of resistant metastases likely results from de novo somatic mutations secondary to inherent tumor genetic instability. We hypothesize that intertumoral genetic differences introduce immunosuppressive mechanisms, such as inducing myeloid-derived suppressor cell differentiation, that dampen existing antitumor immune responses. As all metastases share one host immune system, we investigated the local and systemic effects of intertumoral heterogeneity on antitumor immunity and PD-1 immunotherapy response in mice with multiple, synchronous melanoma metastases. To recapitulate intertumoral heterogeneity, we simultaneously injected PD-1 blockade-resistant YUMM 1.7 and its UVB-irradiated, PD-1 sensitive, derivative YUMMER 1.7 melanoma cell lines into opposite flanks of the same C57BL/6J mouse. We previously demonstrated that tumor-specific de novo somatic mutations, in combination with the host immune response, generate distinct microenvironments in synchronous YUMMER (immunologically active) and YUMM (immunologically inactive) tumors. Our novel murine synchronous melanoma model demonstrated that the presence of immunologically inactive YUMM tumors changes systemic antitumor immunity, including the induction of host splenic myelopoiesis. This phenomenon is accompanied by an increase in YUMMER intratumoral M-CSF (macrophage colony stimulating factor) concentration and an influx of immunosuppressive PD-L1+ (PD-1 ligand) macrophages into YUMMER tumors as assessed by flow cytometry and Luminex analyses. Furthermore, compared to control CD8 T cells, synchronous YUMMER-infiltrating CD8 T cells exhibit reduced functionality, with increased surface PD-1 persistence and failure to respond to PD-1 blockade. As a result, the presence of YUMM tumors are able to confer PD-1 blockade resistance to the normally PD-1 sensitive YUMMER tumors. Our results suggest that intertumoral heterogeneity leads to a “reverse abscopal effect,” where immunologically “cold” tumors can alter systemic myeloid immune responses to suppress antitumor CD8 T cell function in immunologically “hot” tumors. As such, the addition of myeloid-altering agents to PD-1 immunotherapy are likely to improve treatment efficacy and prevent acquired therapy resistance in patients with metastatic melanoma. Citation Format: Shuyang S. Qin, Booyeon J. Han, Alexander C. Chacon, Alexa D. Melucci, Alyssa R. Williams, Rachel Jewell, Minsoo Kim, David C. Linehan, Scott A. Gerber, Peter A. Prieto. "Reverse abscopal effect": Immunologically cold tumors alter systemic immunity and confer PD-1 resistance to immunologically hot tumors in synchronous melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1605.

e21564 Background: Less than half of metastatic melanoma patients experience objective responses to first line anti-PD-1 immune checkpoint inhibition (αPD-1 ICI). Omega-3 fatty acids in dietary fish oil (FO) shift myeloid and T-cell populations to anti-inflammatory, pro-resolution phenotypes in immunity and cancer. We investigated whether dietary FO supplementation could augment tumor microenvironment (TME) immune profiles and improve ICI response in melanoma. Methods: C57-BL6/J mice were injected with YUMM 1.7 melanoma in the flank. Mice were fed control chow (CTL) or omega-3 rich (FO) chow (10% w/w, 30%kcal/kcal) starting 7 days post tumor implantation. Intraperitoneal αPD1 or IgG2a vehicle were injected q3-4 days starting day 12. To assess the temporal flux of TME immune populations, tumors from mice in analogous experiments were assessed for growth, harvested on day 12, day 22, or day 32, and characterized via flow cytometry. Results: In multiple models, FO decreased YUMM tumor growth vs. CTL by day 32 (&gt; 21% for all models). FO halved tumor associated macrophage content, including M2 macrophages (Arg1+, CD206+, CD68+, F480+), at day 12. Conversely, FO increased M2 macrophages at day 22, leveling even with CTL at day 32. At all three timepoints, FO increased TME monocytes (Ly6C+, Ly6G-). In the T cell compartment, FO decreased TME CD4 and CD8 T cell content (including PD1+ CD4 and PD1+ CD8) at day 12, an effect which was reversed by day 22 (increased CD4 and CD8s vs CTL). FO increased PD-L1+ CD4 T cells and CD8+ T cells, including PD1+ CD8 T cells, on day 32. When fish oil diets were started on day 12 (concurrent with immunotherapy), FO decreased tumor volume, increased monocytes (and PD-L1+ monocytes), and increased CD4 and PD1+ CD8 T cells by day 32, all less significantly than when starting diets 5 days prior to initiation of immunotherapy. All results significant (p &lt; 0.05) by 2-way ANOVA or t-test unless specified. Food eaten, water imbibed, and weight change did not vary between groups. Conclusions: Fish oil impairs in vivo murine melanoma tumor growth with and without αPD-1 ICI. Fish oil modulated myeloid and T cell lineages via delaying M2 macrophage and CD4 / CD8 T-cell appearance in the tumor microenvironment, with concomitant increases in monocytes and key T cell effectors involved in αPD-1 ICI responsiveness by the time decrements in tumor growth were noted. Dietary fish oil may benefit patients with advanced melanoma treated with αPD-1 immunotherapy; further study in humans is warranted.

INTRODUCTION: Anti–PD-1 immune checkpoint inhibition (αPD-1 ICI) treatment responses remain suboptimal in metastatic melanoma. We hypothesized that omega-3 fatty acids in dietary fish oil (FO) could ablate myeloid-induced T-cell suppression in the tumor microenvironment (TME) and improve ICI response in melanoma. METHODS: C57-BL/6J mice were injected with YUMM 1.7 melanoma in the flank. Mice were fed control chow (CTL) or omega-3 rich (FO) chow (30%kcal/kcal) starting day (D) 7. Intraperitoneal αPD1 or IgG2a vehicle were injected every 3 to 4 days starting D12. To assess the temporal flux of TME immune populations, tumors from mice in analogous experiments were assessed for growth and harvested on D12, D22, or D32. All listed results significant (p < 0.05) by 2-way ANOVA or t-test. RESULTS: In multiple models of solo and heterogeneous melanoma (metastatic model with a second melanoma cell line injected synchronously), FO decreased YUMM tumor growth vs CTL by D32 (>21% for all models). On D12, FO halved tumor-associated and M2 macrophage content, decreasing TME CD4 (CTLA-4+) and CD8 T cells. Conversely, by D22 FO increased M2 macrophages, CD4, and CD8 T cells, with PD-L1+ CD4s and PD1+ CD8s remaining increased at D32. At all three timepoints, FO increased TME monocytes and did not change NK cells. CONCLUSION: Fish oil impairs murine melanoma growth with and without αPD-1 ICI, delaying initial macrophage and CD4/ CD8 T-cell appearance in the TME while promoting cytotoxic T-cell persistence. Dietary FO may represent a cost-effective adjunct to αPD-1 immunotherapy in melanoma; further study in humans is warranted.

Comment on: Reconstruction of Pharyngolaryngeal Defects with the Ileocolon Free Flap: A Comprehensive Review and How to Optimize OutcomesArch Plast Surg 2022; 49(03): 378-396DOI: 10.1055/s-0042-1748652

We have seen major advances in cancer treatment through the use of immunotherapy and molecularly targeted therapy, and treatment regimens combining these strategies are now being tested in clinical trials across a range of cancer types. The initial rationale for this was empiric—hoping to complement the high response rates generally observed with molecularly targeted therapy with more durable responses seen with immunotherapy. However, there is now strong scientific evidence to support these combinations, as molecularly targeted therapy may induce favorable changes in the tumor immune microenvironment, thereby facilitating enhanced responses to immunotherapy. Nonetheless, nuances exist in such approaches and will be discussed herein. Ultimately, optimal combination strategies with targeted therapy and immunotherapy rely on a deep understanding of the mechanisms of response and resistance to these treatment modalities in isolation as well as in combination.

Angiosarcomas are a rare subtype of soft-tissue sarcomas originating from the vascular endothelium. Both retroperitoneal and omental angiosarcomas tend to be aggressive and rapidly fatal if not amenable to early intervention. In this report, we describe an unusual case of high-grade angiosarcoma with cytology-negative hemorrhagic ascites and diffuse omental invasion. Multiple investigations into the origin of the hemorrhagic ascites, including cytological analysis, tumor marker measurements, serum-ascites albumin gradient calculation and frozen section pathological examination, failed to reveal a diagnosis. We conclude that malignancy should be considered in the differential diagnosis in the presence of suspicious cytology-negative hemorrhagic ascites and concomitant retroperitoneal and abdominal findings.

Abstract Background Paraneoplastic syndromes are rare manifestations of underlying malignancies, most commonly small cell lung cancer and invasive ductal carcinoma of the breast. Case We present a case of anti‐AMPA receptor paraneoplastic limbic encephalitis, a specific paraneoplastic syndrome, in a woman with neuroendocrine breast cancer. Her condition improved with surgical resection and chemotherapy. Conclusion This is the first known case of anti‐AMPA receptor paraneoplastic limbic encephalitis in a patient with primary neuroendocrine breast cancer.

Background: Cholangiocarcinoma (CCA) is the second most common primary liver malignancy, with recent increasing incidence. While surgical resection offers the hope for cure, prognosis remains poor in part due to high rates of unresectability, recurrence, and poor response to conventional chemotherapy. Thus, new systemic therapies are needed. Unfortunately, few preclinical models exist for testing new targeted or immune based therapies. In humans, CCA is characterized by a dense fibro-inflammatory stroma, believed central in inhibiting effective adaptive immunity to the malignancy. Here we present our findings from a murine model of CCA, and characterize the immune tumor microenvironment (TME) for immunomodulatory interventions. Methods: Mice with targeted hepatic KRAS activation and loss of P53 (KP) were induced into developing CCA through exposure for 4 weeks of 5-Diethoxycarbonyl-1.4-dihydrocollidine (DDC) chow. Disease was tracked with high frequency ultrasonography (US). Histology and immunohistochemistry (IHC) staining was performed of mouse and human CCA for stromal and immunophenotypic markers. Immune compartments (bone marrow, spleen, peripheral blood, and single cell digest suspension of CCA tumor and normal liver) were processed for flow cytometry analysis. Genomic analysis of tumor and normal liver from littermate controls was performed through RNA sequencing, and qRT-PCR. In-vitro assays of isolated myeloid leukocytes were performed to demonstrate function and tropism. Results: IHC of human tumors showed dense infiltration with tumor associated neutrophils (TANs) and tumor associated macrophages (TAMs). This myeloid infiltrate similarly present in the KP mice tumors. Flow cytometric analysis further demonstrated increased mobilization of TAMs and TAMs in mouse tumor, bone marrow, and spleen compared to littermate controls (Figure). This was consistent with increased expression of chemokines implicated in myelopoisis and mobilization of myeloid leukocytes. Interestingly, both human and murine tumors demonstrated T cell infiltration. However, this was accompanied by elevated checkpoint markers on T cells and cytokines associated with immunosuppression, notably Pd-1l, Pd-1, Ctla4, Tgf-β, and Il10. Conclusion: Taken together, these data portray the CCA TME as an immunosuppressive environment, preventing effective anti-tumor response. Though the presence of effector T cells closely infiltrated within the tumor offers the hope for possible response to immunotherapy through checkpoint inhibition, understanding the interplay between TAN and TAM populations and effective adaptive response remains an area of active investigation. Intervening on the myeloid infiltrate through chemokine inhibition or repolarization strategies, may facilitate additional antitumoral behavior by effector leukocytes.

Background: Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) are the number one and two primary hepatic malignancies (PHM), and are estimated to account for over 30,000 this year. Unfortunately, prognosis is poor for these tumors, in part due to resistance to conventional chemotherapy. Part of this resistance is conferred by the tumor stroma, believed central in establishing a hypoperfused milieu, which subsequently reduces small molecule diffusion and chemotherapeutic efficacy. For both PHM, the tumor stroma has been correlated with survival. We present our experience with a murine model of PHM, as well as ultrasonographic techniques for interrogating tumor perfusion in vivo. Methods: A murine model of HCC and CAA with targeted KrasG12D and loss of p53 in the liver (Kras-p53) previously described was used to model the human disease. High resolution ultrasonography (US) facilitated disease monitoring. Non-targeted microbubble US contrast coupled with VEVO CQ analysis was used to quantify tumor perfusion indexes compared to normal adjacent parenchyma. Normal adjacent parenchyma was validated with littermate controls. Post mortem immunohistochemistry (IHC) and histology was performed to quantify tumor collagen and hyaluronic acid (HA) deposition. Extracellular matrix metabolism were assessed by RNA-seq and qRT-PCR, comparing collagen, HA, fibroblast activation markers, and matrix metalloproteinases. Statistical analysis were performed with with paired Wilcoxon test, Chi-square, and Mann-Whitney U. Results: Disease detection through US occured with mean tumor size 2.1 mm. Overtime, volumetric analysis demonstrated accelerated growth of tumors with progressive intratumoral hypoperfusion as measured through contrasted evaluation. Larger tumors (greater than 10 mm), often developed cystic degeneration. Intratumoral perfusion was statistically significantly reduced compared to normal adjacent parenchyma (P < 0.05) (figure). Histology showed a dense collagen and HA infiltrate (figure). IHC showed a paucity of CD31+ staining, a marker for endothelial cells, in tumors compared to normal tissue, recapitulating the human disease. A board certified pathologist confirmed the presence of predominantly HCC, CCA, or mixed type tumors. Genomic analysis revealed significant increases in HA, collagen metabolism, and markers of fibroblast activating gene expression (P < 0.001). Conclusion: In this model of HCC and CCA, autochthonous tumors demonstrate a progressive desmoplastic remodeling of the tumor stroma, with commensurate development of tissue hypoperfusion. Going forward, this model will facilitate the evaluation of stromal targeted agents, such as hyaluronidases, with the purpose of rendering primary hepatic malignancies vulnerable to small molecule systemic therapy.

Abstract Background: Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related mortality, with dismal 5-year prognosis of 8%, in part due to poor response to available therapies. Thus, new systemic cancer control therapies are in dire need. Semaphorin 4D (SEMA4D) is a soluble and membrane bound glycoprotein that binds its cognate Plexin B1/B2 receptors, expressed on monocytic and granulocytic leukocytes. Prior work has shown that increased expression of SEMA4D and Plexin B in resected PDAC patients is associated with lymph node and distant metastasis, as well as a worse prognosis. Additionally, SEMA4D blockade has conferred improved immune checkpoint blockade response in murine models of colorectal carcinoma and head and neck squamous cell carcinoma via abrogation of myeloid-derived suppressor cell recruitment and function, as well as enhanced T-cell recruitment and activity within the tumor microenvironment. Here we study the effects of SEMA4D blockade in a murine model of PDAC. Methods: C57b/6 mice were orthotopically injected with murine PDAC line (KP2) derived from KRASG12D,TP53Flox/Wt;P48-Cre autochthonous tumors. Mice were treated with Folfirinox (5-FU, irinotecan, oxaliplatin, weekly), immune checkpoint blockade (ICB) (anti-PD1, anti-CTLA-4 mAbs bi-weekly), and anti-SEMA4D mAB (biweekly). Peripheral blood and tumor-infiltrating leukocytes from patients with PDAC undergoing pancreaticoduodenectomy were assessed for Plexin B1 via flow cytometry. Results: Human PDAC demonstrates penetration of Plexin B1 positive leukocytes, most notably tumor-associated macrophages, neutrophils, and monocytes. Mice injected with KP2 developed tumors detectable via high-frequency ultrasound and exhibited longer survival when treated with the combination of Folfirinox, ICB, and anti-SEMA4D antibody, compared to Folfirinox alone, Folfirinox plus ICB, or Folfirinox plus anti-SEMA4D antibody. Conclusions: Plexin B1+ myeloid subsets penetrate human PDAC tumors, and treatment with SEMA4D-blocking antibody improved response to ICB in combination with standard-of-care Folfirinox in preclinical murine studies. Future work will focus on understanding the immune mechanism of improved therapeutic response, as well as further characterization of the prevalence and prognostic ramifications of the SEMA4D-Plexin B axis in PDAC. Citation Format: Luis I. Ruffolo, Katherine M. Jackson, Nicholas Ullman, Alexander Chacon, Alexa Melucci, Paul Burchard, Mary Georger, Rachel Jewell, Peter Prieto, Brian Belt, Crystal Mallow, Elizabeth Evans, Terrence Fisher, Maurice Zauderer, Christina Wu, Brian Olson, Gregory B. Lesinski, David C. Linehan. Targeting the semaphorin 4D-plexin B axis to augment FOLFIRINOX in a murine model of pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B48.

Abstract Synchronous metastatic melanoma, clinically defined as multiple lesions diagnosed within 6 months, has a poor prognosis. Despite recent advances in systemic immunotherapy, a majority of patients fail to respond or exhibit lesion-specific responses. While intertumoral heterogeneity has been clinically associated with lesion-specific therapeutic responses, no clear mechanism has been identified, largely due to the scarcity of preclinical models. We developed a novel murine synchronous melanoma model that recapitulates clinical intertumoral heterogeneity. We show that genetic differences between tumors generate distinct tumor immune microenvironments (TIME). These TIMEs can independently upregulate PD-1/PD-L1 expression in response to ongoing anti-tumor immunity and the presence of interferon-gamma. The simultaneous presence of multiple tumors can additionally alter the TIME of each tumor. As such, our model provides a unique approach to investigate the effects of intertumoral heterogeneity on mechanisms of immunotherapy resistance.

dissected at the end of liver parenchymal transection and sectioned with endovascular stapler.Operative time was 370 minutes.No transfusion was needed.There were no postoperative complications, being discharged on day 3. Final pathologic report confirmed the diagnosis of complicated HCAs.Conclusion: Pure laparoscopic liver resection can be currently proposed as surgical approach for complicated HCA in experienced centers.

Loria, Anthony MD; Zambrano, Dennis BA; Buda, Alexandra BS; Juviler, Peter MD; Prieto, Peter A. MD, MPH Author Information

Abstract Treatment with molecularly-targeted therapy has revolutionized cancer care, including BRAF/MEK-targeted melanoma therapy. However responses are heterogenous and frequently not long-lasting. Novel strategies to target resistance are needed. We studied a cohort of patients with resectable metastatic melanoma treated with neoadjuvant BRAF/MEK-targeted therapy (n=52) and noted a strong sexual dimorphism in response to treatment, with female patients demonstrating significantly higher rates of a major pathologic response (MPR) (p=0.0001). RNA sequencing of tumors demonstrated enrichment of androgen-related genes in those failing to achieve MPR. Pre-clinical studies validated these findings, with significantly increased tumor growth in male vs female mice treated with BRAF/MEK inhibitors (BRAF/MEKi) (p=0.0005). Androgen receptor (AR) expression was upregulated in tumors of BRAF/MEKi-treated mice, and modulation of AR signaling via AR-blockade or castration was associated with significantly slower tumor growth (p=0.0001 and p=0.00004, respectively). Together, these results have important implications in the context of treatment with BRAF/MEKi-targeted therapy.