Jaime L. Schneider

Malfunction of autophagy, the process that mediates breakdown and recycling of intracellular components in lysosomes, has been linked to a variety of human diseases. As the number of pathologies associated with defective autophagy increases, emphasis has switched from the mere description of the status of autophagy in these conditions to a more mechanistic dissection of the autophagic changes. Understanding the reasons behind the autophagic defect, the immediate consequences of the autophagic compromise and how autophagy changes with the evolution of the disease has become a 'must,' especially now that manipulation of autophagy is being considered as a therapeutic strategy. Here, we comment on some of the common themes that have emerged from such detailed analyses of the interplay between autophagy and disease conditions.

The activity of chaperone-mediated autophagy (CMA), a catabolic pathway for selective degradation of cytosolic proteins in lysosomes, decreases with age, but the consequences of this functional decline in vivo remain unknown. In this work, we have generated a conditional knockout mouse to selectively block CMA in liver. We have found that blockage of CMA causes hepatic glycogen depletion and hepatosteatosis. The liver phenotype is accompanied by reduced peripheral adiposity, increased energy expenditure, and altered glucose homeostasis. Comparative lysosomal proteomics revealed that key enzymes in carbohydrate and lipid metabolism are normally degraded by CMA and that impairment of their regulated degradation contributes to the metabolic abnormalities observed in CMA-defective animals. These findings highlight the involvement of CMA in regulating hepatic metabolism and suggest that the age-related decline in CMA may have a negative impact on the energetic balance in old organisms.

The population is aging at a rate never seen before in human history. As the number of elderly adults grows, it is imperative we expand our understanding of the underpinnings of aging biology. Human lungs are composed of a unique panoply of cell types that face ongoing chemical, mechanical, biological, immunological, and xenobiotic stress over a lifetime. Yet, we do not fully appreciate the mechanistic drivers of lung aging and why age increases the risk of parenchymal lung disease, fatal respiratory infection, and primary lung cancer. Here, we review the molecular and cellular aspects of lung aging, local stress response pathways, and how the aging process predisposes to the pathogenesis of pulmonary disease. We place these insights into context of the COVID-19 pandemic and discuss how innate and adaptive immunity within the lung is altered with age.

In this Review—intended as an introduction to the topic of hepatic autophagy for clinical scientists—the authors describe the different types of hepatic autophagy, their role in maintaining homeostasis in a healthy liver and the contribution of autophagic malfunction to liver disease. Studies performed in the liver in the 1960s led to the identification of lysosomes and the discovery of autophagy, the process by which intracellular proteins and organelles are degraded in lysosomes. Early studies in hepatocytes also uncovered how nutritional status regulates autophagy and how various circulating hormones modulate the activity of this catabolic process in the liver. The intensive characterization of hepatic autophagy over the years has revealed that lysosome-mediated degradation is important not only for maintaining liver homeostasis in normal physiological conditions, but also for an adequate response of this organ to stressors such as proteotoxicity, metabolic dysregulation, infection and carcinogenesis. Autophagic malfunction has also been implicated in the pathogenesis of common liver diseases, suggesting that chemical manipulation of this process might hold potential therapeutic value. In this Review—intended as an introduction to the topic of hepatic autophagy for clinical scientists—we describe the different types of hepatic autophagy, their role in maintaining homeostasis in a healthy liver and the contribution of autophagic malfunction to liver disease.

Chaperone-mediated autophagy (CMA), a cellular process that contributes to protein quality control through targeting of a subset of cytosolic proteins to lysosomes for degradation, undergoes a functional decline with age. We have used a mouse model with liver-specific defective CMA to identify changes in proteostasis attributable to reduced CMA activity in this organ with age. We have found that other proteolytic systems compensate for CMA loss in young mice which helps to preserve proteostasis. However, these compensatory responses are not sufficient for protection against proteotoxicity induced by stress (oxidative stress, lipid challenges) or associated with aging. Livers from old mice with CMA blockage exhibit altered protein homeostasis, enhanced susceptibility to oxidative stress and hepatic dysfunction manifested by a diminished ability to metabolize drugs, and a worsening of the metabolic dysregulation identified in young mice. Our study reveals that while the regulatory function of CMA cannot be compensated for in young organisms, its contribution to protein homeostasis can be handled by other proteolytic systems. However, the decline in the compensatory ability identified with age explains the more severe consequences of CMA impairment in older organisms and the contribution of CMA malfunction to the gradual decline in proteostasis and stress resistance observed during aging.

Abstract Purpose: ctDNA offers a promising, noninvasive approach to monitor therapeutic efficacy in real-time. We explored whether the quantitative percent change in ctDNA early after therapy initiation can predict treatment response and progression-free survival (PFS) in patients with metastatic gastrointestinal cancer. Experimental Design: A total of 138 patients with metastatic gastrointestinal cancers and tumor profiling by next-generation sequencing had serial blood draws pretreatment and at scheduled intervals during therapy. ctDNA was assessed using individualized droplet digital PCR measuring the mutant allele fraction in plasma of mutations identified in tumor biopsies. ctDNA changes were correlated with tumor markers and radiographic response. Results: A total of 138 patients enrolled. A total of 101 patients were evaluable for ctDNA and 68 for tumor markers at 4 weeks. Percent change of ctDNA by 4 weeks predicted partial response (PR, P < 0.0001) and clinical benefit [CB: PR and stable disease (SD), P < 0.0001]. ctDNA decreased by 98% (median) and >30% for all PR patients. ctDNA change at 8 weeks, but not 2 weeks, also predicted CB (P < 0.0001). Four-week change in tumor markers also predicted response (P = 0.0026) and CB (P = 0.022). However, at a clinically relevant specificity threshold of 90%, 4-week ctDNA change more effectively predicted CB versus tumor markers, with a sensitivity of 60% versus 24%, respectively (P = 0.0109). Patients whose 4-week ctDNA decreased beyond this threshold (≥30% decrease) had a median PFS of 175 days versus 59.5 days (HR, 3.29; 95% CI, 1.55–7.00; P < 0.0001). Conclusions: Serial ctDNA monitoring may provide early indication of response to systemic therapy in patients with metastatic gastrointestinal cancer prior to radiographic assessments and may outperform standard tumor markers, warranting further evaluation.

Keratin intermediate filaments (KIFs) form a fibrous polymer network that helps epithelial cells withstand external mechanical forces. Recently, we established a correlation between the structure of the KIF network and its local mechanical properties in alveolar epithelial cells. Shear stress applied across the cell surface resulted in the structural remodeling of KIF and a substantial increase in the elastic modulus of the network. This study examines the mechanosignaling that regulates the structural remodeling of the KIF network. We report that the shear stress-mediated remodeling of the KIF network is facilitated by a twofold increase in the dynamic exchange rate of KIF subunits, which is regulated in a PKC zeta and 14-3-3-dependent manner. PKC zeta phosphorylates K18pSer33, and this is required for the structural reorganization because the KIF network in A549 cells transfected with a dominant negative PKC zeta, or expressing the K18Ser33Ala mutation, is unchanged. Blocking the shear stress-mediated reorganization results in reduced cellular viability and increased apoptotic levels. These data suggest that shear stress mediates the phosphorylation of K18pSer33, which is required for the reorganization of the KIF network, resulting in changes in mechanical properties of the cell that help maintain the integrity of alveolar epithelial cells.

Sumoylation is a type of post-translational modification that is implicated in the regulation of numerous cellular events. However, its role in the function of human sperm has not yet been characterized.In this study, both immunofluorescence and electron microscopy revealed that small ubiquitin-like modifiers (SUMO) SUMO1 and SUMO2/3 were highly enriched in the neck area of human sperm that is associated with the redundant nuclear envelope and were also detectable in the flagella and some head regions. Similar localization patterns of SUMO were also observed in mouse and fly sperm. Nonmotile, two-tailed, curled tailed, misshapen, microcephalic (small head) and aciphalic (no head) sperm exhibited abnormally high levels of sumoylation in their neck and tail regions relative to normal sperm. Numerous sumoylated proteins, ranging from 20 to 260 kDa, were detected via western blotting and identified by mass spectrometry, and 55 SUMO targets that were present specifically in human sperm, and not in the control fraction, corresponded to flagella proteins, proteins involved in the maturation and differentiation of sperm, heat shock proteins and important glycolytic and mitochondrial enzymes. The targets that were identified included proteins with specific functions in germ cells and sperm, such as heat shock-related 70-kDa protein 2, outer dense fiber protein 3, A-kinase anchor proteins 3 and 4, L-lactate dehydrogenase C, sperm protein associated with the nucleus on the X chromosome B/F, valosin-containing protein, seminogelins, histone H4 and ubiquitin. Coimmunoprecipitation experiments confirmed the sumoylation of semenogelin and indicated that some sperm proteins are modified by sumoylation and ubiquitination simultaneously.Numerous proteins are modified by sumoylation in human sperm; excessive sumoylation is a marker of defective spermatozoa.

Autophagy, the process of degrading intracellular components in lysosomes, plays an important role in the central nervous system by contributing to neuronal homeostasis. Autophagic failure has been linked to neurologic dysfunction and a variety of neurodegenerative diseases. Recent investigation has revealed a novel role for autophagy in the context of mental illness, namely in schizophrenia. This article summarizes the phenomenology, genetics, and structural/histopathological brain abnormalities associated with schizophrenia. We review studies that demonstrate for the first time a connection between autophagy malfunction and schizophrenia. Transcriptional profiling in schizophrenia patients uncovered a dysregulation of autophagy-related genes spatially confined to a specific area of the cortex, Brodmann Area 22, which has been previously implicated in the positive symptoms of schizophrenia. We also discuss the role of autophagy activators in schizophrenia and whether they may be useful adjuvants to the traditional antipsychotic medications currently used as the standard of care. In summary, the field has progressed beyond the basic concept that autophagy impairment predisposes to neurodegeneration, to a mechanistic understanding that loss of autophagy can disrupt neuronal cell biology and predispose to mood disorders, psychotic symptoms, and behavioral change.

<h2>Abstract</h2><h3>Introduction</h3> <i>ROS1</i> fusions are oncogenic drivers in 1% to 3% of NSCLCs. The activity of immune checkpoint inhibitor (ICI) monotherapy or in combination with chemotherapy (chemotherapy with ICI [chemo-ICI]) in these tumors and their immunophenotype have not been systematically described. <h3>Methods</h3> In this multi-institutional retrospective study, tumor programmed death-ligand 1 (PD-L1) expression and tumor mutational burden (TMB) were evaluated in patients with <i>ROS1-</i>rearranged NSCLC. Time-to-treatment discontinuation (TTD) and objective response rate (ORR) (Response Evaluation Criteria in Solid Tumors [RECIST] version 1.1) were calculated for patients treated with ICI or chemo-ICI in the metastatic setting. <h3>Results</h3> A total of 184 patients were identified. Among 146 assessable cases, PD-L1 expression was less than 1% in 60 (41%), 1% to 49% in 35 (24%), and greater than or equal to 50% in 51 tumors (35%). Of 100 (92%) TMB-assessable tumors, 92 had less than 10 mutations per megabase. TMB was significantly lower for <i>ROS1-</i>rearranged tumors (n = 97) compared with tumors with <i>EGFR</i> (n = 1250) or <i>KRAS</i> alterations (n = 1653) and all other NSCLC tumors (n = 2753) evaluated with Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (median TMB = 2.6 versus 3.5, 7.0, and 6.1 mutations per megabase, <i>p</i> < 0.001). Among patients treated with ICI, median TTD was 2.1 months (95% confidence interval [CI]: 1.0–4.2 mo; n = 28) and ORR 13% (2 of 16 RECIST-assessable; 95% CI: 2%–38%). Among patients treated with chemo-ICI, median TTD was 10 months (95% CI: 4.7–14.1 mo; n = 11) and ORR 83% (5 of 6 RECIST-assessable; 95% CI: 36%–100%). There was no difference in PD-L1 expression (<i>p</i> = 0.91) or TMB (<i>p</i> = 0.83) between responders and nonresponders. <h3>Conclusions</h3> Most <i>ROS1-</i>rearranged NSCLCs have low PD-L1 expression and TMB. The activity of ICI in these tumors is modest. In contrast, chemo-ICI can achieve meaningful activity.

Acquired drug resistance remains a major problem across oncogene-addicted cancers. Elucidation of mechanisms of resistance can inform rational treatment strategies for patients relapsing on targeted therapies while offering insights into tumor evolution. Here, we report acquired MET amplification as a resistance driver in a ROS1-rearranged lung adenocarcinoma after sequential treatment with ROS1 inhibitors. Subsequent combination therapy with lorlatinib plus capmatinib, a MET-selective inhibitor, induced intracranial and extracranial tumor response. At relapse, sequencing of the resistant tumor revealed a MET D1246N mutation and loss of MET amplification. We performed integrated molecular analyses of serial tumor and plasma samples, unveiling dynamic alterations in the ROS1 fusion driver and MET bypass axis at genomic and protein levels and the emergence of polyclonal resistance. This case illustrates the complexity of longitudinal tumor evolution with sequential targeted therapies, highlighting challenges embedded in the current precision oncology paradigm and the importance of developing approaches that prevent resistance.

The importance of cellular quality-control systems in the maintenance of neuronal homoeostasis and in the defence against neurodegeneration is well recognized. Chaperones and proteolytic systems, the main components of these cellular surveillance mechanisms, are key in the fight against the proteotoxicity that is often associated with severe neurodegenerative diseases. However, in recent years, a new theme has emerged which suggests that components of protein quality-control pathways are often targets of the toxic effects of pathogenic proteins and that their failure to function properly contributes to pathogenesis and disease progression. In the present mini-review, we describe this dual role as ‘saviour’ and ‘victim’ in the context of neurodegeneration for chaperone-mediated autophagy, a cellular pathway involved in the selective degradation of cytosolic proteins in lysosomes.

The central nervous system (CNS) is a common site of progression among patients with ROS1-rearranged lung cancer receiving crizotinib. We conducted a phase 2 study to evaluate the intracranial efficacy of lorlatinib in patients with ROS1-rearranged lung cancer who developed CNS-only progression on crizotinib.Patients with metastatic ROS1-rearranged lung cancer with CNS-only progression on crizotinib received lorlatinib 100 mg daily. The primary end point was intracranial disease control rate at 12 weeks per modified Response Evaluation Criteria in Solid Tumors version 1.1. Secondary end points included intracranial and extracranial progression-free survival, intracranial objective response rate, and safety/tolerability.A total of 16 patients were enrolled between November 2016 and January 2019. Nine patients (56%) had received prior CNS radiation, with a median of 10.9 months between radiation and lorlatinib. At 12 weeks, the intracranial disease control rate was 100% and intracranial objective response rate was 87%. While on study, the complee intracranial response rate was 60%. With median follow-up of 22 months, seven patients experienced disease progression, including five patients with CNS relapse. The median intracranial and extracranial progression-free survivals were 38.8 months (95% confidence interval: 16.9-not reported) and 41.1 months (95% confidence interval: 17.6-not reported), respectively. Molecular analysis of plasma or tissue from patients with extracranial progression on lorlatinib revealed ROS1 G2032R (n = 1), ROS1 L2086F (n = 1), and CCDC6-RET fusion plus ROS1 G2032R (n = 1). The safety profile of lorlatinib was consistent with prior studies. There were 11 patients (69%) who required dose reduction, including one patient who discontinued treatment for grade 3 edema. No grade greater than or equal to 4 adverse events were observed.Lorlatinib induced durable intracranial responses in patients with ROS1-rearranged NSCLC and prior isolated CNS progression on crizotinib.

&lt;p&gt;Supplementary Table S3&lt;/p&gt;

&lt;p&gt;Supplementary Table S5&lt;/p&gt;

&lt;p&gt;Supplementary Table S4&lt;/p&gt;

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

&lt;p&gt;Supplementary Figures&lt;/p&gt;

&lt;p&gt;Supplementary Methods&lt;/p&gt;

&lt;p&gt;Supplementary Table S2&lt;/p&gt;

&lt;div&gt;AbstractPurpose:&lt;p&gt;ctDNA offers a promising, noninvasive approach to monitor therapeutic efficacy in real-time. We explored whether the quantitative percent change in ctDNA early after therapy initiation can predict treatment response and progression-free survival (PFS) in patients with metastatic gastrointestinal cancer.&lt;/p&gt;Experimental Design:&lt;p&gt;A total of 138 patients with metastatic gastrointestinal cancers and tumor profiling by next-generation sequencing had serial blood draws pretreatment and at scheduled intervals during therapy. ctDNA was assessed using individualized droplet digital PCR measuring the mutant allele fraction in plasma of mutations identified in tumor biopsies. ctDNA changes were correlated with tumor markers and radiographic response.&lt;/p&gt;Results:&lt;p&gt;A total of 138 patients enrolled. A total of 101 patients were evaluable for ctDNA and 68 for tumor markers at 4 weeks. Percent change of ctDNA by 4 weeks predicted partial response (PR, &lt;i&gt;P&lt;/i&gt; &lt; 0.0001) and clinical benefit [CB: PR and stable disease (SD), &lt;i&gt;P&lt;/i&gt; &lt; 0.0001]. ctDNA decreased by 98% (median) and &gt;30% for all PR patients. ctDNA change at 8 weeks, but not 2 weeks, also predicted CB (&lt;i&gt;P&lt;/i&gt; &lt; 0.0001). Four-week change in tumor markers also predicted response (&lt;i&gt;P&lt;/i&gt; = 0.0026) and CB (&lt;i&gt;P&lt;/i&gt; = 0.022). However, at a clinically relevant specificity threshold of 90%, 4-week ctDNA change more effectively predicted CB versus tumor markers, with a sensitivity of 60% versus 24%, respectively (&lt;i&gt;P&lt;/i&gt; = 0.0109). Patients whose 4-week ctDNA decreased beyond this threshold (≥30% decrease) had a median PFS of 175 days versus 59.5 days (HR, 3.29; 95% CI, 1.55–7.00; &lt;i&gt;P&lt;/i&gt; &lt; 0.0001).&lt;/p&gt;Conclusions:&lt;p&gt;Serial ctDNA monitoring may provide early indication of response to systemic therapy in patients with metastatic gastrointestinal cancer prior to radiographic assessments and may outperform standard tumor markers, warranting further evaluation.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;AbstractPurpose:&lt;p&gt;ctDNA offers a promising, noninvasive approach to monitor therapeutic efficacy in real-time. We explored whether the quantitative percent change in ctDNA early after therapy initiation can predict treatment response and progression-free survival (PFS) in patients with metastatic gastrointestinal cancer.&lt;/p&gt;Experimental Design:&lt;p&gt;A total of 138 patients with metastatic gastrointestinal cancers and tumor profiling by next-generation sequencing had serial blood draws pretreatment and at scheduled intervals during therapy. ctDNA was assessed using individualized droplet digital PCR measuring the mutant allele fraction in plasma of mutations identified in tumor biopsies. ctDNA changes were correlated with tumor markers and radiographic response.&lt;/p&gt;Results:&lt;p&gt;A total of 138 patients enrolled. A total of 101 patients were evaluable for ctDNA and 68 for tumor markers at 4 weeks. Percent change of ctDNA by 4 weeks predicted partial response (PR, &lt;i&gt;P&lt;/i&gt; &lt; 0.0001) and clinical benefit [CB: PR and stable disease (SD), &lt;i&gt;P&lt;/i&gt; &lt; 0.0001]. ctDNA decreased by 98% (median) and &gt;30% for all PR patients. ctDNA change at 8 weeks, but not 2 weeks, also predicted CB (&lt;i&gt;P&lt;/i&gt; &lt; 0.0001). Four-week change in tumor markers also predicted response (&lt;i&gt;P&lt;/i&gt; = 0.0026) and CB (&lt;i&gt;P&lt;/i&gt; = 0.022). However, at a clinically relevant specificity threshold of 90%, 4-week ctDNA change more effectively predicted CB versus tumor markers, with a sensitivity of 60% versus 24%, respectively (&lt;i&gt;P&lt;/i&gt; = 0.0109). Patients whose 4-week ctDNA decreased beyond this threshold (≥30% decrease) had a median PFS of 175 days versus 59.5 days (HR, 3.29; 95% CI, 1.55–7.00; &lt;i&gt;P&lt;/i&gt; &lt; 0.0001).&lt;/p&gt;Conclusions:&lt;p&gt;Serial ctDNA monitoring may provide early indication of response to systemic therapy in patients with metastatic gastrointestinal cancer prior to radiographic assessments and may outperform standard tumor markers, warranting further evaluation.&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;Supplementary Table S3&lt;/p&gt;

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

&lt;p&gt;Supplementary Table S2&lt;/p&gt;

&lt;p&gt;Supplementary Methods&lt;/p&gt;

&lt;p&gt;Supplementary Table S4&lt;/p&gt;

&lt;p&gt;Supplementary Figures&lt;/p&gt;

&lt;p&gt;Supplementary Table S5&lt;/p&gt;

Abstract There is a longstanding desire to take therapeutic advantage of dysregulated metabolic states in cancer. While it has been appreciated that lung tumors rewire their cellular metabolic networks to support unrestrained proliferation, metabolic vulnerabilities have largely not been explored in the context of specific onco-genotypes. This represents a major gap in our understanding of how different oncogenic drivers in non-small cell lung cancer (NSCLC) confer reliance on discrete metabolic networks to sustain tumor growth. The goals of this project are (1) to investigate metabolic dependencies in distinct molecular subtypes of lung cancer and (2) to elucidate how metabolic reprogramming drives resistance to targeted therapy. Using patient-derived cell culture models and tumor specimens collected from patients with ALK-positive (ALK+) NSCLC, we identified that lung tumors with ALK rearrangements harbor a unique metabolic signature marked by reliance on anabolic nucleotide pathways. A phosphoproteomic screen in ALK+ patient-derived cells identified a novel metabolic target of ALK signaling, GUK1, the only known enzyme responsible for GDP synthesis. We show that ALK binds to and phosphorylates GUK1 and that ALK-mediated GUK1 phosphorylation augments GDP/GTP nucleotide biosynthesis. Steady-state and tracing metabolomic studies demonstrate that ALK inhibition and GUK1 phosphomutant are epistatic in guanine nucleotide production. Molecular dynamic modeling suggests that phosphorylation of GUK1 alters the dynamics of active site closure to enhance substrate processivity and protects GUK1 from a non-catalytic confirmation. Introduction of phosphomutant GUK1 into ALK+ patient-derived cell lines results in decreased tumor proliferation in vitro and in vivo in xenograft models. Spatially resolved mass spectrometry imaging of tumor specimens from ALK+ patients demonstrates significant enrichment of guanine nucleotides in ALK+ and phospho-GUK1+ tumor cells. We identified that other oncogenic fusion proteins regulate GUK1 phosphorylation, highlighting the need to further characterize GUK1 as a metabolic liability in NSCLC. Furthermore, a subset of patient-derived cell lines with resistance to ALK tyrosine kinase inhibitors (TKIs) exhibits increased expression and phosphorylation of GUK1, indicating that regulation of this metabolic enzyme may play a role in mediating acquired resistance. We anticipate these studies will pave the way for the development of new therapeutic approaches by exploiting metabolic vulnerabilities in oncogene-driven lung cancers. Citation Format: Jaime Laurel Schneider, Kiran Kurmi, Ishita Dhiman, Roberta Colapietro, Shakchhi Joshi, Christian Johnson, Satoshi Yoda, Joao Paulo, Daniela Ruiz, Sylwia Stopka, Gerard Baquer, Jessica Lin, Kevin Haigis, Nathalie Agar, Steven Gygi, Aaron Hata, Marcia Haigis. GUK1 is a novel metabolic liability in oncogene-driven lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1156.

Introduction: In several cancers circulating tumor DNA (ctDNA) is emerging as a promising technology for assessing early response to treatment and predicting overall clinical benefit. Currently, in gastrointestinal (GI) cancers, assessment of treatment response utilizes imaging and serum tumor markers. However, standard tumor markers may lack adequate sensitivity and specificity and imaging is typically performed several months into treatment. We hypothesize that ctDNA may offer a more accurate method of assessing early response to treatment and predicting outcomes for metastatic GI tract cancers. Methods: We evaluated changes in ctDNA and standard tumor markers to predict treatment response and clinical benefit in metastatic GI cancer patients receiving chemotherapy and/or targeted therapy. A total of 139 consecutive patients with a confirmed metastatic GI cancer diagnosis (70 colorectal, 26 pancreatic, 17 gastroesophageal, and 18 biliary) had pretreatment and 4-week plasma samples available for ctDNA analysis. To assess ctDNA levels, one or more mutations identified by tumor profiling were evaluated for ctDNA by droplet digital PCR (ddPCR), and the change in mutant allele fraction was measured in ctDNA at 4 weeks relative to baseline. Patients were excluded if there was insufficient tumor for testing (n = 2), no variants detected on tumor profiling (n = 12), effective ddPCR assay could not be designed (n = 5), or if they had undetectable baseline ctDNA levels (n = 18). For patients who also had elevated tumor markers (CEA and/or CA19-9) prior to treatment and at 4 weeks, the percent change in marker level was calculated. ctDNA and tumor markers were compared to RECIST response and PFS. Results: Of 102 patients with evaluable ctDNA data, 68 received chemotherapy, 23 targeted therapy, and 11 combination chemotherapy and targeted therapy. The average change in ctDNA by 4 weeks was -91% for patients with PR, -66% for patients with clinical benefit (CB; PR + SD), and -20% for PD (P = .002 for PR vs. SD/PD, P = .003 for CB vs. PD). By contrast, in 48 patients who also had evaluable standard tumor markers (CEA or CA19-9) at 4 weeks, the average change in tumor markers was -30% for PR, +2% for CB, and +24% for PD (P = .12 PR vs. SD/PD, P = .50 for CB vs. PD). Of 24 patients with PR, all but one had a ctDNA decrease of > 50% by 4 weeks (sensitivity 96%, specificity 41%). Failure to achieve a ctDNA decrease >50% by 4 weeks had an NPV of 97% for PR. Patients with ctDNA decrease >50% had median PFS of 161 days vs. 64 days for <50% decrease (P = .01). Conclusion: Monitoring change in ctDNA may provide an early predictor of response and CB to therapy across GI cancers. ctDNA may predict response and clinical benefit more accurately than standard tumor markers. Further prospective studies of real-time ctDNA monitoring to predict the effects of therapy are warranted.

Abstract Background: Patients with gastrointestinal (GI) malignancies undergoing treatment are surveyed for disease relapse with radiographic imaging and measurement of standard tumor markers. However, there is a need for more sensitive techniques to detect early cancer progression. Measurement of circulating tumor DNA (ctDNA) is emerging as an important diagnostic tool that can provide real-time information about disease response during treatment. In this study, we evaluated whether dynamic monitoring of ctDNA may predict cancer progression earlier than standard methods in patients with GI malignancies undergoing targeted therapy. Methods: Tumor and blood specimens were obtained from patients treated at the Massachusetts General Hospital (MGH) under IRB-approved studies. Tumor biopsies were performed at the time of diagnosis and were subjected to the MGH standard molecular diagnostics panel for 104 known cancer genes. Blood samples for ctDNA analysis were collected at baseline at the start of targeted therapy, and one more more driver mutations were followed longitudinaly at two week intervals starting one month after treatment. ctDNA was extracted from plasma using QIAGEN-based protocols and amplified by digital droplet PCR (ddPCR) using primers for tumor-specific point mutations. Tumor markers and CT scans were part of routine clinical care and procured using standard procedures at MGH. RECIST measurements, if available, were obtained in a blinded fashion using the Tumor Imaging Metrics Core. Results: We studied 44 patients with GI malignancies undergoing treatment with targeted therapies. Represented cancer types included colorectal (53%), gastroesophageal (21%), biliary (20%), and pancreatic cancers (6%). We correlated the % change in ctDNA mutant allele fraction (MAF) or standard tumor markers 4 weeks after treatment initiation with radiographic response by RECIST. Tumor-specific MAF decreased by 88% in patients who went on to show partial response (PR) or stable disease (SD), compared to an increase by 51% in patients shown to have progressive disease (PD) (p=1.8x10-5). All patients who achieved PR, except one, had a reduction in ctDNA levels by at least 93% or more. In contrast, standard tumor markers did not predict response as there was no significant difference in the % change at 4 weeks in the PR/SD group compared to PD (p=0.64). Conclusions: Real-time monitoring of patient-specific tumor mutations by ctDNA analysis has the potential to predict response to targeted therapy earlier than standard surveillance mechanisms. Integration of liquid biopsies into medical decision-making may allow for earlier identification of cancer progression and consequent modification of therapeutic approach. However, more studies are needed to determine whether incorporation of ctDNA analysis into clinical practice would impact patient outcomes. Citation Format: Jaime L. Schneider, Aparna Parikh, Mehlika Rethinam, Brandon Nadres, Emily Van Seventer, Heather Shahzade, Ryan B. Corcoran. Circulating tumor DNA as a tool for predicting response to targeted therapy in gastrointestinal malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1575.

9049 Background: ROS1 fusions are oncogenic drivers in various cancers types, including 1-3% of non-small cell lung cancers (NSCLCs). Immunotherapy approvals for NSCLC include ROS1-rearranged carcinomas, but the activity of immune checkpoint inhibition (ICI) as monotherapy or in combination with chemotherapy (chemo-ICI) therapy, as well as the immunophenotypic characteristics of these tumors, have not been described in a large data set. Methods: In this multi-institutional study, patients with ROS1-rearranged NSCLC were identified retrospectively. Tumor PD-L1 expression and tumor mutational burden (TMB) were assessed as part of routine clinical care. In patients who received ICI monotherapy or chemo-ICI in the metastatic setting, time to treatment discontinuation (TTD) and objective response rate (ORR; RECIST v. 1.1) were calculated. TTD was assessed with Kaplan-Meier methods; patients remaining on treatment were censored at last follow up. Results: 184 patients with ROS1-rearranged NSCLC were identified. Among 146 PD-L1 evaluable cases, PD-L1 expression was &lt; 1% in 60 (41%), 1-49% in 35 (24%) and ≥50% in 51 (35%) tumors. Ninety-two of 100 (92%) TMB-evaluable tumors had &lt; 10 mutations/megabase (mut/Mb). TMB was significantly lower for ROS1-rearranged NSCLCs (n = 97) vs. ROS1-wild type tumors (n = 5,380) evaluated with next-generation sequencing using MSK-IMPACT (median 2.6 vs. 5.9 mut/Mb, p &lt; 0.001). Twenty-eight patients received ICI monotherapy and 11 patients received chemo-ICI. The median TTD was 2.1 months (95% CI: 1.0-4.2; n = 28) for single-agent ICI therapy and 10 months (95% CI: 4.7-14.1; n = 11) for chemo-ICI therapy. The ORR was 13% (2/16 RECIST-evaluable; 95% CI: 2-38%) for ICI monotherapy and 83% (5/6 RECIST-evaluable; 95% CI: 36-100%) for chemo-ICI therapy. There was no difference in PD-L1 tumor expression (p = 0.9) or TMB (p = 0.8) between responders and non-responders and no correlation between PD-L1 tumor expression (rho = 0.16, p = 0.6) or TMB (rho = 0.03, p = 0.9) and maximum change in sum of target lesions. Conclusions: Most ROS1-rearranged NSCLCs have low or no PD-L1 expression and low TMB. The activity of checkpoint inhibitor monotherapy is disappointing in ROS1-driven NSCLC. In contrast, combination chemoimmunotherapy can achieve clinically meaningful activity.