Potential Biomarkers Of Clinical Response Research Articles

Neural Correlates of the DEEPP (Anti-suicidal Response to Ketamine in Treatment-Resistant Bipolar Depression) Study: Protocol for a Pilot, Open-Label Clinical Trial.

Suicide is among the top 10 leading causes of death worldwide. Of people who died by suicide, the majority are diagnosed with depression. It is estimated that 25%-60% of people with bipolar depression (BD) will attempt suicide at least once, and 10%-15% will die by suicide. Several treatments, such as lithium, clozapine, electroconvulsive therapy, and cognitive behavioral therapy, have been shown to be effective in treating suicidality. However, these treatments can be difficult to tolerate or may take months to take effect. Ketamine, a glutamate N-methyl-D-aspartate antagonist, has been shown to have rapid antisuicidal effect and antidepressant qualities, and is thus a promising intervention to target acute suicidality in patients with BD. However, the biological mechanism underlying its therapeutic action remains poorly understood. Enhancing our understanding of underlying mechanisms of action for ketamine's effectiveness in reducing suicidality is critical to establishing biological markers of treatment response and developing tailored, personalized interventions for patients with BD. This is an open-label clinical trial to test the safety and feasibility of repeated ketamine infusions to treat acute suicidality. The primary objective is to test the safety and feasibility of ketamine intervention. The secondary objective is to examine ketamine's potential neurophysiological mechanisms of action by assessing cortical excitation and inhibition to determine potential biomarkers of clinical response. Other objectives are to evaluate the effect of ketamine on acute suicidality and other clinical outcomes, such as depressive symptoms and quality of life, to inform a future larger trial. This open-label clinical trial aims to test the safety and feasibility of repeated ketamine infusions in patients with BD for suicidality and to assess ketamine's neurophysiological effects. A sterile form of racemic ketamine hydrochloride will be administered over a 40-minute intravenous infusion 2 times per week on nonconsecutive days for 4 weeks (8 sessions). We will recruit 30 adults (24-65 year olds) over 2 years from an academic psychiatric hospital in Toronto, Canada. This study is currently ongoing and actively recruiting participants. So far, 5 participants have completed the trial, 1 is currently in active treatment, and 8 participants are on the waitlist to be screened. We anticipate initial results being available in the fall of 2023. This proposal was presented as a poster presentation at the Research to Reality Global Summit on Psychedelic-Assisted Therapies and Medicine, held in May 2022 in Toronto, Canada. Developing effective interventions for acute suicidality in high-risk populations such as those with BD remains a major therapeutic challenge. Ketamine is a promising treatment due to its rapid antidepressant and antisuicidal effects, but its underlying neurophysiological mechanisms of action remain unknown. ClinicalTrials.gov NCT05177146; https://clinicaltrials.gov/ct2/show/NCT05177146. DERR1-10.2196/41013.

Clonal Architecture and Variant Allele Frequency Predict Clinical Outcomes and Drug Response in Acute Myeloid Leukemia

Introduction: AML is an aggressive cancer that develops from the sequential accumulation and clonal expansion of somatic mutations in hematopoietic stem and progenitor cells. Recent next-generation sequencing (NGS) studies of AML have correlated mutations with clinical outcomes and response to targeted therapies. Additionally, emerging reports have suggested that increased clonal heterogeneity and mutation burden tend to correlate with worse survival outcomes. However, due to previous cohort sizes, the architecture of clonal evolution and variant allele frequency (VAF) of recurrent mutations have yet to be robustly correlated with response to therapy or with more granular risk stratification. To address previous limitations, we combined available datasets of sequenced AML to model features of clonality and determine their correlations with clinical outcomes and drug sensitivity. Methods: A systematic literature review was performed to identify cohorts of clinically annotated and genetically profiled adult AML. Studies were included if: (i) their sequencing panel targeted at least 30 of the most commonly mutated genes, (ii) censored overall survival data was reported, and (iii) data were publicly available. An additional cohort of patients profiled at Stanford was also included. Leveraging statistical learning methods and robust clonal modeling algorithms (PyClone and ClonEvol), we performed a meta-analysis of the clonal architecture of mutations, their temporal relationships, sensitivity to drugs, and correlation with outcomes in AML. Results: A total of 12 studies were aggregated into a uniformly annotated database comprising 2,987 AML patient samples profiled with an array of DNA sequencing modalities (2,884 with VAFs) and ex vivo drug screening results (nsamples = 562; ndrugs = 122); survival outcomes were available for 2,606 patients. To investigate broad features of leukemia evolution, we used clonal modeling algorithms to infer clonal architecture. Interestingly, patients exhibiting linear evolution (sequential mutations in the same clone) displayed worse outcomes compared to those with branched architecture (distinct subclonal populations). Additionally, mutational burden and clonal heterogeneity only stratified patients with branched structure. These results motivated us to understand how the temporal acquisition of mutations might further stratify outcomes. Using dynamic VAF thresholds, we identified novel high-risk patient populations for 15 recurrently mutated genes. Greater VAF was associated with statistically significant improved survival in genotypes such as GATA2mut and WT1mut and with worse outcomes for patients with NRAS and NF1 mutations. Next, we leveraged VAFs to infer the temporal ordering of individual mutations and functional mutation categories. Patients where NRAS mutations occurred before GATA2 mutations showed a significant correlation with worse outcomes. We also observed that patients in which (i) DNA methylation mutations occurred before those in tumor suppressors and (ii) splicing factor mutations occurred before RTK/RAS signaling components showed significantly shortened overall survival. These results indicate that patients with the same genotype can be stratified by the timing of mutations in the clonal evolution of their leukemia. Finally, we used linear regression between drug sensitivity and VAF to identify several mutations which predict drug sensitivity exclusively in a VAF-dependent manner. Increased WT1 VAF correlated with sensitivity to ABT-737 and elevated FLT3-TKD VAF predicted sensitivity to cabozantinib, among other clinically notable drug-gene relationships. These results suggest potential biomarkers for clinical response to emerging targeted agents. Conclusions: We show that VAF can identify novel high-risk patient populations at the individual mutation level (e.g. BCOR and NF1) and can also be leveraged to stratify outcomes based on inferring the temporal ordering of mutations (e.g. NRAS and GATA2). Our observation that patients with leukemias exhibiting branched evolution showed improved survival compared to linear evolution was also striking and warrants further experimental and clinical validation. Incorporating these results with our findings of drug sensitivity validate the clinical utility of integrating clonal analysis into the molecular evaluation and treatment of AML. Disclosures Majeti: Zenshine Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Kodikaz Therapeutic Solutions Inc.: Membership on an entity's Board of Directors or advisory committees; Stanford University: Patents & Royalties: pending patent application on CD93 CAR ; Coherus BioSciences: Membership on an entity's Board of Directors or advisory committees; BeyondSpring Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Forty-Seven Inc.: Divested equity in a private or publicly-traded company in the past 24 months; Gilead Sciences, Inc.: Patents & Royalties: inventor on patents related to CD47 cancer immunotherapy; CircBio Inc.: Research Funding.

Single-Cell Cytokine Analysis of LV20.19 Bispecific CAR T-Cell Products from a Phase I Clinical Trial

Introduction Anti CD19 CAR T-cell therapy is a breakthrough immunotherapeutic approach for relapsed, refractory B-cell malignancies. Despite initial excitement, long term progression free survival with single antigen targeted CD19 CAR T-cells range from 30-40% in aggressive B-cell NHL. Loss of target due to selective pressure against CD19 can lead to antigen downregulation and development of a CD19-negative clonal population. To overcome this resistance mechanism, we recently completed a Phase 1 anti-CD19, anti-CD20 (LV20.19) CAR T-cell trial demonstrating the safety of 2.5x10e6 cells/kg dose in patients with relapsed, refractory B-cell lymphomas (in press in Nature Medicine). We report below single cell cytokine studies from final LV20.19 CAR T-cell products using the Isoplexis single-cell proteomics device. Methods LV20.19 CAR T-cells were manufactured onsite using the CliniMACS Prodigy device on a fixed 14-day manufacturing platform with IL-2 for cell expansion (NCT03019055). Isoplexis cytokine analysis was limited to the cohort of patients treated at goal dose of 2.5x10e6 cells/kg (n=16). Leftover LV20.19 CAR T-cells from the original product were thawed, CD4 and CD8 T cells were enriched by immunomagnetic sorting, and the T-cell subsets separately stimulated with CD19-engineered K562 stimulators overnight. The stimulated cells were loaded onto single-cell, Adaptive Immune Isocode chips, and the chips read for 18 hours in an Isolight instrument to assess single-cell production of 32 individual cytokines. Isospeak software was used to analyze the single-cell data. Results 15 patients had adequate samples for analysis. Results were stratified by response (complete responders (CR) versus partial responders (PR) or progressive disease (PD)). The Isoplexis device was utilized to generate a polyfunctional (i.e., ability to produce more that 1 cytokine) index score (PSI) that has been previously reported (Rossi et al. Blood 2018) to correlate with CAR T-cell treatment outcomes. At Day 28 after LV20.19 CAR T-cell therapy, there were 11 CR patients and 4 PR/PD patients. CD4+ LV20.19 CAR T-cells from complete responders had a non-statistically significant decreased PSI score versus CD4+ cells from PR/PD patients (Figure 1A, p=0.38). The PSI score among CD8+ T-cells was similar (p=0.71) (Figure 1A). The increased PSI score among CR patients' CD4+ LV20.19 CAR T-cells was driven primarily by cells producing effector (IFNg and TNFb) cytokines. On a singular cytokine level, compared to PR/PD patient cells, CR patient CAR T-cells produced significantly higher levels of CCL-11, GM-CSF and IL-17A (Figure 1B; p<0.05). Notably, CR patients had dramatically higher percentages of individual CD4+ cells that secreted these 3 cytokines as compared to PR/PD patients (Figure 1B). Conclusion CD4+ LV20.19 CAR T-cells in responding patients trended towards a higher PSI than non-responding patients. At the single cell level, differences in the PSI among CD4+ LV20.19 CAR T-cells from responding patients were driven primarily by a few cytokines including IL-17A, CCL11, GM-CSF, IFNg, and TNFb, which may be key to clinical response. While our results are limited by sample size and only 4 PR/PD patients in this cohort, identification of cytokines correlating with response will allow selective modulation to improve clinical outcomes while limiting CRS. Ongoing studies with LV20.19 CAR T-cells will further delineate the role of IL-17A, GM-CSF and CCL-11 production as potential biomarkers of clinical response with bispecific CAR T-cell products. Disclosures Johnson: Cell Vault: Research Funding; Miltenyi Biotec: Research Funding. Fenske:Medical College of Wisconsin: Current Employment. Hamadani:Sanofi Genzyme, AstraZeneca: Speakers Bureau; Janssen R&D; Incyte Corporation; ADC Therapeutics; Celgene Corporation; Pharmacyclics, Omeros, AbGenomics, Verastem, TeneoBio: Consultancy; ADC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Takeda Pharmaceutical Company; Spectrum Pharmaceuticals; Astellas Pharma: Research Funding. Dropulic:Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties: CAR-T immunotherapy. Schneider:Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties. Hari:GSK: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Incyte Corporation: Consultancy; BMS: Consultancy; Amgen: Consultancy. Shah:Miltenyi Biotec: Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Incyte: Consultancy; TG Therapeutics: Consultancy; Verastim: Consultancy; Lily: Consultancy, Honoraria; Cell Vault: Research Funding; Kite Pharma: Consultancy, Honoraria.

Fc receptor-like 5 and anti-CD20 treatment response in granulomatosis with polyangiitis and microscopic polyangiitis.

BACKGROUNDBaseline expression of FCRL5, a marker of naive and memory B cells, was shown to predict response to rituximab (RTX) in rheumatoid arthritis. This study investigated baseline expression of FCRL5 as a potential biomarker of clinical response to RTX in granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA).METHODSA previously validated quantitative PCR–based (qPCR-based) platform was used to assess FCRL5 expression in patients with GPA/MPA (RAVE trial, NCT00104299).RESULTSBaseline FCRL5 expression was significantly higher in patients achieving complete remission (CR) at 6, 12, and 18 months, independent of other clinical and serological variables, among those randomized to RTX but not cyclophosphamide-azathioprine (CYC/AZA). Patients with baseline FCRL5 expression ≥ 0.01 expression units (termed FCRL5hi) exhibited significantly higher CR rates at 6, 12, and 18 months as compared with FCRL5lo subjects (84% versus 57% [P = 0.016], 68% versus 40% [P = 0.02], and 68% versus 29% [P = 0.0009], respectively).CONCLUSIONOur data taken together suggest that FCRL5 is a biomarker of B cell lineage associated with increased achievement and maintenance of complete remission among patients treated with RTX and warrant further investigation in a prospective manner.FUNDINGThe analysis for this study was funded by Genentech Inc.

Cereblon gene variants and clinical outcome in multiple myeloma patients treated with lenalidomide

Carfilzomib-lenalidomide-dexamethasone (KRd) therapy has yielded promising results in patients with newly diagnosed multiple myeloma (NDMM). Cereblon (CRBN) is the direct molecular target of lenalidomide and genetic polymorphisms in CRBN have been associated with lenalidomide efficacy. In this study, we assessed the correlation of five single nucleotide variants (SNVs) in the CRBN gene with clinical response and outcomes in patients with NDMM administered KRd therapy with lenalidomide maintenance, achieving favorable trial endpoints in a prospective Phase II study (NCT01402284). Of the observed SNVs, no associations with KRd therapy response were found in this patient cohort, although strong trends in hypoalbuminemia grade and hyperbilirubinemia grade emerged across the CRBN rs1672753 genotype (P = 0.0008) and the rs1714327 genotype (P = 0.0010), respectively. Our results do not provide conclusive support for the predictive utility of CRBN gene polymorphisms as potential biomarkers of clinical response to lenalidomide-based therapy in our patient population. However, these findings remain to be validated in prospective studies using larger patient populations.

Abstract 569: Expression and modulation by IFN of HLA class I APM components in melanoma: Potential biomarkers of clinical response to checkpoint inhibitors

Abstract Purpose: The goals of this study are i) to analyze the expression of human leukocyte antigen class I (HLA-I) antigen processing machinery (APM) components in surgically removed melanoma tumors, ii) to assess their modulation by Interferons (IFNs) and iii) to determine their value as predictive biomarkers of the clinical response to immunotherapy with checkpoint inhibitors (CI). Methods: The expression levels of HLA class I APM components [HLA-I (ABC) chains, beta(2)microglobulin (β2m), Calnexin, Calreticulin, ERp57, Tapasin, TAP1-2, LMP2, LMP7, LMP10] were evaluated by staining with mAbs and flow cytometry in melanoma cells isolated from surgical biopsies from 16 patients with advanced melanoma. In addition the modulation of these molecules by IFN-γ or IFN-α2 (IFNs) was evaluated. Results: The frequency of cells expressing HLA-I/β2m was >90% in 15/16 Mel tumors. In 1 case extracellular HLA-I/β2m molecules were not detected. The expression levels of extracellular HLA class I APM molecules were quite heterogeneous. Based on Mean Fluorescence Intensity (MFI) of HLA-I/β2m, Mel segregated into 2 groups with high (MFI >200, n=8) and low (MFI<200, n=8) expression levels. In 11 cases (5 from the high and 6 from the low expression groups) we could explore the modulatory activity of IFNs. In vitro exposure to IFNs globally enhanced the expression levels of HLA-I/β2m, allowing the transition from the low to the highexpression group in 1 case. The distribution of expression defects in the intracellular APM components resulted intensely heterogeneous. Highly defective (<25%) expressions were detected notably for LMP2 (12/16), TAP1 (13/16), TAP2 (10/16) and Tapasin (14/16). In 10 cases we could explore the beneficial modulatory activity of IFNs on defective APM components. IFNs could restore the expression of LMP2 (4/10), TAP1 (6/10), and TAP2 (7/8), but not that of Tapasin. Seven patients were treated with CI (either anti-PD1 or anti-CTLA4). Four of them had clinical responses (PR or CR); in 3 of them Mel expressed high levels of HLA-I/β2m. Conversely, Mel from all 3 patients that did not respond to CI expressed low HLA-I/β2m levels which could not be upregulated by IFNs. Highly defective expression of APM components, not responsive to IFN restoration, was detected in all 3 Mel from non-responders. Conclusions: We report a wide heterogeneous distribution of HLA-I/β2m expression levels and deficits of APM components in Melanoma, partially restorable by IFNs. Our data support the possibility that defects in HLA expression, not responsive to IFN restoration, may predict low responsiveness to CI. We provide rationale to incorporate the evaluation of HLA-I/β2m and APM in clinical immunotherapy studies with CI. Citation Format: Lidia Giraudo, Loretta Gammaitoni, Bortolot Valentina, Ilenia Iaia, Giulia Cattaneo, Paolo Becco, Susanna Gallo, Alessandro Zaccagna, Alberto Pisacane, Luca Crotto, Soldano Ferrone, Massimo Aglietta, Fabrizio Carnevale Schianca, Dario Sangiolo. Expression and modulation by IFN of HLA class I APM components in melanoma: Potential biomarkers of clinical response to checkpoint inhibitors [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 569.

Identifying a potential biomarker for anti-PD-1 immunotherapy in patients with advanced stage, surgically-resectable endometrial cancer.

5590 Background: The FDA approval of pembrolizumab for patients with MSI-H or dMMR tumors has led to the treatment of a select cohort of endometrial cancer (EC) patients. We sought to ascertain tumor immune modulatory effects in the front-line setting for advanced stage III/IV EC patients regardless of MSI-H or dMMR. The primary objective was to determine the safety of preoperative and maintenance pembrolizumab. The secondary objective was to examine pembrolizumab-induced changes in peripheral immune effector phenotype in order to identify potential biomarkers of clinical response. Methods: In an open label, single-arm Phase I trial, 8 EC patients were treated with 2 doses of preoperative pembrolizumab IV prior to surgery followed by chemotherapy and 4 doses of pembrolizumab IV. As an initial study, pre- and post-treatment (on the day of surgery) peripheral blood was collected from 3 patients as well as a healthy control and processed for high-dimensional single-cell mass cytometry (CyTOF) using an optimized antibody panel. Results: Six of 8 patients completed the treatment. One patient had rapid cancer progression and another had an exacerbation of comorbidities. Peripheral blood from 3 patients with pathological response were then immunoprofiled using CyTOF. Data analysis revealed that the frequencies of CD8+ T cells, B cells and CD56hiCD16- NK cells were lower, whereas the frequency of CD14+CD16-HLA-DRhi classical monocytes was higher in the cancer patients compared to controls. Cancer patients had lower frequencies of circulating CD4+ and CD8+ naïve T cells but higher frequencies of effector CD8+ and CD4+ T cells. Notably, the median expression of Granzyme B in CD8+ and CD4+ T cells was higher and median expression of signal regulatory protein (SIRP), CD172a-b on monocytes was lower for cancer patients compared to control. The frequencies of NK and myeloid cells expressing CD137(4-1BB), PD-1+NK cells, and PD-L1+DCs were greater in post-compared to pre-pembrolizumab. Conclusions: This is the first trial to evaluate the use of neoadjuvant pembrolizumab in advanced stage EC patients. Here, we present peripheral immune correlative data and show an increase in markers of activation in patients with pathologic responses to pembrolizumab. Additional data from this ongoing study will help us to identify candidate predictive biomarkers. Clinical trial information: NCT02630823.

MiR‑145‑5p is associated with pathological complete response to neoadjuvant chemotherapy and impairs cell proliferation by targeting TGFβR2 in breast cancer.

Cancer patients who better benefit from neoadjuvant chemotherapy (NeoCh) are those who achieve a successful pathological complete response (pCR) represented by the absence of residual disease. Unfortunately, no highly sensitive and specific tumor biomarkers for predicting the clinical response to NeoCh have yet been defined. The aim of the present study was to ascertain whether miR‑145‑5p could discriminate between pCR and no‑pCR in triple‑negative breast cancer patients that received a cisplatin/doxorubicin‑based neoadjuvant treatment. miR‑145‑5p expression was determined in breast tumors by quantitative RT‑PCR. Our data showed that miR‑145‑5p had a significant low expression (P<0.005) in patients that achieved pCR in comparison to the non‑responder group. Kaplan Meier analysis indicated that low levels of miR‑145‑5p were associated with increased disease‑free survival. In addition, receiver operating characteristic (ROC) curve analysis suggested that miR‑145‑5p is a good predictor of pCR (P<0.003, AUC=0.7899, 95% CI, 0.6382‑0.9416). Quantitative RT‑PCR expression analysis also revealed that miR‑145‑5p was downregulated in four breast cancer cell lines relative to normal cells. To study the functions of miR‑145‑5p, its expression was restored in triple‑negative MDA‑MB‑231 cells and its effects in cell proliferation were evaluated by MTT assays and in apoptosis using Annexin V experiments. Data revealed that ectopic expression of miR‑145‑5p resulted in a significant inhibition of cell proliferation and also induced apoptosis. Moreover, miR‑145‑5p led to sensitization of breast cancer cells to cisplatin therapy. In addition, western blot assays indicated that miR‑145‑5p downregulated the TGFβR2 protein. In conclusion, miR‑145‑5p could be a potential biomarker of clinical response to NeoCh in triple‑negative breast cancer. Functionally miR‑145‑5p may regulate cell proliferation, at least in part, by targeting TGFβR2.

Abstract 4346: Integrated whole genome profiling of the immune tumour interaction identifies predictive biomarkers of checkpoint inhibitor response in metastatic cancer

Abstract Immune checkpoint inhibitors are fast becoming a key therapy in the medical oncologist's toolbox across a diverse array of tumour types. The excitement surrounding the utility of these emerging agents is marred by the heterogeneous response observed among patients, highlighting the urgent need for clinically applicable predictive biomarkers. Significant progress has been made in identifying potentially relevant biomarkers including mutation burden and immune infiltration. However, it is still unclear which biomarkers are most predictive across various disease types, and how to integrate various markers of response and resistance for clinical interpretation. We hypothesized that comprehensive genomic profiling, combined with clinical data, would reveal differences in the immuno-oncologic phenotype that could be used as biomarkers for response to checkpoint inhibitor therapy. The Personalized OncoGenomics (POG) study at BC Cancer performs whole genome and transcriptome sequencing of metastatic disease across a diverse array of cancer types to comprehensively characterize cancers and inform clinical therapeutic decision-making. Here we performed in-depth genomic profiling of 64 POG patients who received immune checkpoint inhibitors encompassing multiple disease types including skin, lung, breast, pancreatic and colorectal cancers and sarcomas. Single nucleotide variants, structural variants, copy number alterations, and RNA expression derived from whole genomic and transcriptomic data were used to characterize genomic instability, neoantigen landscape, immune infiltration, and other potential biomarkers of clinical response to checkpoint inhibitors. This collection of putative immune-oncologic biomarkers were integrated into a multivariate model to stratify markers of the tumour immune response. Our analysis shows limited association between PD-L1 expression and checkpoint inhibitor response, consistent with the inadequate effectiveness of this as a universal marker, and shows stronger association between signatures of T cell infiltration based on RNA-Seq data. We also observed patients with low mutation burden that respond to checkpoint inhibitors and show high levels of immune infiltration, and cases with high mutation burden that harbour mechanisms of resistance including reduction in predicted neoantigen diversity. Additional mechanisms of therapeutic resistance observed in post treatment biopsies highlight disruption of antigen presentation and JAK1 mutations in resistant tumours. Our study helps to define which features distinguish patients most likely to respond to checkpoint inhibitors, and uses these features in selecting patients for immunotherapy treatment within the POG clinical trial. Citation Format: Hillary Pearson, Laura Williamson, Erin Pleasance, Scott Brown, Emma Titmuss, Martin Jones, Stuart Zong, Payal Sipahimalani, Yussanne Ma, Steve Jones, Robert Holt, Marco Marra, Janessa Laskin. Integrated whole genome profiling of the immune tumour interaction identifies predictive biomarkers of checkpoint inhibitor response in metastatic cancer [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 4346.

Dynamics of Heat Shock Protein 70 Serum Levels As a Predictor of Clinical Response in Non-Small-Cell Lung Cancer and Correlation with the Hypoxia-Related Marker Osteopontin.

Hypoxia mediates resistance to radio(chemo)therapy (RT) by stimulating the synthesis of hypoxia-related genes, such as osteopontin (OPN) and stress proteins, including the major stress-inducible heat shock protein 70 (Hsp70). Apart from its intracellular localization, Hsp70 is also present on the plasma membrane of viable tumor cells that actively release it in lipid vesicles with biophysical characteristics of exosomes. Exosomal Hsp70 contributes to radioresistance while Hsp70 derived from dying tumor cells can serve as a stimulator of immune cells. Given these opposing traits of extracellular Hsp70 and the unsatisfactory outcome of locally advanced lung tumors, we investigated the role of Hsp70 in the plasma of patients with advanced, non-metastasized non-small-cell lung cancer (NSCLC) before (T1) and 4–6 weeks after RT (T2) in relation to OPN as potential biomarkers for clinical response. Plasma levels of Hsp70 correlate with those of OPN at T1, and high OPN levels are significantly associated with a decreased overall survival (OS). Due to a therapy-induced reduction in viable tumor mass after RT Hsp70 plasma levels dropped significantly at T2 (p = 0.016). However, with respect to the immunostimulatory capacity of Hsp70 derived from dying tumor cells, patients with higher post-therapeutic Hsp70 levels showed a significantly better response to RT (p = 0.034) than those with lower levels at T2. In summary, high OPN plasma levels at T1 are indicative for poor OS, whereas elevated post-therapeutic Hsp70 plasma levels together with a drop of Hsp70 between T1 and T2, successfully predict favorable responses to RT. Monitoring the dynamics of Hsp70 in NSCLC patients before and after RT can provide additional predictive information for clinical outcome and therefore might allow a more rapid therapy adaptation.

Composite biomarkers defined by multiparametric immunofluorescence analysis identify ALK-positive adenocarcinoma as a potential target for immunotherapy

ABSTRACTAnaplastic lymphoma kinase (ALK) inhibitors have been successfully developed for non-small cell lung carcinoma (NSCLC) displaying chromosomal rearrangements of the ALK gene, but unfortunately resistance invariably occurs. Blockade of the PD-1-PD-L1/2 inhibitory pathway constitutes a breakthrough for the treatment of NSCLC. Some predictive biomarkers of clinical response to this therapy are starting to emerge, such as PD-L1 expression by tumor/stromal cells and infiltration by CD8+ T cells expressing PD-1. To more effectively integrate all of these potential biomarkers of clinical response to immunotherapy, we have developed a multiparametric immunofluorescence technique with automated immune cell counting to comprehensively analyze the tumor microenvironment of ALK-positive adenocarcinoma (ADC). When analyzed as either a continuous or a dichotomous variable, the mean number of tumor cells expressing PD-L1 (p = 0.012) and the percentage of tumor cells expressing PD-L1 were higher in ALK-positive ADC than in EGFR-mutated ADC or WT (non-EGFR-mutated and non-KRAS-mutated) NSCLC. A very strong correlation between PD-L1 expression on tumor cells and intratumoral infiltration by CD8+ T cells was observed, suggesting that an adaptive mechanism may partly regulate this expression. A higher frequency of tumors combining positive PD-L1 expression and infiltration by intratumoral CD8+ T cells or PD-1+CD8+ T cells was also observed in ALK-positive lung cancer patients compared with EGFR-mutated (p = 0.03) or WT patients (p = 0.012). These results strongly suggest that a subgroup of ALK-positive lung cancer patients may constitute good candidates for anti-PD-1/-PD-L1 therapies.

Biomarkers of MS therapies

As more therapies become available for the treatment of MS, developing predictive biomarkers of treatment response is an unmet need. Potential immunological, pharmacogenomics and proteomic biomarkers of MS therapies will be reviewed. We have recently identified two HLA-class II haplotypes as potential biomarkers of clinical response to glatiramer acetate(GA): HLA DR 15+DQ6+; DR17-DQ2− was associated with a favorable response (71%) where as HLA DR15-DQ6-;DR17+DQ2+ was associated with a poor response. A simplified binary predictor (AUC 0.81-0.85) of response to GA involving an increase in IL-10 or IL-4, and reduction in IL18, caspase 1, or TNF-a at 3–6 months into treatment will be discussed. We have also reported significantly higher IL-17A levels at Month 6 ( p =0.036) in relapsing patients treated with IFNβ-1b for 12 months while BDNF levels were significantly higher at Month 3 ( p =0.028) in relapse-free subjects. Change from baseline in IL-4 levels inversely correlated with disability scores whereas change from baseline in IL-10/IFN-gamma ratio inversely correlated with occurrence of relapses. CXCR3+CD8+ T-cells tended to be higher but declined with treatment in relapse-free compared with relapsing subjects. Promising potential biomarkers of response to Natalizumab and its PML complication will be discussed.

NRAS mutation: A potential biomarker of clinical response to immune-based therapies in metastatic melanoma (MM).

9019 Background: NRAS mutant (mut) MM comprises a distinct, molecularly defined cohort of this disease that appears to have a poor prognosis compared to other genetic subsets. In contrast to BRAF mut MM, there are no effective small molecule inhibitors specifically targeting NRAS. Immune based therapy (IT) has become a mainstay in MM treatment, especially in BRAF wild type (WT) patients (pts). Biomarkers to predict which pts will benefit from IT have not been validated. The goal of this study was to evaluate whether genetic subtype (specifically NRAS) has a role in predicting benefit from IT in BRAF WT MM. Methods: We identified 173 pts from 3 institutions who underwent clinical genotyping (exome or PCR based) for NRAS and BRAF mutation from 1/09 – 8/12 and were treated with IT (defined as IL-2, ipilimumab (ipi), or anti-PD-1 (nivolumab, MK3475)/ PD-L1 (MPDL3280A)). Only BRAF WT pts were included. Primary endpoints were response rate (RR) and clinical benefit rate (CBR), defined as RR + stable disease lasting &gt;24 wks to IT (best response as assessed by treating clinicians in any line of IT). Secondary endpoints were overall survival (OS) and progression-free survival (PFS) from first line IT. Results: Of the 173 pts, 59 had NRAS mut MM compared to 114 WT/WT (no mutation in NRAS or BRAF). Improved clinical outcomes were seen in the NRAS mut compared to WT/WT cohort in terms of RR (32% vs. 18%, p=0.042), and CBR (49% vs. 30%, p=0.012). Improvements in PFS and OS did not reach statistical significance. By specific IT, NRAS mutation predicted benefit compared to WT/WT for anti-PD-1/PD-L1 (RR 78% vs. 19%, p=0.002; CBR 78% vs. 29%, p=0.013, n=30) and ipi (RR 18% vs. 12%, p=0.315; CBR 41% vs. 22%, p=0.018, n=137). No significant differences were observed with IL-2 (RR 33% vs. 28%, p=0.730; CBR 33% vs. 39%, p=0.741, n=33). Conclusions: This study demonstrates that pts with NRAS mut MM achieve increased clinical benefit from IT compared to pts with BRAF/NRAS WT MM. A larger, prospective analysis is necessary to validate and expand on these results, including those with BRAF mut and KIT mut MM. However, our data suggest that NRAS mutation status may be a biomarker of response to IT in MM and that molecularly targeted immunotherapy may be feasible.

Methotrexate polyglutamates as a marker of patient compliance and clinical response in psoriasis: a single-centre prospective study

Methotrexate is activated by the sequential addition of glutamic acid residues to form methotrexate polyglutamates (MTXPG(1-5)). MTXPG(1-5) inhibit enzymes of the folate-purine-pyrimidine pathways, and longer-chain MTXPG(3-5) species are more active. To determine the pattern of erythrocyte MTXPG(1-5) in patients initiated on oral methotrexate for psoriasis, and to investigate the potential utility of MTXPGs as markers of compliance and/or clinical response. This was a single-centre, prospective study of 55 adult patients with chronic plaque psoriasis initiated on weekly oral methotrexate. Erythrocyte MTXPG(1-5) concentrations were measured (at weeks 4, 8, 12, 24 and 52) using high-performance liquid chromatography. Methotrexate responders achieved ≥ 50% improvement in Psoriasis Area and Severity Index or physician's global score of 'clear'/'nearly clear' at 24 weeks. MTXPG levels were measured in 14-33 patients at each time point. All MTXPG(1-5) species were detected at week 4 of therapy. Steady state for long-chain MTXPG(3-5) and total MTXPG(1-5) was achieved by week 24. MTXPG(3) emerged as the predominant MTXPG species (from week 12 onwards) and reflected overall polyglutamate status (correlating strongly with MTXPG(2-5) , MTXPG(3-5) and MTXPG(4-5) ; R = 0·76-0·95, P < 1·55 × 10(-5)). Age, renal function and sex were not significant determinants of MTXPG(3) concentration. No significant association was identified between MTXPG and adverse events or responder status. This is the first study to demonstrate the prospective accumulation of MTXPG(1-5) in patients with psoriasis. The detection of MTXPGs early in therapy and the establishment of a steady state with continuous treatment may offer measuring of MTXPG as a test to monitor patient compliance with therapy. Larger studies are required to determine the role of MTXPG as a potential biomarker of clinical response.

Role of Human Nucleoside Transporters in the Uptake and Cytotoxicity of Azacitidine and Decitabine

The nucleoside analogs 5-azacytidine (azacitidine) and 5-aza-2′-deoxycytidine (decitabine) are active against acute myeloid leukemia and myelodysplastic syndromes. Cellular transport across membranes is crucial for uptake of these highly polar hydrophilic molecules. We assessed the ability of azacitidine, decitabine, and, for comparison, gemcitabine, to interact with human nucleoside transporters (hNTs) in Saccharomyces cerevisiae cells (hENT1/2, hCNT1/2/3) or Xenopus laevis oocytes (hENT3/4). All three drugs inhibited hCNT1/3 potently (K i values, 3–26 μM), hENT1/2 and hCNT2 weakly (K i values, 0.5–3.1 mM), and hENT3/4 poorly if at all. Rates of transport of [3H]gemcitabine, [14C]azacitidine, and [3H]decitabine observed in Xenopus oocytes expressing individual recombinant hNTs differed substantially. Cytotoxicity of azacitidine and decitabine was assessed in hNT-expressing or hNT-deficient cultured human cell lines in the absence or presence of transport inhibitors where available. The rank order of cytotoxic sensitivities (IC 50 values, μM) conferred by hNTs were hCNT1 (0.1) > hENT1 (0.3) ≫ hCNT2 (8.3), hENT2 (9.0) for azacitidine and hENT1 (0.3) > hCNT1 (0.8) ⋙ hENT2, hCNT2 (>100) for decitabine. Protection against cytotoxicity was observed for both drugs in the presence of inhibitors of nucleoside transport, thus suggesting the importance of hNTs in manifestation of toxicity. In summary, all seven hNTs transported azacitidine, with hCNT3 showing the highest rates, whereas hENT1 and hENT2 showed modest transport and hCNT1 and hCNT3 poor transport of decitabine. Our results show for the first time that azacitidine and decitabine exhibit different human nucleoside transportability profiles and their cytotoxicities are dependent on the presence of hNTs, which could serve as potential biomarkers of clinical response.

Phase II Study of a HER-2/Neu (HER2) Intracellular Domain (ICD) Vaccine Given Concurrently with Trastuzumab in Patients with Newly Diagnosed Advanced Stage Breast Cancer.

Abstract HER2 is a tumor antigen in breast cancer and several trials have demonstrated that breast cancer patients can be immunized against this protein. We have developed HER2 peptide based vaccines that are aimed at eliciting CD4+ Th1 tumor antigen specific T cell responses. Th1 effectors provide immunologic memory, enhance cross priming which will allow the elaboration of tumor specific CD8+ T cells, and stimulate epitope spreading which we have shown to be a potential biomarker of clinical response. 52 patients will be enrolled with the primary objective to determine relapse free survival after active immunization. Eligible patients are newly diagnosed with Stage III (B or C) or Stage IV breast cancer and begin vaccination within 6 months of starting maintenance trastuzumab. This interim report will present data on the first 25 patients enrolled; 21 stage IV and 4 locally advanced patients. The vaccine is well tolerated with all adverse events (AE) being Grade I or 2. The most common AE is injection site reaction. Moreover, the combination of HER2 vaccination with trastuzumab did not result in additive cardiac toxicity in these patients. Immune responses were evaluated by IFN-gamma ELISPOT. To date, 88% of patients immunized developed significant immunity to the components of the ICD vaccine. The majority, 75%, developed robust immunity to the HER2 protein. Our group has recently demonstrated that a broadening of immunity throughout the HER2 protein, to components of the protein that weren't in the vaccine, i.e. epitope spreading, may be associated with improved survival in vaccinated patients. 63% of immunized patients demonstrated evidence of intramolecular epitope spreading. We questioned whether such high frequencies of homing Type 1 T cells might modulate the immunosuppressive tumor microenvironment, so we evaluated whether circulating serum immunosuppressive cytokines were impacted by immunization. TGF-beta is an immunosuppressive cytokine secreted by tumor stroma and regulatory T cells. We found that the levels of serum TGF-beta decreased significantly in the majority of patients after vaccination. We further analyzed the correlation between the change of serum levels of TGF-beta post vaccination and HER2 ICD vaccine-induced T cell responses. We found that the greater the magnitude of the HER2 specific T cell response, as demonstrated by IFN-gamma secretion, the greater the decrease in serum TGF-beta (p=0.0045, r=0.742). The correlation between the increased epitope spreading T cell response and decreased levels of TGF-beta was even more significant (p=0.0003). The median overall survival has not been reached with 100% of patients alive at this time. Relapse free survival data will be presented. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5102.