Putative Biomarkers Of Response Research Articles

Neoadjuvant immunotherapy for dMMR and pMMR colorectal cancers: therapeutic strategies and putative biomarkers of response.

Approximately 15% of locally advanced colorectal cancers (CRC) have DNA mismatch repair deficiency (dMMR), resulting in high microsatellite instability and a high tumour mutational burden. These cancers are frequently sensitive to therapy with immune-checkpoint inhibitors (ICIs) in the metastatic setting. This sensitivity seems to be even more pronounced in locally advanced disease, and organ preservation has become a realistic aim in ongoing clinical trials involving patients with dMMR rectal cancer. By contrast, metastatic CRCs with proficient DNA mismatch repair (pMMR) are generally resistant to ICIs, although a proportion of locally advanced pMMR tumours seem to have a high degree of sensitivity to ICIs. In this Review, we describe the current and emerging clinical evidence supporting the use of neoadjuvant ICIs in patients with dMMR and pMMR CRC, and the potential advantages (based on a biological rationale) of such an approach. We discuss how neoadjuvant 'window-of-opportunity' trials are being leveraged to progress biomarker discovery and we provide an overview of potential predictive biomarkers of response to ICIs, exploring the challenges faced when evaluating such biomarkers in biopsy-derived samples. Lastly, we describe how these discoveries might be used to drive a rational approach to trialling novel immunotherapeutic strategies in patients with pMMR CRC, with the ultimate aim of disease eradication and the generation of long-term immunosurveillance.

Elucidating cellular response to treatment with viral immunotherapies in pediatric high-grade glioma and medulloblastoma

HSV G207, a double-stranded, DNA virus, and the polio:rhinovirus chimera, PVSRIPO, a single positive-strand RNA virus, are viral immunotherapies being used to treat pediatric malignant brain tumors in clinical trials. The purpose of this work is to elucidate general response patterns and putative biomarkers of response. Multiple pediatric high-grade glioma and medulloblastoma cell lines were treated with various multiplicities of infection of G207 or PVSRIPO. There was a significant inverse correlation between expression of one HSV cellular receptor, CD111, and the lethal dose of 50% of cells (LD50) of cells treated with G207 (r = -0.985, P<0.001) but no correlation between PVSRIPO cellular receptor expression (CD155) and LD50. RNA sequencing of control cells and cells treated for 8 and 24 h revealed that there were few shared differentially expressed (DE) genes between cells treated with PVSRIPO and G207: GCLM, LANCL2, and RBM3 were enriched whilst ADAMTS1 and VEGFA were depleted. Likewise, there were few shared DE genes enriched between medulloblastoma and high-grade glioma cell lines treated with G207: GPSM2, CHECK2, SEPTIN2, EIF4G2, GCLM, GDAP1, LANCL2, and PWP1. Treatment with G207 and PVSRIPO appear to cause disparate gene enrichment and depletion suggesting disparate molecular mechanisms in malignant pediatric brain tumors.

Uncovering drug repurposing candidates for head and neck cancers: insights from systematic pharmacogenomics data analysis

Effective treatment options for head and neck squamous cell carcinoma (HNSCC) are currently lacking. We exploited the drug response and genomic data of the 28 HNSCC cell lines, screened with 4,518 compounds, from the PRISM repurposing dataset to uncover repurposing drug candidates for HNSCC. A total of 886 active compounds, comprising of 418 targeted cancer, 404 non-oncology, and 64 chemotherapy compounds were identified for HNSCC. Top classes of mechanism of action amongst targeted cancer compounds included PI3K/AKT/MTOR, EGFR, and HDAC inhibitors. We have shortlisted 36 compounds with enriched killing activities for repurposing in HNSCC. The integrative analysis confirmed that the average expression of EGFR ligands (AREG, EREG, HBEGF, TGFA, and EPGN) is associated with osimertinib sensitivity. Novel putative biomarkers of response including those involved in immune signalling and cell cycle were found to be associated with sensitivity and resistance to MEK inhibitors respectively. We have also developed an RShiny webpage facilitating interactive visualization to fuel further hypothesis generation for drug repurposing in HNSCC. Our study provides a rich reference database of HNSCC drug sensitivity profiles, affording an opportunity to explore potential biomarkers of response in prioritized drug candidates. Our approach could also reveal insights for drug repurposing in other cancers.

HGG-24. PRECLINICAL EFFICACY OF THE BRAIN PENETRANT CYCLIN-DEPENDENT KINASE INHIBITOR ZOTIRACICLIB IN PEDIATRIC DIFFUSE MIDLINE GLIOMAS

Children diagnosed with diffuse midline gliomas (DMG), including diffuse intrinsic pontine glioma (DIPG), have extremely poor outcomes with a median overall survival of 9–12 months from initial diagnosis. Standard-of-care is limited to focal radiation therapy, given the paucity of effective targeted therapies for DMG. To identify effective drugs for treatment of children diagnosed with DMG, we investigated the brain-penetrating multi cyclin-dependent kinase inhibitor Zotiraciclib (ZTR/TG02). ZTR has demonstrated encouraging response rates and a benign safety profile in phase 1 trials of adults with high-grade glioma. It is thought to achieve its anti-cancer activity mainly by transcription disruption, a previously described vulnerability of DMGs, by inhibiting multiple cyclin-dependent kinases 9 and 7 (CDK9, 7). We found that ZTR robustly reduces viability of different patient derived DMG cells in a dose-dependent manner, with a median IC50 of 201 nM across eight tested cell lines (range 11–1258 nM, 72 hrs). Consistently, we observed loss of RNA polymerase II phosphorylation after 24 hours of treatment, indicating effective CDK9 inhibition at low drug concentrations and after short incubation time. This effect was followed by depletion of short-lived proteins including MYC and the anti-apoptotic factor MCL-1. Putative biomarkers of response and resistance were identified in silico using DepMap data analysis. To assess the safety profile of ZTR, we exposed our zebrafish model to various drug concentrations and found the drug to be safe at IC50 molarity. Ongoing in vitro and in vivo studies evaluating the efficacy of ZTR in combination with promising combination therapies for more effective treatment of children with DMG are also underway.

Putative biomarkers of response to anti-PD-1 therapy in metastatic castration-resistant prostate cancer (mCRPC).

155 Background: Metastatic castration resistant prostate cancer (mCRPC) is a heterogeneous disease in which molecular stratification is needed to improve clinical outcomes. Targeting programmed cell death protein 1 (PD-1) elicits durable antitumor responses in multiple cancer types. Putative biomarkers predictive of response to anti-PD-1 therapies include programmed death-ligand-1 (PD-L1), tumour mutational burden (TMB), T cell-inflamed 18-gene expression profile (GEP), and DNA repair defects. Objective: To study potential biomarkers of response in mCRPC to anti-PD-1 therapy (PD-L1, GEP, mismatch repair (MMR) protein), other biomarkers of interest including BRCA2, PALB2, CDK12, PTEN, TP53, SOX2, and to determine association with clinical outcomes. Methods: The study population included 100 men with mCRPC treated at the Royal Marsden Hospital with available fresh mCRPC biopsy tissue. All men had received at least one line of therapy with a next generation hormonal agent and one line of taxane-based chemotherapy. Clinical characteristics and outcomes were extracted from medical records. mCRPC biopsies were assayed by whole exome sequencing (WES), targeted next generation sequencing (NGS), RNA sequencing (RNAseq), GEP score (Nanostring), PD-L1 immunohistochemistry (IHC; DAKO 22C3 assay), SOX2 IHC, ATM IHC. Correlations among these biomarkers and clinical outcomes were assessed. Results: The median age of patients was 68.0 years; 46/84 (54.8%) had de novo metastatic disease at diagnosis and 24/100 (24.0%) patients had visceral disease. Median follow-up from mCRPC biopsy was 56.2 months. The prevalence of loss of protein expression by IHC and/or pathogenic mutation by NGS of MMR was 7/100 (7%). Loss of PTEN and ATM by IHC was 29/100 (29%) and 13/100 (13%) respectively. SOX2 expression (defined as expression in &gt;5% of cells) was 27/100 (27%). The prevalence of TP53 mutation was 25/100 (25%); deleterious alterations of BRCA2 was 9/100 (9%), CDK12 3/100 (3%) and PALB2 1/100 (1%). PDL1 and GEP results were available for 70 and 93 samples respectively. PD-L1 was expressed (combined positive score ≥1) in 24 (33%) mCRPC biopsies; 23 (26%) had high GEP scores (&gt; -0.318). PD-L1 and GEP expression were positively correlated (Phi 0.63). No other biomarkers showed strong correlations. Of 5 samples with MMR loss for which PD-L1 was available, 1 (20%) had PD-L1 ≥1; one of the CDK12 samples had PD-L1 ≥1 (33%). 0/6 BRCA2 mutated biopsies expressed PD-L1 (0%). Based on univariate analysis, PD-L1 expression [HR: 1.75 (1.00;3.06), p=0.045], high GEP score [HR: 2.00 (1.18;3.39), p=0.0083] and SOX2 expression [HR: 1.81 (1.12;2.94), p=0.015] were associated with worse overall survival (OS). No other biomarkers associated with OS. Conclusions: PD-L1 IHC expression was detected in 33% of mCRPC patients and associated with high GEP score. Higher PD-L1, GEP, and SOX2 expression were associated with poor prognosis.

Abstract LB-075: CDK12-mutated lethal prostate cancers: How hot are these tumors

Abstract Background: Genomic analyses describing the molecular landscape of primary prostate cancer (PCa) and metastatic castration-resistant prostate cancer (mCRPC) have identified recurrent CDK12 alterations in 2-4% of primary PCa and 4-11% of mCRPC. These aberrations, as well as high lymphocyte tumor infiltration, could be clinically relevant as putative biomarkers of response to immunotherapies. However, there are few studies looking into whether CDK12 biallelic mutant cases are immune ‘hot'. Here, we describe a cohort of patients with CDK12 aberration and studied the tumor infiltration of those cancers. Methods: Patients with mCRPC and available diagnostic archival and/or CRPC tumor samples were evaluated. Mutation analysis involved custom designed targeted sequencing on MiSeq sequencer. Loss of heterozygosity (LOH) was assessed for the CDK12 cases from exome sequencing using ASCAT, which include single copy lost or copy neutral LOH. Tumor infiltrating lymphocytes were assessed initially with CD3 IHC, using a deep learning-based AI analysis approach. We then subtyped the TILs using a multiplex IF approach, classifying lymphocytes using CD4, CD8 and FOXP3 positivity. Results: Overall 913 samples (between Feb/15 and Oct/19) were sequenced by targeted NGS, 42 patients presented with pathogenic alterations in CDK12 (4.6%), 27 had bi-allelic alterations, 14 mono-allelic, 1 likely biallelic. In these cases, we identified 39 frameshift alterations, 10 missense mutations mainly involving the kinase domain; with 5 having concomitant LOH. One case showed deep deletion of CDK12 and five presented with additional aberrations in other DNA repair related genes. CDK12 biallelic alterations were present in all 3 cases with both diagnostic and matched mCRPC samples available. We studied T cell infiltration in 100 (23 CDK12 biallelic alterations, 7 monoallelic, 70 controls) samples selected from within the initial targeted NGS cohort. Median intratumoral CD3+ cell density was significantly higher in CDK12 biallelic loss samples compared to matched controls in diagnostic biopsies (271.4 vs 104.683 cells/mm2, p=0.026). A similar trend was seen in mCRPC samples (142.130 vs 51.75 cells/mm2, p=0.36). Intratumoral CD4+ cell infiltration was again significantly higher in CDK12 biallelic loss samples compared to controls (98.864 vs 6.188 cells/mm2, p=0.014). Conclusions: In our analysis we show that a proportion of mCRPC patients harbor defects in CDK12, these are often alterations involving both alleles, likely present from the time of diagnosis. A majority of these CDK12 altered cancers has high CD3 infiltration compared to controls. We envision that these CDK12 aberrant ‘hot' tumors could represent a subset of mCRPC likely to respond to immune-checkpoint inhibition. Citation Format: Bora Gurel, Pasquale Rescigno, Wei Yuan, Rita Pereira, Mateus Crespo, Mattia Rediti, Ines Figueiredo, Maialen Barrero, Diletta Bianchini, Maria D. Fenor de la Maza, Khobe Chandran, Juliet Carmichael, Alec Paschalis, Adam Sharp, George Seed, Ruth Riisnaes, Claudia Bertan, Suzanne Carreira, Johann S. De Bono. CDK12-mutated lethal prostate cancers: How hot are these tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-075.

Immunofocus-PD-L1 high throughput quantitative next generation sequencing assay.

e13521 Background: Immunohistochemical (IHC) assays are presently used as the gold standard predictive tests for immunotherapy but are compromised due to a number of potential variables. Comparative studies have demonstrated differing levels of PD-L1 staining between assays which appears independent of the antibody binding epitope. Secondly, inter-reader reliability even between expert pathologists is problematic particularly for assessment of PD-L1 positive immune cell populations. Methods: To improve predictive testing for anti PD-L1/PD1 immunotherapies we have developed and validated a Next Generation Sequencing Platform, Immunofocus, able to perform high-throughput quantitative PD-L1 gene expression levels in routine diagnostic PWET biopsies. We applied Immunofocus to a cohort of 130 NSCLCs and compared PD-L1 gene expression levels with PD-L1 IHC scores generated using the VENTANA PD-L1 (SP142) Assay. The PD-L1 IHC assessment was carried out double blinded by an independent laboratory. PD-L1 IHC scores were calculated using an algorithm combining tumour proportion score (TPS) with a PD-L1 positive immune cell (IC) score and immune cell area. Results: An exceptionally high degree of correlation was observed between the NGS PD-L1 levels with the combined PD-L1 IHC scores (P &lt; 0.001). Therapeutic cut points for NGS PD-L1 levels were identified corresponding to PD-L1 IHC defined clinical cut points. Notably, ~20% of patients with negative PD-L1 IHC scores showed high NGS PD-L1 expression levels. We hypothesize that these cases represent false negatives and identify a cohort of patients who have shown significant response rates to anti-PD-L1/PD-directed immunotherapies. Conclusions: The Immunofocus NGS PD-L1 assay has potential to greatly improve patient selection for immunotherapy by removing the IHC assay variables and inter-reader variability which compromise current PD-L1 IHC tests while also providing standardized high throughput in the clinical setting. Immunofocus is able to integrate gene expression with somatic mutation analysis allowing capture of networks regulating the immune-checkpoint including for example adaptive and innate resistance pathways, JAK1/2 pathways, differential MHC expression, TEFF gene signature, neoantigen surrogates such as DDR defects and TMB. The integration of NGS PD-L1 expression with other putative biomarkers of response is presently ongoing to further improve prediction of response.

Proteomic phenotyping of metastatic melanoma reveals putative signatures of MEK inhibitor response and prognosis

BackgroundGenotyping of melanomas is used to identify patients for treatment with BRAF and MEK inhibitors, but clinical responses are highly variable. This study investigated the utility of protein expression phenotyping to provide an integrated assessment of gene expression programs in BRAF/NRAS melanoma which would be useful for prognosis and may predict response to MEK inhibition.MethodsMass spectrometry profiling of early passage cell lines established from Stage III cutaneous melanomas was conducted. Basal protein expression was correlated with in vitro response to the MEK inhibitor, selumetinib. Protein expression in a cohort of 32 drug naïve BRAF/NRAS metastatic melanoma specimens was examined. The prognostic utility of a subset of these proteins and mRNA transcripts from a separate cohort was determined.ResultsUnsupervised analysis of basal cell line protein abundances delineated response to selumetinib, but BRAF/NRAS genotype did not. Resistance was associated with functions including cell motility, cell adhesion and cytoskeletal organization. Several of these response biomarkers were observed in lymph node biospecimens and correlated with melanoma-specific survival. Loss of ICAM-1 protein and mRNA expression was a strong prognosticator of diminished survival in BRAF/NRAS mutant melanoma.ConclusionsThese results demonstrate the utility of proteomic phenotyping to identify both putative biomarkers of response to MEK inhibition and prognostication associated with metastatic melanoma.

Abstract B158: Exploiting IGF1R pathway activation as a therapeutic strategy for E-cadherin deficient breast cancers

Abstract Insulin-like growth factor I (IGF1) plays an important role in breast cancer initiation and progression due to its regulation of cell proliferation, migration, and invasion. These characteristics make IGF1R an attractive therapeutic target; however, only a subset of patients show beneficial response to IGF1R inhibitors in clinical trials. To identify putative biomarkers of response to IGF1, we performed a proteomic screen in 21 breast cancer cell lines stimulated with IGF1. Using a novel integrated computational and experimental approach, we identified levels of E-cadherin (CDH1), a major component of the adherens junction (AJ), as a repressor of IGF1R signaling. CDH1 is genetically lost in invasive lobular breast cancer (ILC), a subtype of breast cancer that accounts for ~10-15% of total cases. E-cadherin is also downregulated in estrogen receptor (ER)-negative breast cancers that have undergone epithelial to mesenchymal transition. Consistent with the proteomic screen, knockdown of E-cadherin enhanced IGF1-induced activation of IGF1R and downstream Akt and ERK1/2 phosphorylation, and this enhanced cell cycle progression. Disruption of the AJ alone with an E-cadherin monoclonal antibody also enhanced IGF1R signaling. Further, we have shown a direct interaction of IGF1R and E-cadherin using in situ proximity ligation assay indicating a physical regulation of E-cadherin on IGF1R signaling. Supporting the clinical relevance of our observations, we found increased expression of IGF1 ligand and increased IGF1R phosphorylation in E-cadherin deficient ER+ ILC tumors in The Cancer Genome Atlas (TCGA) compared to ER+ invasive ductal carcinomas (IDC). We also discovered a correlation between mRNA expression and the activation of the IGF gene signature in (1) a cohort of ER+ ILC tumors compared to ER+ IDC and (2) a subset of ER-negative tumors within TCGA. We have previously published that IGF1R inhibitors are effective in inhibiting growth of ER-negative breast cancer cell lines and xenograft models in combination with chemotherapy. Thus, we focused our studies on understanding the utility of IGF1R pathway inhibition in ER+ ILC as a therapeutic strategy. Our data indicate that IGF1R and Akt pathway inhibitors are effective in inhibiting growth in ER+ ILC cell lines and ex vivo tumor culture, and synergize with standard-of-care anti-ER targeted therapy. Therefore, we hypothesize that loss of E-cadherin potentiates IGF1R signaling to enhance breast cancer progression and that loss of E-cadherin expression in ILC and ER-negative tumors may highlight those susceptible to IGF1R inhibition. Citation Format: Alison M. Nagle, Cemal Erdem, Kevin Levine, Christos Sotiriou, Otto Metzger, D Lans Taylor, Timothy Lezon, Steffi Oesterreich, Adrian V. Lee. Exploiting IGF1R pathway activation as a therapeutic strategy for E-cadherin deficient breast cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B158.

Putative Biomarkers of Response to Treatment in Breast Cancer Patients: A Pilot Assay

ABSTRACTIdentifying tumor biomarkers associated with clinical behavior in breast cancer patients may allow higher accuracy in the selection of treatment. Different types of cells were determined in the primary tumors of stage I, II, and III of breast cancer patients, who were assigned to one of the two groups: (1) disease-free or (2) relapsed/progressed, at 5 years after primary treatment. We studied 32 tumor samples. CD4+ lymphocytes and CD44+CD24−/low cells (cancer stem cells) showed a significant association with clinical outcome at 5 years of primary treatment, while CD8+, Foxp3+, CD34+, and myeloid-derived suppressor cells did not show any association. Coincident with the results of individual analysis, we identified CD4+ cells and CD44+CD24−/low cells as good predictors of long-term clinical outcome in a logistic regression.

A comparative proteomic approach for identification of putative biomarkers of neoadjuvant chemotherapy resistance in breast cancer.

e22123 Background: Neoadjuvant chemotherapy is a standard treatment for locally advanced breast cancer however chemoresistance can be a major obstacle in ER+ cancers. Using comparative proteomic approaches (antibody microarray/AbMA and 2D-PAGE with MALDI-TOF/TOF MS) to investigate a pilot series of breast cancer samples we recently identified 14-3-3 theta/tau, tBID and BcL-XL as putative biomarkers of response to neoadjuvant chemotherapy (Hodgkinson et al. J Prot 2012, 75:1276-1283 and 75:2745-2752). Here we aimed to analyse further samples using the AbMA approach and to re-analyse the combined data. Methods: Samples from chemoresistant and chemosensitive breast cancers were selected following anthracycline-taxane chemotherapy and 4 experiments were performed using ductal ER+ tumours. Differential protein expression was compared between chemoresistant and chemosensitive samples using the Panorama XPRESS Profiler725 AbMA kit. The combined data from 9 AbMA assays and 3 2D-PAGE/MS experiments was then analysed using Ingenuity Pathway Analysis (IPA; Ingenuity Systems). Results: 89 differentially expressed proteins (DEPs) were seen in the 4 further AbMA experiments. Sixteen proteins (DR4, E2F6, ILK, MeCP2, MyD88, PI19INK4d, PRMT2, ID, FANCD2, FAKpTyr577, Pinin, Zyxin, 14-3-3 theta/tau, BcL-xL, Pancytokeratin and RIP) were found in at least 2/4 experiments. In the combined dataset (12 experiments from 2 proteomic platforms), 8 DEPs were seen in at least 3 experiments. These were 14-3-3 theta, 14-3-3 epsilon, 14-3-3 gamma, Bcl-xl, Bid, Phosphokinase B, Vimentin and FAK. 121 DEPs from the combined data were analysed using IPA; 12 DEPs were mapped onto the PI3K/AKT pathway. Conclusions: We have identified at least 8 proteins which could play a role in chemoresistance and clinical validation of these putative biomarkers is currently underway.

A Cancer Research UK two-stage multicenter phase II study of imatinib in the treatment of patients with c-kit positive metastatic uveal melanoma (ITEM).

8523 Background: The median overall survival (OS) for metastatic uveal melanoma is less than 6 months with a median progression free survival(PFS) of 3 months. No systemic or regional therapy has shown a survival advantage over best supportive care. Despite pre-clinical evidence suggesting anti-tumour activity for the KIT tyrosine kinase inhibitor imatinib, there are several negative phase II trials in unselected patients. Our primary aim was to test the efficacy of imatinib in patients with prospectively-tested c-kit immunopositive metastatic uveal melanoma. Secondary aims included assessment of toxicity and patient recruitment. Methods: A phase II UK multicentre single-arm, two-stage Gehan design recruited 25 evaluable patients receiving imatinib 400mg OD until progression/unacceptable toxicity. Primary efficacy outcome was PFS at 3 months. Secondary outcomes were OS, overall PFS, disease response (RECIST) and toxicity. Prospective sample collection for putative biomarkers was included. Results: After 16.6 months 37 patients were screened, with 25 were registered and included in final efficacy analyses. The sample included PS 0-1 patients with a median age of 63 yrs and a median 9.3 months from diagnosis of metastatic disease. 82% had high LDH levels (&gt;460IU/L), 65% had no previous treatment for metastatic disease and 8% did not have liver involvement by metastasis. Preliminary final results indicate the estimated proportion of patients progression free at 3 months is 0.24 (95% CI, 0.09 to 0.45). Median PFS and OS were 12.0 weeks (95% CI,11.6 to 14.3) and 29.6 weeks (95% CI, 19.3 to 61.0) respectively. Two patients had confirmed PR with response duration of 93 and 112 weeks, respectively, despite the absence of mutations in exons 11, 13, and 17 of the c-KIT gene. Conclusions: The trial successfully recruited to target in this rare disease area failing to convincingly show improved PFS in a selected cohort of c-kit immunopositive patients. Both patients with PR experienced long periods of disease control. Response was not dependent upon the presence of activating mutations in KIT. Further translational studies are ongoing to determine putative biomarkers of response.