Potential Predictive Biomarkers Of Response Research Articles

Immune features are associated with response to neoadjuvant chemo-immunotherapy for muscle-invasive bladder cancer

Neoadjuvant cisplatin-based chemotherapy is standard of care for muscle-invasive bladder cancer (MIBC). Immune checkpoint inhibition (ICI) alone, and ICI in combination with chemotherapy, have demonstrated promising pathologic response (<pT2) in the neoadjuvant setting. In LCCC1520 (NCT02690558), a phase 2 single-arm trial of neoadjuvant chemo-immunotherapy (gemcitabine and cisplatin plus pembrolizumab; NAC-ICI) for MIBC, 22/39 patients responded (pathologic downstaging as primary outcome), as previously described. Here, we report post-hoc correlative analyses. Treatment was associated with changes in tumor mutational profile, immune gene signatures, and RNA subtype switching. Clinical response was associated with an increase in plasma IL-9 from pre-treatment to initiation of cycle 2 of therapy. Tumors harbored diverse predicted antigen landscapes that change across treatment and are associated with APOBEC, tobacco, and other etiologies. Higher pre-treatment tumor PD-L1 and TIGIT RNA expression were associated with complete response. IL-8 signature and Stroma-rich subtype were associated with improved response to NAC-ICI versus neoadjuvant ICI (ABACUS trial, NCT02662309). Plasma IL-9 represents a potential predictive biomarker of NAC-ICI response, while tumor IL-8 signature and stroma-rich subtype represent potential predictive biomarkers of response benefit of NAC-ICI over neoadjuvant ICI. Future efforts must include additional independent biomarker discovery and validation, ultimately to improve the selection of patients for ICI-related treatments.

Abstract PO3-14-12: SIRT3 as a new potential predictive biomarker for response to CDK4/6 inhibition in ER+/HER2- metastatic breast cancer patients

Abstract Background: Several prospective, randomized clinical trials showed that the CDK4/6 inhibitor palbociclib in combination with either letrozole or fulvestrant significantly improves progression-free survival (PFS) in patients with ER+/HER2- metastatic breast cancer (MBC). However, in some cases, there is development of endocrine resistance and ultimate disease progression. Molecular analysis performed in tissue specimens collected in the PALOMA-3 study, comparing fulvestrant and Palbociclib versus fulvestrant placebo, demonstrated that one of the genes associated with an increased clinical benefit is SIRT3 (Sirtuin 3) which is thought to control mitochondrial integrity and metabolism (Cristofanilli M. et al., Lancet Oncol. 2016). This study aimed to evaluate in preclinical models the predictive role of SIRT3 in ER+/HER2- breast tumors. Methods: Using lentiviruses we generated MCF-7 and T47D cells stably expressing either control shRNA (sh ctr) or shRNA targeting SIRT3 (shSIRT3). With these models, we studied cell viability, tumor growth, apoptosis, and autophagy in MCF-7 cells treated with fulvestrant and palbociclib as a single treatment or in combination, these either alone or in combination with shSIRT3 (knockdown). Nude mice were used for our in vivo studies, and sh ctr or shSIRT3 MCF7 cells were injected. Results: We showed in vitro that the response to palbociclib was significantly associated with levels of SIRT3 expressionas high or low in ER+ cells (p&amp;lt; 0.0001). Furthermore, we found that irreversible cell growth inhibition mediates the beneficial effect of palbociclib in SIRT3 high expressing cellscompared to low expression (p&amp;lt; 0.001).Then, we evaluated the mechanistic activity of SIRT3. We showed thatSIRT3-low expression cells induced enhanced sensitivity to palbociclib by targeting the reactive oxygen species (ROS)/autophagy axis:(i) SIRT3 regulated the mitochondrial homeostasis (e.g. glucose, lactate, pyruvate, succinates), (ii) ROS levels where lower in sh crt comparing and shSIRT3 in treated and untreated cells (p&amp;lt; 0.001), (iii) in terms of senescence,a higher level of positive cells for β-gal activity was found (p≤0.05) and (iv) for autophagy, our western blot analysis showed cleaved LC3 proteins. Our in vivo studies showed that SIRT3 regulated treatment response to palbociclib with a significant decrease in tumor weight and tumor volume (p&amp;lt; 0.01) and importantly, after immunohistochemistry analysis, we validated the role of SIRT3 in regulating the proteins involved in the autophagy/senescence balance, was mainly in epithelial cells compared to stromal cells. Conclusion: Our preclinical studies demonstrated that elevated SIRT3 expression levels sensitizes ER+/HER2- breast tumors to palbociclib treatment. This study suggested that SIRT3 expression could represent a potential predictive biomarker in HR+ MBC patients treated with Palbociclib. Citation Format: Maroua Manai, Ghada Sahraoui, Raoudha Doghri, Lorenzo Gerratana, Paolo D’Amico, Youbin Zhang, Jeannine Donahue, Ami Shah, Carolina Reduzzi, Wenan Qiang, Massimo Cristofanilli. SIRT3 as a new potential predictive biomarker for response to CDK4/6 inhibition in ER+/HER2- metastatic breast cancer patients [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-14-12.

Somatostatin receptor 1 (SSTR1) expression as a potential predictive biomarker for response to androgen receptor signaling inhibitor (ARSI) therapy in metastatic castration-resistant prostate cancer (mCRPC).

180 Background: Overcoming ARSI resistance is an unmet need for mCRPC patients. Known resistance mechanisms include genetic alterations augmenting androgen receptor (AR) function (e.g., amplifications of the AR gene and its enhancer and AR mutations) and treatment-emergent evolution from an AR-driven to an AR-indifferent phenotype such as small cell neuroendocrine prostate cancer. Other mechanisms remain unexplored. Methods: To phenotype ARSI-resistant prostate cancer, we compared the RNA-seq profiles of paired metastatic biopsies from 31 mCRPC patients obtained before the initiation of first-line abiraterone or enzalutamide, and upon radiographic progression. The patient samples were obtained from two independent cohorts, the Stand Up To Cancer/Prostate Cancer Foundation (SU2C/PCF) West Coast Prostate Cancer Dream Team (WCDT) and a multi-institutional cohort from the Netherlands sequenced through the Hartwig Medical Foundation. We first evaluated RNA-seq data to identify the most differentially expressed genes and then performed survival analysis in the retrospective WCDT cohort (n=115, including 54 ARSI-naïve and 61 ARSI-experienced tumors) to assess the association between gene expression and overall survival, defined as the time interval from biopsy to death from any cause. Results: In the analysis of paired RNA-seq data from 31 mCRPC patients, SSTR1 was identified as the most significantly altered gene; SSTR1 expression was consistently decreased in post-treatment, ARSI-resistant tumors (log2[fold change]=-2.5, p=1.08×10-16). SSTR1 encodes somatostatin receptor 1, a G-protein coupled receptor that, when activated by the endogenous peptide hormone somatostatin or exogenous somatostatin analogues, mediates anti-proliferative, anti-migratory, and anti-secretory effects on target cells. Retrospective analysis in the 115 WCDT patients demonstrated that low SSTR1 expression was associated with worse overall survival ( p=0.047); ARSI treatment improved survival for patients with SSTR1-high (≥ median) tumors (HR=0.29, p=0.0002) more significantly than those with SSTR1-low (&lt; median) tumors (HR=0.67, p=0.17), with an interaction ( SSTR1 expression * ARSI treatment) towards statistical significance ( p=0.0598). A differential survival benefit of ARSI therapy with respect to SSTR1 expression was observed only among the 61 ARSI-experienced patients. Conclusions: SSTR1 expression is a potential predictive biomarker for response to ARSI in mCRPC, with higher SSTR1 expression predicting better response. This observation could be explained by an anti-tumor effect of SSTR1 signaling. Further work is warranted to prospectively validate these findings and to investigate SSTR1 as an actionable drug target to overcome ARSI resistance.

Genetic Polymorphisms and Tumoral Mutational Profiles over Survival in Advanced Colorectal Cancer Patients: An Exploratory Study.

Colorectal cancer is a common disease, both in Chile and worldwide. The most widely used chemotherapy schemes are based on 5-fluorouracil (5FU) as the foundational drug (FOLFOX, CapeOX). Genetic polymorphisms have emerged as potential predictive biomarkers of response to chemotherapy, but conclusive evidence is lacking. This study aimed to investigate the role of genetic variants associated with 5FU-based chemotherapy on therapeutic response, considering their interaction with oncogene mutations (KRAS, NRAS, PI3KCA, AKT1, BRAF). In a retrospective cohort of 63 patients diagnosed with metastatic colorectal cancer, a multivariate analysis revealed that liver metastases, DPYD, ABCB1, and MTHFR polymorphisms are independent indicators of poor prognosis, irrespective of oncogene mutations. BRAF wild-type status and high-risk drug-metabolism polymorphisms correlated with a poor prognosis in this Chilean cohort. Additionally, findings from the genomics of drug sensitivity (GDSC) project demonstrated that cell lines with wild-type BRAF have higher IC50 values for 5-FU compared to BRAF-mutated cell lines. In conclusion, the genetic polymorphisms DPYDrs1801265, ABCB1rs1045642, and MTHFRrs180113 may serve as useful biomarkers for predicting a poor prognosis in patients undergoing 5-fluorouracil chemotherapy, regardless of oncogene mutations.

Amivantamab plus lazertinib in osimertinib-relapsed EGFR-mutant advanced non-small cell lung cancer: a phase 1 trial

Patients with epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer (NSCLC) often develop resistance to current standard third-generation EGFR tyrosine kinase inhibitors (TKIs); no targeted treatments are approved in the osimertinib-relapsed setting. In this open-label, dose-escalation and dose-expansion phase 1 trial, the potential for improved anti-tumor activity by combining amivantamab, an EGFR-MET bispecific antibody, with lazertinib, a third-generation EGFR TKI, was evaluated in patients with EGFR-mutant NSCLC whose disease progressed on third-generation TKI monotherapy but were chemotherapy naive (CHRYSALIS cohort E). In the dose-escalation phase, the recommended phase 2 combination dose was established; in the dose-expansion phase, the primary endpoints were safety and overall response rate, and key secondary endpoints included progression-free survival and overall survival. The safety profile of amivantamab and lazertinib was generally consistent with previous experience of each agent alone, with 4% experiencing grade ≥3 events; no new safety signals were identified. In an exploratory cohort of 45 patients who were enrolled without biomarker selection, the primary endpoint of investigator-assessed overall response rate was 36% (95% confidence interval, 22–51). The median duration of response was 9.6 months, and the median progression-free survival was 4.9 months. Next-generation sequencing and immunohistochemistry analyses identified high EGFR and/or MET expression as potential predictive biomarkers of response, which will need to be validated with prospective assessment. ClinicalTrials.gov identifier: NCT02609776.

Combined PARP and WEE1 inhibition triggers anti-tumor immune response in BRCA1/2 wildtype triple-negative breast cancer

Novel therapeutic strategies that can effectively combine with immunotherapies are needed in the treatment of triple-negative breast cancer (TNBC). We demonstrate that combined PARP and WEE1 inhibition are synergistic in controlling tumour growth in BRCA1/2 wild-type TNBC preclinical models. The PARP inhibitor (PARPi) olaparib combined with the WEE1 inhibitor (WEE1i) adavosertib triggered increases in anti-tumour immune responses, including STING pathway activation. Combinations with a STING agonist resulted in further improved durable tumour regression and significant improvements in survival outcomes in murine tumour models of BRCA1/2 wild-type TNBC. In addition, we have identified baseline tumour-infiltrating lymphocyte (TIL) levels as a potential predictive biomarker of response to PARPi, WEE1i and immunotherapies in BRCA1/2 wild-type TNBC.

Potential predictive biomarker for response to radiotherapy and CXCL12 inhibition in glioblastoma in the phase I/II GLORIA trial.

2048 Background: Standard of care (SOC) treatment achieves poor clinical outcomes in patients with glioblastoma (GBM), particularly in the absence of MGMT promoter hypermethylation. Preclinical models suggest that GBM recurrence is facilitated by CXCL12-mediated recruitment of bone marrow-derived cells capable of vasculogenesis after radiotherapy (RT). We have recently reported favorable safety and feasibility data of the phase I/II GLORIA trial, which combines RT and the CXCL12-neutralizing L-RNA aptamer olaptesed pegol (NOX-A12) in patients with newly diagnosed, incompletely resected or unresected GBM lacking MGMT promoter hypermethylation (NCT04121455). Here we report on clinical outcomes and their correlation with potential biomarkers. Methods: 10 patients with newly diagnosed incompletely resected (n=8) or biopsied (n=2) GBM with ECOG≤2, age≥18 and without MGMT promoter hypermethylation were enrolled. All patients received standard RT (60 Gy in 30 fractions or 40.05 Gy in 15 fractions) and escalating dose levels of continuous (24/7) i.v. infusions of NOX-A12 for 26 weeks. While the previously reported primary endpoint was safety, secondary efficacy endpoints included progression-free survival (PFS) and overall survival (OS). Biomarker assessment was performed with a six-plex immunofluorescence staining (CODEX) of neuropathologically confirmed tumor areas of pretreatment tissues for CXCL12, CD31 (endothelial cells), GFAP (glioma cells), CD68 (macrophages, microglia), aSMA (pericytes), Ki-67 (proliferating cells) and a nuclear marker. An independent patient cohort treated with SOC (n=15) matched by clinical and histopathological features was used for comparisons of efficacy and biomarker assessment. Results: Median PFS of the GLORIA cohort was 5.7 (range 1.9–8.5) months and the median OS was 12.7 (4.7–18.4) months. Biomarker analyses revealed that a higher frequency of CXCL12 expressing endothelial and glioma cells (EG12 score) significantly correlated with PFS (r=0.87; p=0.005) in patients treated with RT and NOX-A12, but not with SOC (r=-0.10; p=0.724). GLORIA patients with a high EG12 score (median classifier) had a significantly longer PFS than those with lower scores (3.0 vs. 6.0 months; p=0.031) and a trend towards prolonged OS (11.1 vs. 15.8 months; p=0.075). These correlations were not seen in the reference cohort treated with SOC (PFS: 6.0 vs. 4.6 months; p=0.502; OS: 10.0 vs. 9.6 months; p=0.243). Conclusions: We show superior clinical efficacy of RT and NOX-A12 in patients with high frequency of CXCL12 expressing endothelial and glioma cells, suggesting the use of the EG12 score as a novel predictive biomarker for CXCL12-directed therapies in GBM. Clinical trial information: NCT04121455 .

Identification of novel phosphoproteomic biomarkers in patients with advanced hepatocellular carcinoma (HCC).

e15155 Background: HCC is one of the leading causes of cancer deaths worldwide. Despite increasing systemic therapy options for patients with advanced HCC, there are currently no predictive biomarkers to guide therapy selection. Phosphoproteomics has been successfully used to identify accurate predictive biomarkers in other cancers from frozen clinical samples (Dokal et al., 2021 ASCO Annual Meeting). In this study, we test the feasibility of biomarker discovery using phospho- and whole-proteomics analyses on formalin fixed and paraffin embedded (FFPE) tru-cut liver biopsies from patients with HCC. Methods: FFPE tru-cut biopsies (n = 29) were obtained from separate cohorts of 15 HCC patients [tumor (T)] and 14 patients with chronic liver disease with varying levels of cirrhosis, fibrosis or steatosis but without cancer [non-tumor (NT)]. T samples were from advanced HCC patients with varied aetiology (chronic viral hepatitis, alcohol or NAFLD). Proteins were extracted from 10 x 10µm sections; crosslinks reversed before digestion to peptides; and multiple clean-up/enrichment steps before analysis. Peptides were quantified by mass spectrometry and performance assessed using multi-variate and enrichment analyses. Results: Overall, 4978 phosphopeptides and 3721 proteins were quantified across all samples. Multi-variate analyses based on the relative expression of phosphopeptides or proteins showed a clear separation between T and NT samples, as well as phenotype-distinct separation (i.e according to differentiation) within the T group. Subsequent testing also verified that the method could reproducibly measure these HCC-differentiating phosphorylation sites (and proteins). Proteins associated with HCC were regulated as expected in T vs NT, e.g. ASS1 (log2-fold change (FC) = -1.75, p &lt; 0.001), BDH2 (log2-FC = -1.97, p &lt; 0.001). Known drug targets were found to be significantly upregulated in poorly-differentiated HCC, e.g. PARP1 (log2-FC = 1.7, p &lt; 0.001) and AKT1 (log2-FC = 1.5, p &lt; 0.001), as well as previously described phosphorylation sites, e.g. p-CDK1 T14 (log2-FC = 5.95, p = 0.007) and p-MCM2 S139 (log2-FC = 2.98, p = 0.046). Kinase substrate enrichment analysis (Casado et al., Science Signaling 2013) showed increased activity for several kinases in T vs NT, including A-RAF (z-score = 3.2, p &lt; 0.001), MAPK3 (z-score = 2.1, p = 0.022) and TNK2 (z-score = 2.1, p = 0.003). Conclusions: We successfully adapted (phospho-)proteomics to FFPE tru-cut biopsy specimens, enabling reproducible identification of novel and known (phospho-)proteins that distinguished differentiation status. A prospective study utilising this phosphoproteomic analysis pipeline is now underway, recruiting patients with advanced HCC receiving first line standard of care therapy with immune checkpoint inhibitor combinations or tyrosine kinase inhibitors to identify potential predictive biomarkers of response.

Abstract P5-02-14: Identification of mechanisms of acquired resistance to ribociclib plus endocrine therapy using baseline and end-of-treatment circulating tumor DNA samples in the MONALEESA-2, -3, and -7 trials

Abstract Background: Genetic alterations that contribute to resistance to therapy may be acquired during treatment (tx) for hormone receptor−positive/human epidermal growth factor receptor−negative (HR+/HER2−) advanced breast cancer (ABC). A previous pooled analysis of circulating tumor DNA (ctDNA) in MONALEESA (ML)-2, -3, and -7 identified potential predictive biomarkers for response and resistance to ribociclib (RIB) at baseline (BL). Here, we describe an analysis of paired BL and end of treatment (EOT) samples from ML-2, -3, and -7 to identify acquired mechanisms that may impact resistance to RIB + endocrine therapy (ET) vs placebo (PBO) + ET. Methods: ML-2 (NCT01958021), ML-3 (NCT02422615), and ML-7 (NCT02278120) evaluated efficacy and safety of RIB + ET vs PBO + ET in pre- and postmenopausal patients (pts) with HR+/HER2− ABC treated in first-line (1L) and second-line (2L) settings. Plasma samples were collected at cycle 1 day 1 (C1D1; prior to first therapy exposure) and at EOT (± 28 days of recorded progression). ctDNA was sequenced using a targeted next-generation sequencing panel of ≈550 genes. Genes with an alteration frequency of &amp;gt;5% at EOT, regardless of their frequency at BL, were included. Tumor mutational burden (TMB) was assessed by tx arm; a TMB cutoff of 10 mutations/MB was used to categorize pts as TMB high vs low. To assess differences in the presence of alterations, a McNemar test was performed on paired samples and adjusted (adj) for multiple testing using the false discovery rate (FDR). A Bayesian mixed effects model was used to account for ctDNA fraction and trial and to test for tx-specific resistance by including a tx × visit interaction term. Results: A total of 905 paired samples from ML-2, -3, and -7 were included in this analysis, 441 and 464 samples from pts treated with RIB + ET and PBO + ET, respectively. Overall, 17 genes had an alteration frequency of &amp;gt;5% at EOT. The ctDNA fraction was higher at EOT vs C1D1 in both the RIB (P=.037) and PBO (P=.033) arms. The frequency of alterations in RB1 (10.4% vs 2.0%), ATM (11.3% vs 8.4%), FAT1 (4.8% vs 3.0%), and FAT3 (5.0% vs 2.5%) was higher at EOT vs C1D1 in the RIB arm (FDR-adj P&amp;lt;.10). Alterations in ESR1 were also higher at EOT vs C1D1 in both the RIB (26.3% vs 9.1%) and PBO arms (28.9% vs 5.4%) (FDR-adj P&amp;lt;.0001). Conversely, alterations in GATA3 were higher at EOT in the PBO arm (FDR-adj P=.11). These results were consistent after adjusting for ctDNA fraction. The most common ESR1 mutations were D538G, Y537S/N/C/D, E380Q, and L536H/P/R. Tx × visit interaction effects were observed for RB1 in the RIB arm and GATA3 in the PBO arm, suggesting tx-specific resistance. A tx × visit interaction for ESR1 was also observed, suggesting a larger relative increase in ESR1 mutations with PBO vs RIB. The percentage of pts with high TMB (&amp;gt;10) at EOT increased from 1.1% to 5.7% in the RIB arm and from 1.7% to 3% in the PBO arm. After accounting for ctDNA fraction and trial, a larger numerical increase in TMB was observed for RIB (odds ratio [OR], 9.0; 95% CI, 2.9-32.7) vs PBO (OR, 2.1; 95% CI, 0.7-6.5); however, the model did not support a differential tx effect. Conclusions: This comprehensive analysis of pooled samples from ML-2, -3, and -7 identified acquired gene alterations in pts with HR+/HER2− ABC treated with 1L or 2L RIB + ET or PBO + ET. The frequency of several genes known to contribute to resistance (ESR1, RB1, ATM, FAT1, and FAT3) was higher at EOT vs C1D1 in pts treated with RIB + ET, while ESR1 and GATA3 alterations were higher at EOT vs C1D1 in pts treated with PBO + ET. This paired dataset of BL and EOT samples from pts with HR+/HER2− ABC treated with a CDK4/6 inhibitor and ET is the largest to date and could be used to validate and confirm acquired resistance mechanisms with low alteration frequency. Citation Format: Fabrice Andre, Nadia Solovieff, Faye Su, Aditya Bardia, Patrick Neven, Yoon-Sim Yap, Debu Tripathy, Yen-Shen Lu, Dennis Slamon, Stephen K. Chia, Mukta Joshi, Arunava Chakravartty, Agnes Lteif, Tetiana Taran, Carlos Arteaga. Identification of mechanisms of acquired resistance to ribociclib plus endocrine therapy using baseline and end-of-treatment circulating tumor DNA samples in the MONALEESA-2, -3, and -7 trials [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-14.

A pilot trial evaluating the rapid sequencing of approved therapies in patients (pts) with advanced clear cell renal cell carcinoma (ccRCC).

TPS751 Background: Five doublet combinations incorporating immune checkpoint inhibitor (ICI) with or without tyrosine kinase inhibitor (TKI) are now approved for the first line treatment (tx) of advanced ccRCC. In COSMIC-313, the triplet combination with cabozantinib (CABO)-ipilimumab (IPI)-nivolumab (NIVO) demonstrated promising efficacy compared to IPI-NIVO in pts with intermediate/poor risk disease, but definitive overall survival (OS) benefit has yet to be shown, and is associated with higher incidence of adverse events. Currently, the optimal first /subsequent lines of therapies remain to be defined, and predictive biomarker to guide optimal tx approach is urgently needed. Pre-clinical studies allude to the ability of TKIs, such as CABO, to promote an immune-permissive environment and thus enhance ICI response. This open-label, single-arm, investigator-initiated pilot trial will evaluate the rapid sequencing from CABO to IPI-NIVO with the hypothesis of enhanced efficacy when compared to IPI-NIVO doublet, and potentially lower toxicity when compared to the concurrent triplet combination. Correlative studies, before and after administration of each systemic therapy, will be performed with the goal of identifying potential predictive biomarkers of response and resistance. Methods: Advanced, ccRCC pts will be treated with CABO 60mg PO once daily (QD) for 12 weeks (W) followed by IPI 1mg/kg + NIVO 3mg/kg IV every (Q)3W for 4 cycles. Subsequent therapy is dependent on response, and can be NIVO 480mg IV Q4W (CR/PR/SD), lenvatinib 18mg + everolimus 5mg PO QD (PD on both prior regimens), or re-initiation of CABO 60mg PO QD (CR/PR/SD at CABO but PD on IPI-NIVO). Key inclusion criteria are histologically confirmed, metastatic or unresectable RCC with predominantly clear cell component (sarcomatoid differentiation &lt;50%), tx naïve in the advanced disease setting, and measurable disease per RECIST 1.1 criteria. IMDC or MSKCC favorable, intermediate, and poor risk groups are included. Pts with active CNS disease or contraindications to CABO, IPI, and/or NIVO are excluded. Tumor biopsies and blood/urine samples will be collected for correlative studies. The primary endpoint is efficacy of the sequential approach, based on overall response rate (ORR) as assessed by investigator per RECIST 1.1. The ORR will be estimated by a two-sided 95% confidence interval. The trial plan to enroll 20 pts, which will allow us to estimate a range of plausible ORRs from 0.42 to 0.62 with a precision of ±0.23. Additional efficacy endpoints (ORR per modified RECIST for immune response, duration of response, progression free survival, OS), safety, pt reported outcomes, correlative biomarkers and their association with clinical outcome as determined by tumor immune/genomic profiling will also be evaluated. The trial is currently enrolling pts. Clinical trial information: NCT05188118 .

Clinical efficacy and biomarker analysis of pucotenlimab (HX008) in patients with previously treated advanced mismatch repair-deficient or microsatellite instability-high solid tumors: A single-arm, multicenter, phase 2 study.

799 Background: Pucotenlimab is a humanized IgG4 anti-PD-1 monoclonal antibody with an engineered Fc domain. We aim to assess the efficacy and safety of pucotenlimab, and potential predictive biomarkers of response in patients with previously treated advanced microsatellite instability-high (MSI-H) or mismatch repair-deficient (dMMR) solid tumors. Methods: In this single-arm, multi-center, phase 2 study, eligible patients had locally advanced or metastatic, centrally confirmed MSI-H or dMMR solid tumors treated with at least one prior systemic therapy. Patients received pucotenlimab 200mg every 3 weeks until disease progression or unacceptable toxicity. The primary endpoint was overall response rate (ORR) assessed by the independent review committee (IRC) per Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. Besides, patients’ PD-L1 expression, blood neutrophil to lymphocyte ratio (NLR), germline HLA-I genotyping, loss of heterozygosity (LOH) of HLA-I genes, tumor mutational burden (TMB), and genetic alterations in the tumor revealed by next-generation sequencing were investigated to identify potential predictors of efficacy. Results: 100 patients with MSI-H or dMMR solid tumors were enrolled. Colorectal cancer (n=71) and gastric cancer (n=10) were the most common cancer types. The median follow-up duration was 22.5 months (range: 0.3-37.4) at data cutoff (December 4, 2021). The ORR was 49.0% (95% CI 38.86%-59.20%), and the 12-month progression-free survival (PFS) rate was 56.1% (95% CI 45.63%-65.24%) based on IRC’s assessment. Both the median PFS and median duration of response were not reached. Pucotenlimab was well tolerated. Grade ≥3 treatment-related adverse events (TRAEs) were reported in 18 patients, most commonly anemia, leukocytopenia, and hypertension. For the biomarker analysis, patients with NLR &lt;4 at the second treatment cycle, TMB≥32.5 muts/Mb (the lower 35th TMB-percentile), or mutations in the KMT2D gene showed significantly higher ORR. Conclusions: Pucotenlimab demonstrated durable antitumor activity and manageable safety for previously treated patients with advanced MSI-H/dMMR solid tumors. KMT2D gene mutation, along with NLR and TMB, warrants further investigation as predictive biomarkers. Clinical trial information: NCT03704246 .

Targeting ATR in Cancer Medicine.

As a key component of the DNA Damage Response, the Ataxia telangiectasia and Rad3-related (ATR) protein is a promising druggable target that is currently widely evaluated in phase I-II-III clinical trials as monotherapy and in combinations with other rational antitumor agents, including immunotherapy, DNA repair inhibitors, chemo- and radiotherapy. Ongoing clinical studies for this drug class must address the optimization of the therapeutic window to limit overlapping toxicities and refine the target population that will most likely benefit from ATR inhibition. With advances in the development of personalized treatment strategies for patients with advanced solid tumors, many ongoing ATR inhibitor trials have been recruiting patients based on their germline and somatic molecular alterations, rather than relying solely on specific tumor subtypes. Although a spectrum of molecular alterations have already been identified as potential predictive biomarkers of response that may sensitize to ATR inhibition, these biomarkers must be analytically validated and feasible to measure robustly to allow for successful integration into the clinic. While several ATR inhibitors in development are poised to address a clinically unmet need, no ATR inhibitor has yet received FDA-approval. This chapter details the underlying rationale for targeting ATR and summarizes the current preclinical and clinical landscape of ATR inhibitors currently in evaluation, as their regulatory approval potentially lies close in sight.

Identification of HMGA2 as a predictive biomarker of response to bintrafusp alfa in a phase 1 trial in patients with advanced triple-negative breast cancer.

We report the clinical activity, safety, and identification of a predictive biomarker for bintrafusp alfa, a first-in-class bifunctional fusion protein composed of the extracellular domain of TGFβRII (a TGF-β "trap") fused to a human IgG1 mAb blocking PD-L1, in patients with advanced triple-negative breast cancer (TNBC). In this expansion cohort of a global phase 1 study, patients with pretreated, advanced TNBC received bintrafusp alfa 1200 mg every 2 weeks intravenously until disease progression, unacceptable toxicity, or withdrawal. The primary objective was confirmed best overall response by RECIST 1.1 assessed per independent review committee (IRC). As of May 15, 2020, a total of 33 patients had received bintrafusp alfa, for a median of 6.0 (range, 2.0-48.1) weeks. The objective response rate was 9.1% (95% CI, 1.9%-24.3%) by IRC and investigator assessment. The median progression-free survival per IRC was 1.3 (95% CI, 1.2-1.4) months, and median overall survival was 7.7 (95% CI, 2.1-10.9) months. Twenty-five patients (75.8%) experienced treatment-related adverse events (TRAEs). Grade 3 TRAEs occurred in 5 patients (15.2%); no patients had a grade 4 TRAE. There was 1 treatment-related death (dyspnea, hemolysis, and thrombocytopenia in a patient with extensive disease at trial entry). Responses occurred independently of PD-L1 expression, and tumor RNAseq data identified HMGA2 as a potential biomarker of response. Bintrafusp alfa showed clinical activity and manageable safety in patients with heavily pretreated advanced TNBC. HMGA2 was identified as a potential predictive biomarker of response. NCT02517398.

Validation of a non-oncogene encoded vulnerability to exportin 1 inhibition in pediatric renal tumors

Malignant rhabdoid tumors (MRTs) and Wilms' tumors (WTs) are rare and aggressive renal tumors of infants and young children comprising ∼5% of all pediatric cancers. MRTs are among the most genomically stable cancers, and although WTs are genomically heterogeneous, both generally lack therapeutically targetable genetic mutations. Comparative protein activity analysis of MRTs (n= 68) and WTs (n= 132) across TCGA and TARGET cohorts, using metaVIPER, revealed elevated exportin 1 (XPO1) inferred activity. Invitro studies were performed on a panel of MRT and WT cell lines to evaluate effects on proliferation and cell-cycle progression following treatment with the selective XPO1 inhibitor selinexor. Invivo anti-tumor activity was assessed in patient-derived xenograft (PDX) models of MRTs and WTs. metaVIPER analysis identified markedly aberrant activation of XPO1 in MRTs and WTs compared with other tumor types. All MRT and most WT cell lines demonstrated baseline, aberrant XPO1 activity with invitro sensitivity to selinexor via cell-cycle arrest and induction of apoptosis. Invivo, XPO1 inhibitors significantly abrogated tumor growth in PDX models, inducing effective disease control with sustained treatment. Corroborating human relevance, we present a case report of a child with multiply relapsed WTs with prolonged disease control on selinexor. We report on a novel systems-biology-based comparative framework to identify non-genetically encoded vulnerabilities in genomically quiescent pediatric cancers. These results have provided preclinical rationale for investigation of XPO1 inhibitors in an upcoming investigator-initiated clinical trial of selinexor in children with MRTs and WTs and offer opportunities for exploration of inferred XPO1 activity as a potential predictive biomarker for response. This work was funded by CureSearch for Children's Cancer, Alan B. Slifka Foundation, NIH (U01 CA217858, S10 OD012351, and S10 OD021764), Michael's Miracle Cure, Hyundai Hope on Wheels, Cannonball Kids Cancer, Conquer Cancer the ASCO Foundation, Cycle for Survival, Paulie Strong Foundation, and the Grayson Fund.

The amount of DNA combined with TP53 mutations in liquid biopsy is associated with clinical outcome of renal cancer patients treated with immunotherapy and VEGFR-TKIs

BackgroundDespite the increasing number of treatment options, reliable prognostic/predictive biomarkers are still missing for patients affected by metastatic clear cell renal cell carcinoma (mccRCC).MethodsPatients with mccRCC undergoing standard first line treatment were enrolled. Blood (12 ml) was drawn at treatment baseline and circulating free DNA (cfDNA) was extracted from plasma. Next-generation sequencing (NGS) was performed on cfDNA using the Oncomine Pan-Cancer Cell-Free Assay and clinical outcomes were correlated with liquid biopsy findings.ResultsA total of 48 patients were enrolled, 12 received immunotherapy and 36 received a vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitor (TKI). A cfDNA cut-off of 0.883 ng/μl stratified patients based on progression-free survival (PFS) and overall survival (OS) (p = 0.001 and p = 0.008, respectively). cfDNA amount was also correlated with best response (p = 0.006). Additional cfDNA cut-points divided patients into short, intermediate and long responders, with PFS of 4.87 vs 9.13 vs 23.1 months, respectively (p < 0.001). PFS resulted to be significantly shorter in carriers of mutant TP53 compared to not carriers (p = 0.04). Patients with high cfDNA levels and mutant TP53 have the worst PFS, while patients with low cfDNA amounts and no mutations in TP53 displayed the longest PFS (p = 0.004).ConclusionsThe present study demonstrates that cfDNA and TP53 are potential predictive biomarkers of response in mccRCC to be further explored in larger and/or prospective studies.

What is the status of immunotherapy in thyroid neoplasms?

Immunotherapy has changed the treatment of patients with advanced cancer, with different phase III trials showing durable responses across different histologies. This review focuses on the preclinical and clinical evidence of potential predictive biomarkers of response and efficacy of immunotherapy in thyroid neoplasms. Programmed death-ligand 1 (PD-L1) staining by immunohistochemistry has shown higher expression in anaplastic thyroid cancer (ATC) compared to other subtypes. The tumor mutational burden in thyroid neoplasms is low but seems to be higher in ATC. Immune infiltrates in the tumor microenvironment (TME) differ between the different thyroid neoplasm subtypes. In general, differentiated thyroid cancer (DTC) has a higher number of tumor-associated lymphocytes and regulatory T cells (Tregs), while ATC and medullary thyroid cancer (MTC) display a high density of tumor-associated macrophages (TAMs). Nevertheless, results from clinical trials with immunotherapy as monotherapy or combinations have shown limited efficacy. Further investigation into new strategies aside from anti-cytotoxic T-lymphocyte antigen 4 (CTLA-4)/programmed death 1 (PD-1)/PD-L1 antibodies, validation of predictive biomarkers, and better population selection for clinical trials in thyroid neoplasms is more than needed in the near future.

TRIM26 inhibits hepatitis B virus replication by promoting HBx degradation and TRIM26 genetic polymorphism predicts PegIFNα treatment response of HBeAg‐positive chronic hepatitis B Patients

Hepatitis B virus (HBV) infection is a serious global health burden. TRIM26 has been reported to affect hepatitis C virus replication. To manifest the role of TRIM26 on HBV replication and explore if there are single-nucleotide polymorphisms (SNPs) in TRIM26 associated with response to pegylated interferon-alpha (PegIFNα) treatment in patients with chronic hepatitis B (CHB). We investigated the effect and mechanism of TRIM26 on HBV replication in vitro. The association between SNPs in TRIM26 and PegIFNα treatment response was evaluated in two independent cohorts including 238 and 707 patients with HBeAg-positive CHB. Knockdown of TRIM26 increased, while overexpression of TRIM26 inhibited, HBV replication. Co-immunoprecipitation assays and immunofluorescence showed that TRIM26 interacted and co-localised with HBx. Co-transfection of HBx-HIS and TRIM26-FLAG plasmids in Huh7 cells showed that TRIM26 inhibited the expression of HBx. Furthermore, TRIM26 inhibited HBV replication by mediating HBx ubiquitination degradation, and TRIM26 SPRY domain was responsible for the interaction and degradation of HBx. Besides, IFN increased TRIM26 expression. TRIM26 rs116806878 was associated with response to PegIFNα in two CHB cohorts. Moreover, a polygenic score integrating TRIM26 rs116806878, STAT4 rs7574865 and CFB rs12614 (previously reported to be associated with response to PegIFNα) was related to response to PegIFNα in CHB. TRIM26 inhibits HBV replication; IFN promotes TRIM26 expression. TRIM26 exerts an inhibitory effect on HBx by promoting ubiquitin-mediated degradation of HBx. Furthermore, TRIM26 rs116806878 is a potential predictive biomarker of response to PegIFNα in patients with CHB.

Tumor Mutational Burden Is a Potential Predictive Biomarker for Response to Immune Checkpoint Inhibitors in Patients With Advanced Biliary Tract Cancer.

Patients with advanced biliary tract cancers (BTCs) have a dismal prognosis. This multisite, single-institution study analyzed the efficacy and safety of immune checkpoint inhibitors (ICIs) in patients with advanced BTC. The prospectively maintained institutional database was searched for patients with advanced BTC. Electronic medical records of the patients with advanced BTC treated with an ICI that included programmed death-1 or programmed death-ligand 1 blockers were retrospectively reviewed to obtain data on patient characteristics, tumor characteristics including molecular biomarkers, detailed treatment, response characteristics, survival, and toxicities. The analysis included overall response rate, survival, and correlation between survival and molecular biomarkers. The institutional database query identified 47 patients with advanced BTC who received at least one dose of an ICI; 11 (24%) patients in the first-line setting and the rest of the patients had refractory disease. The median age of the cohort was 62 years, and 51% were female. The overall response rate was 10.6%, with a disease control rate of 53.2%. The median progression-free survival (PFS) and overall survival were 3.6 months and 6.9 months, respectively. Biomarker analysis revealed improved PFS in patients with tumor mutational burden > 5 mutations per megabase (median PFS: 6.4 v 2.2 months; P = .0027). No unexpected adverse events were observed. ICIs are well tolerated and have modest antitumor activity in patients with advanced BTC. The study result supports the exploration of tumor mutational burden as a potential predictive biomarker for response to ICIs in patients with advanced BTC.

Artificial intelligence in digital pathology approach identifies the predictive impact of tertiary lymphoid structures with immune-checkpoints therapy in NSCLC.

9065 Background: The presence of Tertiary Lymphoid Structures (TLS) in multiple cancer types has been recognized as a potential predictive biomarker for response to immune-checkpoint blockade. However, there is no standardized method to quantify their presence. In this context, Artificial Intelligence (AI)-based assessment of histology images may well contribute to improve reproducibility, accuracy and speed of TLS quantification. Methods: We developed an automated workflow for quantification of TLS on digitized H&amp;E slides through A) pixel-level classification of tissue using supervised artificial neural networks model, B) object-level cell classification of candidate TLS regions, C) merging the two approaches for curation and validation of TLS versus non-TLS regions. 433 advanced stage non-small cell lung cancer (NSCLC) patients treated with first or subsequent line of anti-PD-(L)1 single agent at DFCI were included in this study. Results: TLS were detected in 37% (n = 161) of the patients H&amp;E slides, with the highest score of 4.7 TLS per mm2 (interquartile range: Q1 = 0, Q2 = 0, Q3 = 0.03 TLS/mm2). TLS density (per mm2) was significantly higher in surgically resected (n = 246; TLSPOS= 49%) compared to bioptic samples (n = 187; TLSPOS= 21%). No association was observed between TLS and tumor mutational burden (TMB) or PD-L1 protein expression as continuous variables. Among clinically actionable mutations, EGFR (all subtypes) mutated patients (n = 38) had a significantly lower number of TLS compared to patients without EGFR mutations. Patients with ≥ 0.01 TLS/mm2 had a significantly higher objective response rate (32% vs 22%, p = 0.03), a significantly longer median progression-free survival (PFS, 4.8 vs 2.7 months, HR: 0.73, 95% CI: 0.59-0.90, p = 0.004), and a significantly improved median overall survival (OS, 16.5 vs 12.5 months, HR: 0.72, 95% CI: 0.57-0.92, p = 0.008). In multivariable analysis, after adjusting for PD-L1 (≥ vs &lt; 50%), TMB (≥ vs &lt; 10 mu/Mb), sex, age, ECOG score, smoking and line of treatment, TLS/mm2 (≥ vs &lt; 0.01) levels were found to be an independent positive predictive factor for both PFS (HR:0.69, 95% CI: 0.54-0.88, p = 0.003) and OS (HR: 0.70, 95% CI: 0.52-0.93, p = 0.01). Conclusions: These findings suggest that TLS status is an independent predictor of immunotherapy effectiveness in NSCLC, with predictive value similar to that of PD-L1 expression and TMB. This novel AI system has potential for automated identification and quantification of the TLS on digital histopathological slides, and could be utilized in a standard pathology workflow with relative ease. These findings are currently being validated in other solid tumors and cohorts.

Immunotherapy in triple-negative breast cancer: A literature review and new advances.

Triple-negative breast cancer (TNBC) is a highly complex, heterogeneous disease and historically has limited treatment options. It has a high probability of disease recurrence and rapid disease progression despite adequate systemic treatment. Immunotherapy has emerged as an important alternative in the management of this malignancy, showing an impact on progression-free survival and overall survival in selected populations. In this review we focused on immunotherapy and its current relevance in the management of TNBC, including various scenarios (metastatic and early -neoadjuvant, adjuvant-), new advances in this subtype and the research of potential predictive biomarkers of response to treatment.