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Background Anti-Androgen Receptor (AR) therapy holds promise for a subset of AR expressing triple-negative breast cancer (TNBC) patients. However, current AR assays are suboptimal in detecting the dynamic range of AR expression, contributing to its controversial role in TNBC disease prognosis. This study is aimed at evaluating the feasibility of qRT-PCR to sensitively and robustly detect AR mRNA levels for prognostication. Methods mRNA expression profiling was performed on FFPE blocks from a retrospective cohort of 101 TNBC patients using qRT-PCR and compared with AR protein expression by immunohistochemistry . Statistical analyses included Spearman’s rank correlation, Chi-square and Kaplan-Meier analyses. Distant Metastasis Free Survival was used as the end point in survival analysis. Results AR mRNA expression was observed in 34/101 patients (34%) whereas 12/80 cases (15%) were positive by IHC. qRT-PCR could thus detect more AR positive patients as compared to IHC, with 75% (9/12) concordance between the two methods. Co-expression of GATA3 and FOXA1 mRNA was observed in 85 and 88% of AR mRNA positive tumors, respectively. AR mRNA positivity was significantly correlated with age at disease onset ( p  = 0.02), high FOXA1/GATA3 ( p  < 0.05) and distant recurrence. AR mRNA positive patients had poorer DMFS (43%; p  = 0.002). DMFS dropped further to 26% ( p  = 0.006) in AR (+)/high FOXA1/GATA3 patients. AR mRNA expression together with node positivity had the worst DMFS (23%; p  < 0.0001) compared to patients who were either positive for any one of these, or negative for both AR and node status. Low Ki67 mRNA with AR mRNA positivity also had poorer DMFS (39%; p  = 0.001) compared to patients expressing low Ki67 with no AR mRNA expression. Conclusion qRT-PCR was more sensitive and reliable in detecting the dynamic expression levels of AR compared to IHC and this variation could be explained by the higher sensitivity of the former method. High AR mRNA expression was strongly associated with expression of AR protein, high FOXA1/GATA3 mRNA, and with poor prognosis. qRT-PCR was more efficient in detecting the AR positive cases compared to IHC. A distinct signature involving high GATA3/FOXA1, low Ki67, and node positivity in AR mRNA positive tumors correlated with poor prognosis. Thus, AR mRNA screening can serve as an effective prognostic marker along with offering potential targeted therapy options for TNBC.

Dynamic interactions within the tumor micro-environment drive patient response to immune checkpoint inhibitors. Existing preclinical models lack true representation of this complexity. Using a Head and Neck cancer patient derived TruTumor histoculture platform, the response spectrum of 70 patients to anti-PD1 treatment is investigated in this study. With a subset of 55 patient samples, multiple assays to characterize T-cell reinvigoration and tumor cytotoxicity are performed. Based on levels of these two response parameters, patients are stratified into five sub-cohorts, with the best responder and non-responder sub-cohorts falling at extreme ends of the spectrum. The responder sub-cohort exhibits high T-cell reinvigoration, high tumor cytotoxicity with T-cells homing into the tumor upon treatment whereas immune suppression and tumor progression pathways are pre-dominant in the non-responders. Some moderate responders benefit from combination of anti-CTLA4 with anti-PD1, which is evident from better cytotoxic T-cell: T-regulatory cell ratio and enhancement of tumor cytotoxicity. Baseline and on-treatment gene expression signatures from this study stratify responders and non-responders in unrelated clinical datasets. Tumor histocultures have been exploited as tools to predict response to cancer therapy. Here the authors report the development and testing of a tumor histoculture platform to study response to immune checkpoint inhibitors in head and neck squamous cell carcinoma.

Breast and/or ovarian cancer (BOC) are among the most frequently diagnosed forms of hereditary cancers and leading cause of death in India. This emphasizes on the need for a cost-effective method for early detection of these cancers. We sequenced 141 unrelated patients and families with BOC using the TruSight Cancer panel, which includes 13 genes strongly associated with risk of inherited BOC. Multi-gene sequencing was done on the Illumina MiSeq platform. Genetic variations were identified using the Strand NGS software and interpreted using the StrandOmics platform. We were able to detect pathogenic mutations in 51 (36.2%) cases, out of which 19 were novel mutations. When we considered familial breast cancer cases only, the detection rate increased to 52%. When cases were stratified based on age of diagnosis into three categories, ⩽40 years, 40-50 years and >50 years, the detection rates were higher in the first two categories (44.4% and 53.4%, respectively) as compared with the third category, in which it was 26.9%. Our study suggests that next-generation sequencing-based multi-gene panels increase the sensitivity of mutation detection and help in identifying patients with a high risk of developing cancer as compared with sequential tests of individual genes.

BackgroundThe prognosis of ovarian cancer (OvCa) is poor, with a 5-year survival rate in patients of 59.60% (95% CI, 56.06–63.13).1 Personalized treatment regimens could improve treatment outcome and quality of life. We developed the FarcastTM TruTumor Ovarian Cancer histoculture platform to predict response to therapies enabling personalized treatment options for every patient.MethodsFreshly resected OvCa tissue samples (n=10) along with matched blood were collected from consented patients. Tissue explants were generated and distributed into arms and cultured for 72 h. The functional fidelity of immune components was assessed by stimulating these with anti-CD3 (0.01 µg/ml) + Interleukin-2 (IL2, 100 µg/ml), or with Lipopolysaccharides (LPS, 1 µg/ml). Response was characterized through cytokine release assay and flow cytometry (n=3). Tumor cytotoxic response on treatment with Platin (Cisplatin:3.3 µg/ml, or Carboplatin: 37.1 µg/ml) and/or Taxane (Docetaxel:2 µg/ml, or Paclitaxel:2.7 µg/ml), or Nivolumab (132 µg/ml) was evaluated by quantifying the decrease in tumor content and/or increase in the tumor expression of cleaved caspase 3 (CC3).ResultsAt baseline OvCa samples (n=4) exhibited a higher proportion of lymphocytes (79.88±8.05%) than myeloid sub-population, with a high Programmed cell death protein1, PD1+ T-cell population (43.13±11.97%). Baseline immune content in OvCa was lower in comparison to head and neck cancer but similar to renal cell cancer samples. Post-culture explants exhibited preserved tumor morphology with no significant changes across major immune cell sub-populations. Stimulation with anti-CD3+IL2 resulted in notable increase in proliferating (>2.5-fold) and active (>2.8-fold) cytotoxic-T cells across samples. Additionally, anti-CD3+IL2 stimulation led to substantial fold release of Interferon-γ (15.1±7) and Granzyme-B (2.5±1), while LPS stimulation induced higher fold release of Tumor Necrosis Factor-α (11.3±4.6) and Interleukin-10 (5.5±0.8) with respect to control.OvCa cohort (n=7) receiving Platin and Taxane combination treatment, showed variable treatment response with six of the seven samples showing significant decrease in tumor content (p<0.05). The seventh sample that did not show response to combination treatment but exhibited significant fold increase in tumoral CC3 expression (p<0.01) with Cisplatin treatment.Given the high PD1 expression at baseline, we attempted to check efficacy of Nivolumab (n=3). Effective masking of PD1 receptor indicated drug binding to target. Differential T-cell reinvigoration was observed across samples. None of the treated samples, however, exhibited a significant tumor cell cytotoxicity.ConclusionsThe FarcastTM OvCa TruTumor platform facilitates simultaneous investigation with multiple drug treatment regimens enabling personalized treatment decision making for patients.ReferenceMaleki Z, Vali M, Nikbakht HA, Hassanipour S, Kouhi A, Sedighi S, Farokhi R, Ghaem H. Survival rate of ovarian cancer in Asian countries: a systematic review and meta-analysis. BMC cancer 2023;23(1):1–11.Ethics ApprovalThe Institutional Ethics Committee (IEC) from the sample collection centers approved the protocol (protocol # FCB-PROTOCOL-01) and informed consent for participation in the approved study was obtained from every donor.

BackgroundOnly 1–3 % of patients with Head and Neck Squamous Cell Carcinoma (HNSCC) in low- and middle-income countries can afford Nivolumab treatment even though it is approved for recurrent and metastatic disease.1 Low Dose Nivolumab (LDN) has shown similar efficacy compared to Standard Dose Nivolumab (SDN) in renal and lung cancer.2 3 A recent study demonstrated that HNSCC patient sub-cohort treated with chemotherapy (CT) and LDN showed improved progression-free and overall survival compared to sub-cohort treated with CT alone.1 To better understand the added benefit of the combination treatment, it is important to rule out the contribution of CT alone in the responding patients that might not result in a durable response. To address this, we employed the FarcastTM TruTumor, a near native human histoculture platform which can compare multiple treatment response simultaneously for the same patient sample.MethodsFresh surgically resected HNSCC samples (n=20) along with matched blood were collected from consented patients. Thin explants were generated and distributed into arms. These arms were treated in culture with either LDN (7.3 µg/ml) or SDN (132 µg/ml) and CT (Methotrexate (220.8µg/ml)+erlotinib (2.5µg/ml)+celecoxib (0.7µg/ml) or Paclitaxel (2.7µg/ml)+Carboplatin (37.1µg/ml) alone or CT+LDN for 72 hours. The response was evaluated using histopathology, interferon-γ (IFN-g) cytokine release and flow cytometry.ResultsBoth SDN and LDN treatment led to effective masking of Programmed cell death protein 1 (PD1) expression. No significant difference was observed in tumor cytotoxicity response between LDN and SDN in the same samples (n=8). Analysis of immune cell types and activation markers such as CD8+Granzyme-B+, CD8+Ki67+ T cells also showed no significant difference between LDN and SDN treatments. Comparable IFN-g response was observed in 7/8 samples.We next compared response to CT alone or CT+LDN in the same sample (n=12). Ten out of 12 samples did not show a significant difference in tumor content on treatment with CT+LDN compared to CT alone. Five out of these 10 samples showed a significant increase in cleaved caspase expression in tumor, primarily driven by CT treatment. In two samples CT+LDN showed significant drop in tumor content but not in CT or SDN alone, clearly implicating the benefit of combination treatment.ConclusionsThe FarcastTM TruTumor platform thus provides the unique opportunity to identify patients who would truly benefit from treatment with LDN+CT combination. An observational trial is underway to correlate the response observed in our platform with response of the patient in the clinic.ReferencesPatil VM, Noronha V, Menon N, Rai R, Bhattacharjee A, Singh A, Nawale K, Jogdhankar S, Tambe R, Dhumal S, Sawant R, Alone M, Karla D, Peelay Z, Pathak S, Balaji A, Kumar S, Purandare N, Agarwal A, Puranik A, Mahajan A, Janu A, Kumar Singh G, Mittal N, Yadav S, Banavali S, Prabhash K. Low-dose immunotherapy in head and neck cancer: A randomized study. J Clin Oncol. 2023;41(2):222–232Zhao JJ, Kumarakulasinghe NB, Muthu V, Lee M, Walsh R, Low JL, Choo J, Tan HL, Chong WQ, Ang Y, et al. Low-dose Nivolumab in renal cell carcinoma: A real-world experience. 2021;99(3):192–202Yoo SH, Keam B, Kim M, Kim SH, Kim YJ, Kim TM, Kim DW, Lee JS, Heo DS. Low-dose nivolumab can be effective in non-small cell lung cancer: alternative option for financial toxicity. ESMO Open. 2018;3(5):e000332Ethics ApprovalThe Institutional Ethics Committee (IEC) from the sample collection centers approved the protocol (protocol # FCB-PROTOCOL-01) and informed consent for participation in the approved study was obtained from every donor.

Liquid biopsy is increasingly gaining traction as an alternative to invasive solid tumor biopsies for prognosis, treatment decisions, and disease monitoring. Matched tumor‐plasma samples were collected from 180 patients across different cancers with >90% of the samples below Stage IIIB. Tumors were profiled using next‐generation sequencing (NGS) or quantitative PCR (qPCR), and the mutation status was queried in the matched plasma using digital platforms such as droplet digital PCR (ddCPR) or NGS for concordance. Tumor‐plasma concordance of 82% and 32% was observed in advanced (Stage IIB and above) and early (Stage I to Stage IIA) stage samples, respectively. Interestingly, the overall survival outcomes correlated to presurgical/at‐biopsy ctDNA levels. Baseline ctDNA stratified patients into three categories: (a) high ctDNA correlated with poor survival outcome, (b) undetectable ctDNA with good outcome, and (c) low ctDNA whose outcome was ambiguous. ctDNA could be a powerful tool for therapy decisions and patient management in a large number of cancers across a variety of stages. This study examines the tumor‐plasma concordance in seven cancer types across stages. A concordance of 82% and 32% was observed in advanced (Stage IIB and above) and early (Stage I to Stage IIA) stage samples, respectively. Overall survival outcomes correlated well to presurgical/at‐biopsy ctDNA levels.

BackgroundA 3D histo-culture platform provides a near native Tumor immune Micro-Environment (TiME), making it best suited for evaluating response to immunotherapy drugs. Farcast™ TiME is a human 3D tumor histo-culture platform that preserves TiME and maintains functional fidelity of intra-tumoral immune cells (IIC). In this study we investigated the utility of this platform in demonstrating treatment induced Antibody-Dependent Cellular Cytotoxicity (ADCC) mechanism driven by IICs alone versus co-culture with autologous peripheral blood immune cells.MethodsHead and neck squamous cell carcinoma tissue samples (n=5) along with matched blood from consented patients were used in this study. All Peripheral Blood Nucleated Cells (PBNCs) including lymphocytes, monocytes, NK cells and neutrophils were isolated and stained with a tracking dye to distinguish them from IICs. Tumor tissues were processed to generate explants, treated with 184 µg/ml Cetuximab (anti-EGFR) or vehicle control, and cultured with or without PBNCs for 72 hrs. Response was evaluated using flow cytometry and cytokine release assay.ResultsAmount of infiltrated autologous PBNCs showed a strong negative correlation (R2=0.98) with the amount of IICs in the absence of drug treatment. The proportions of infiltrated immune cell sub-populations were similar to the composition of PBNCs added in culture. Cetuximab treatment, however, led to enhanced infiltration of the effector cells for ADCC driven tumor killing, namely NK cells, macrophages, neutrophils, and cytotoxic T cells (CTLs). Notably the unique infiltration pattern of effector cell populations observed in each sample was reflected in the secretion of specific cytokine/chemokines associated with that cell population. NK cell increase (fold change: 1.6 ± 0.8) was observed in all samples with a concomitant increase in MCP-1 secretion (fold change: 1.7 ± 0.9). Granzyme-B expressing NK cells increased (>1.7 fold) in a subset of samples. Samples showing increase in neutrophil infiltration exhibited increased MMP9 secretion, involved in neutrophil infiltration via stromal remodeling. Sample with highest increase in infiltration of CD16+ Monocyte/Macrophages (>2.4 fold) showed maximum increase in Granzyme-B secretion with respect to the untreated arm. Increase in fold secretion (>1.4) of CXCL9/CXCL10 was associated with the sample that showed highest fold increase of Granzyme-B expressing CTL in comparison to untreated arm. IICs alone were not sufficient in eliciting optimal ADCC response.ConclusionsThe study demonstrated ADCC response in the explant/PBNC co-culture platform leading to specific infiltration of effector sub-populations. FarcastTM TiME thus provides a unique platform to explore for heterologous adoptive cell and CAR-T therapies that involve immune cell infiltration.Ethics ApprovalAll samples included in the study were approved by institutional review boards of the centers providing the samples.

PurposeBreast and/or ovarian cancers are among the most common cancers in women across the world. In the Indian population, the healthcare burden of breast and/or ovarian cancers has been steadily rising, thus stressing the need for early detection, surveillance, and disease management measures. However, the burden attributable to inherited mutations is not well characterized.MethodsWe sequenced 1010 unrelated patients and families from across India with an indication of breast and/or ovarian cancers, using the TruSight Cancer panel which includes 14 genes, strongly associated with risk of hereditary breast and/or ovarian cancers. Genetic variations were identified using the StrandNGS software and interpreted using the StrandOmics platform.ResultsWe were able to detect mutations in 304 (30.1%) cases, of which, 56 mutations were novel. A majority (84.9%) of the mutations were detected in the BRCA1/2 genes as compared to non-BRCA genes (15.1%). When the cases were stratified on the basis of age at diagnosis and family history of cancer, the high rate of 75% of detection of hereditary variants was observed in patients whose age at diagnosis was below 40 years and had first-degree family member(s) affected by breast and/or ovarian cancers. Our findings indicate that in the Indian population, there is a high prevalence of mutations in the high-risk breast cancer genes: BRCA1, BRCA2, TP53, and PALB2.ConclusionIn India, socioeconomic inequality limiting access to treatment is a major factor towards increased cancer burden; therefore, incorporation of a cost-effective and comprehensive multi-gene test will be helpful in ensuring widespread implementation of genetic screening in the clinical practice for hereditary breast and/or ovarian cancers.

BackgroundThe traditional method of biomarker identification often fails to capture the complexity of the Tumor Immune Microenvironment (TME) response, particularly in small datasets. SurgeCare’s Stabl framework1 addresses this by using noise injection for reproducible, quantitative, and high-throughput biomarker selection. In this study, we applied the Stabl framework to anti-PD1 response data generated using the Farcast TruTumor ex vivo platform2 to identify a response biosignature.MethodsExplants generated from fresh surgically resected HNSCC samples (n=67) collected from the consented patients were cultured with Nivolumab (132 µg/ml) for 72 hours and response evaluated using multiple assays such as cytokine release, histopathology, flow cytometry, and NanoString based mRNA analysis. Selected features were derived from multimodal assay data using Stabl.The Stabl methodology involved preprocessing omics datasets using variance thresholds, missing value filters, median imputation, and z-score standardization, implemented with sci-kit-learn v1.2.1 Five repetitions of 5-fold Monte Carlo cross-validation were done to ensure stratified sampling. Feature selection was conducted using Knockoffs or Random Permutation with Logistic Regression or Random Forest, along with Adaptive Lasso for penalized regression. These methods identified features distinguishing control (RxA) from anti-PD1 treated (RxB) arms. A responder signature was derived from 23 samples (with complete multimodal data) by correlating selected features with tumor cytotoxicity markers, such as reduced tumor content and/or increased cleaved caspase 3 expression.ResultsFrom a total of 849 multimodal features, 25 were identified by Stabl as effective in distinguishing RxA from RxB, with a predictive power of 0.68 (AUC). A six-feature responder signature was derived from these 25 features by correlating them with tumor cytotoxicity (Spearman, p ≤ 0.08). The features included M1 macrophage, IL1beta signaling, non-T cell immune cell sub-population, activated cytotoxic T-cells (CD8+GranzymeB+), Perforin, and Granzyme B cytokines. UMAP on RxB data using these features revealed three distinct clusters: C1, C2, and C3.C1 (n=8) had the highest responder rate (87.5%), followed by C2 (n=11, 36.4%) and C3 (n=4, 25%).C1 samples showed increased immune infiltration and high non-T cell levels in control, correlating with enhanced tumor cytotoxicity on treatment. C2 had a moderate response profile with high cytokine release but limited immune infiltration. C3 showed low immune infiltration and high tumor content.ConclusionsThe complex response phenotypes observed from the TruTumor ex vivo platform that can generate multi-dimensional datasets combined with the robust Stabl framework can lead to considerable derisking of novel therapies at late pre-clinical stage.ReferencesHédou J, Mari I, Bellan G, et al. Discovery of sparse, reliable omic biomarkers with Stabl. Nat Biotechnol. 2024;42:1581–1593.Basak NP, Jaganathan K, Das B, et al. Tumor histoculture captures the dynamic interactions between tumor and immune components in response to anti-PD1 in head and neck cancer. Nat Commun. 2024;15(1):1585.Ethics ApprovalDonor tissue specimens along with matched blood sample were obtained from consented patients. Institutional Ethics Committee (IEC) from the sample collection centers approved the protocol (protocol # FCB-PROTOCOL-01) and informed consent for participation in the approved study was obtained from every donor.

BackgroundColorectal cancer (CRC) is a significant global health issue. Response rate to immune checkpoint inhibitors (ICI) in MSI-H CRC patients have been encouraging but highly variable, pointing to the need for a better response biomarker for patient selection. We employed the Farcast CRC TruTumor histoculture platform to understand the role and response of immune cell types in the complex Tumor MicroEnvironment (TME) that could identify ICI therapy responders more accurately.MethodsFreshly resected tumor tissue samples along with matched blood were collected from consented patients. Tumor explants were generated and distributed into arms and cultured for 72 h with media replenished every 24h. The response to T-cell stimulation with anti-CD3 (100 ng/mL) + Interleukin-2 (IL-2, 100 IU/mL) and treatment with Nivolumab (132 µg/mL) was evaluated using cytokine release and flow cytometry based immune profiling.ResultsThe CRC TME (n=6) was compared with other indications, namely, Head and Neck Carcinoma (HNSCC: n=10), Renal Cell Carcinoma (RCC: n=10) and Breast Carcinoma (CaBr: n=10). Though the proportion of effector cell populations like (Cytotoxic T-cells (CD8+), Natural killer T cells (CD3+CD56+) and Natural killer cells (CD3-CD56+)) were present in CRC, the activated effector cell (Granzyme B+; GZMB+) proportions were significantly lower. Interestingly, the proportion of Natural Killer (NK) and NK T-cell proportions were higher in CRC and RCC than the other two indications. Additionally, macrophages were biased towards M2 like in CRC compared to other indications. Upon stimulating the tumor fragments with anti-CD3+IL2, CRC samples (n=5) showed only modest increase in the activation (mean value <10%) which was significantly lower (p<0.05) than the other 3 indications.Despite presence of comparable proportions of exhausted CTL population (CD8+PD1+); the target for nivolumab, in CRC (n=4), RCC (n=2) and CaBr (n=3), Nivolumab treatment induced increase in proportion of activated effector immune cells was much more robust in RCC and CaBr compared to CRC. Of the 4 CRC samples treated with nivolumab 2 (S2 and S4) showed more than 1.1-fold increase in GZMB release. Of the two, in S4 there was 3.7-fold increase in IFN-γ secretion with 1.6-fold increase in Perforin release without exhibiting treatment driven tumor cytotoxicity.ConclusionsThe TruTumor platform reproduced the immunosuppressive TME in CRC and points to the presence of dysfunctional or irreversibly exhausted CTLs that restricts the efficacy of ICI monotherapy. It also provides the opportunity to explore combination therapy strategies to overcome the shortcoming of ICI monotherapy for better outcomes.Ethics ApprovalDonor tissue specimens along with matched blood sample were obtained from consented patients. Institutional Ethics Committee (IEC) from the sample collection centers approved the protocol (protocol # FCB-PROTOCOL-01) and informed consent for participation in the approved study was obtained from every donor.