Post-treatment Tumor Samples Research Articles

Intratumoral STING agonist reverses immune evasion in PD-(L)1-refractory Merkel cell carcinoma: mechanistic insights from detailed biomarker analyses

BackgroundAntibodies blocking programmed death (PD)-1 or its ligand (PD-L1) have revolutionized cancer care, but many patients do not experience durable benefits. Novel treatments to stimulate antitumor immunity are needed in...

The Effect of Statins on Markers of Breast Cancer Proliferation and Apoptosis in Women with In Situ or Early-Stage Invasive Breast Cancer.

Statins, inhibitors of HMG-CoA reductase, have been shown to have potential anti-carcinogenic effects through the inhibition of the mevalonate pathway and their impact on Ras and RhoGTAases. Prior studies have demonstrated a reduction in breast tumor proliferation, as well as increased apoptosis, among women with early-stage breast cancer who received statins between the time of diagnosis and the time of surgery. The aim of this study was to evaluate the impact of short-term oral high-potency statin therapy on the expression of markers of breast tumor proliferation, apoptosis, and cell cycle arrest in a window-of-opportunity trial. This single-arm study enrolled 24 women with stage 0-II invasive breast cancer who were administered daily simvastatin (20 mg) for 2-4 weeks between diagnosis and surgical resection. Pre- and post-treatment tumor samples were analyzed for fold changes in Ki-67, cyclin D1, p27, and cleaved caspase-3 (CC3) expression. Out of 24 enrolled participants, 18 received statin treatment and 17 were evaluable for changes in marker expression. There was no significant change in Ki-67 expression (fold change = 1.4, p = 0.597). There were, however, significant increases in the expression of cyclin D1 (fold change = 2.8, p = 0.0003), p27 cytoplasmic (fold change = 3.2, p = 0.025), and CC3 (fold change = 2.1, p = 0.016). Statin treatment was well tolerated, with two reported grade-1 adverse events. These results align with previous window-of-opportunity studies suggesting a pro-apoptotic role of statins in breast cancer. The increased expression of markers of cell cycle arrest and apoptosis seen in this window-of-opportunity study supports further investigation into the anti-cancer properties of statins in larger-scale clinical trials.

Change in tumor microenvironment and PD-L1 expression with rucaparib treatment in triple-negative breast cancer.

e12569 Background: Triple-Negative Breast Cancers (TNBCs) represent a diverse subset of tumors characterized by varied responses to therapies including immunotherapy (IO). While IO is approved in early-stage and advanced TNBC, responses are modest. Modulation of the tumor microenvironment to improve responses to IO is an active area of research. In-vitro and in-vivo studies have shown Poly-ADP Ribose Polymerase inhibitors (PARPi) to increase PD-L1 expression in sporadic, BRCA1 and BRCA2 mutated TNBC cell lines improving response to IO. In addition, TNBCs have diverse mechanisms leading to Homologous Recombination Deficiency and the role of PARPi in sporadic TNBCs is still unclear. Methods: To further investigate the role of PARPi in tumor immuno-modulation we conducted a single-arm, open-label, window of opportunity (WOO) trial in patients with early-stage TNBC (NCT03911453). This study was approved by our institutional review board. Patients with histologically documented stage I-III TNBC were eligible for definitive surgery were enrolled. Patients were treated with 600mg BID rucaparib for 21 days. Pre- and post-treatment tumor biopsies were collected. Post-treatment, patients went to surgery or proceeded with neoadjuvant chemotherapy (NAC) followed by definitive surgery. Our primary endpoint was to assess change in PD-L1 expression. In addition, change in tumor microenvironment was by evaluating change in percentage of B cells, T cells and macrophages was calculated. Results: Twenty patients were enrolled. Nineteen patients completed 21 days and 1 patient went on extended duration PARPi during the COVID19 pandemic. Almost 50% of patients went to surgery while the rest went onto NAC after PARPi therapy. Pre-treatment tumor samples were obtained from the diagnostic core biopsies while post-treatment, fresh frozen and Formalin Fixed Paraffin Embedded were obtained either at time of surgery or a research biopsy prior to NAC initiation. Primary biomarkers data was successfully done in 14 paired samples. Treatment with rucaparib has increased expression of PD-L1 from median of 15% in pre-treatment tumor samples to a median of 26% in post-treatment tumor samples. Table below summarizes our results. Conclusions: Our study revealed that treatment with PARPi, rucaparib, changed the tumor microenvironment as noted by changes in expression of PD-L1, and immune milieu in and around the tumor. This could lead to change in responsiveness to therapies including CKI. This strategy should be further investigated to evaluate improving responses to immunotherapies. Clinical trial information: NCT03911453 . [Table: see text]

Abstract 2023: Multiplex immunofluorescence profiling of the tumor microenvironment and CD73: Activity of neoadjuvant oleclumab in patients with non-small-cell lung cancer in NeoCOAST

Abstract Background: The NeoCOAST study (NCT03794544) investigated the efficacy of neoadjuvant immuno-oncology combinations in patients with resectable non-small-cell lung cancer (NSCLC), using major pathological response (MPR) as the primary endpoint (Cascone T, et al. Cancer Discov 2023;13:2394-411). Patients received a single cycle of the following treatments: durvalumab monotherapy (durva; anti-PD-L1), durva + oleclumab (ole; anti-CD73), or durva + monalizumab (mona; anti-NKG2A). Here we analyze the tumor microenvironment to investigate the mechanism of action of ole. Methods: A cohort of 47 patients (17 durva, 13 durva + mona, 17 durva + ole) with tumor tissue samples from pre-treatment (baseline, n=31) and surgery (post-treatment, n=24; n=8 paired pre- and post-treatment samples) were evaluated. Tumor sections were stained with (1) multiplex immunofluorescence (Panel 1: CD8-PD-L1-PD1-CD68-Ki67-panCK, Panel 2: NKp46-CD20), (2) CD73 immunohistochemistry (IHC), and (3) NKG2A IHC. Stains were analyzed using deep learning algorithms, manual pathology, and digital pathology scoring, respectively. Results: All arms showed similar infiltration of tumors by T cells (CD8), B cells (CD20), NK cells (NKp46), and macrophages (CD68) at the pre-treatment time point (Kruskal-Wallis, p=0.5-0.9 n.s.). Percent CD73+ tumor cells (TCs) at baseline was correlated with increased B cell, T cell, NK cell, and macrophage abundance (Spearman’s Rho=0.4-0.6, p<0.05). Percent CD73+ TC and B cell abundance was higher in patients with MPR vs those without MPR in the durva + ole arm, but not in the durva monotherapy or durva + mona arms. Increased CD8 T cell and NK cell abundance was not associated with MPR in any arm. Moreover, comparison of baseline to post-treatment tumor samples revealed a greater increase in CD8 T cells in the durva + ole arm (FC=2.1, p=0.02), particularly in proliferating activated T cells (CD8+Ki67+PD-1+; FC=8.0, p=0.003), compared to durva alone (FC=1.8, p=0.4 and FC=2.9, p=0.04, respectively). Conclusion: Increased abundance of CD73+ TCs is correlated with high B cells in the tumor microenvironment and associated with patients who experienced MPR in the durva + ole arm. Treatment with neoadjuvant durva + ole results in a greater increase in proliferating CD8 T cells than durva alone. Citation Format: Ina Bisha, Tze Heng Tan, Manuela Weitkunat, Alma Andoni, Iris Dino, Philip Martin, Megha Saraiya, Karma DaCosta, Pallavi Sontakke, Markus Schick, Michael Surace, Jorge Blando, Italia Grenga, Rakesh Kumar, Lara McGrath. Multiplex immunofluorescence profiling of the tumor microenvironment and CD73: Activity of neoadjuvant oleclumab in patients with non-small-cell lung cancer in NeoCOAST [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2023.

Abstract 5280: Minimizes T cells DNA damage induced by PARP inhibitors and enhances its anti-tumor efficacy

Abstract Background: Poly (ADP-ribose) polymerase (PARP) inhibitors have emerged as promising targeted therapies for ovarian and breast cancer. The sustained remission induced by PARP inhibitors largely depends on T cell activity. However, the direct effects of PARP inhibitors on T cells, as well as their precise impact on T cell DNA integrity and subsequent anti-tumor immune responses, have not been fully elucidated. Method & Result: Following treatment with 25 μM olaparib or 15 μM niraparib for 48 hours, T cell viability decreased by 39.20% and 41.89%, respectively. Reduced proliferation and increased cell death were also observed, as indicated by reduced Ki-67 and elevated cleaved Caspase-3 levels. RNA sequencing further revealed a significant enrichment of DNA damage pathways in T cells after olaparib treatment, which was validated by elevated γH2AX levels in T cells using western blots and immunofluorescence. To assess PARP inhibitor-induced DNA damage in clinical settings, paired ovarian cancer samples pre- and post-niraparib monotherapy were obtained from the NANT trial (NCT04507841). Post-treatment tumor samples exhibited a higher number of γH2AX+ T cells compared to their pre-treatment counterparts. To identify the key protein mediating T cell inhibition induced by PARP inhibitors, a whole genomic CRISPR knock-out screening was conducted in human T cells, revealing an enrichment of sgRNAs targeting PARP1 after PARP inhibitor treatment. This outcome was further confirmed by the knockout of PARP1 in human T cells in vitro, leading to the restored viability and reduced levels of DNA damage after olaparib treatment. Importantly, following cytosine base editors (CBEs) of PARP1 in CAR-T cells, improved anticancer activity was observed both in vitro and in two ovarian cancer patient-derived xenografts (PDX) models when combined with PARP inhibitors. Conclusion: This study observed that PARP inhibitors can cause significant DNA damage in human T cells, resulting in cell death, inhibited proliferation, and impairment of anti-cancer immunity. PARP1 was identified as the target for PARP inhibitor-induced T cell death, and its knockout or point mutation led to improved anticancer efficacy of T cells when used in combination with PARP inhibitors. [JH Liu and XF Jiao contributed equally as co-first authors.] Citation Format: Jiahao Liu, Xiaofei Jiao, Wei Mu, Huayi Li, Li Zhu, Xuejiao Zhao, Gordon B. Mills, Qinglei Gao, Yong Fang. Minimizes T cells DNA damage induced by PARP inhibitors and enhances its anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5280.

EXTH-40. PHOTODYNAMIC ONCOLYTIC VIRUS THERAPY FOR CENTRAL NERVOUS SYSTEM MALIGNANCIES

Abstract Oncolytic viruses (OVs) have emerged as a clinical therapeutic modality potentially effective to treat cancers that evade conventional therapies, including central nervous system malignancies. Rationally designed combinatorial strategies can augment the efficacy of OVs by boosting tumor-selective cytotoxicity and modulating the tumor microenvironment (TME). Photodynamic therapy (PDT) of cancer not only mediates direct neoplastic cell death but also primes the TME to sensitize the tumor to secondary therapies. This allows for the combination of two potentially synergistic therapies with broader targets. Here, we created G47Δ-KR, clinical oncolytic herpes simplex virus G47Δ that expresses photosensitizer protein KillerRed (KR). Optical properties and cytotoxic effects of G47Δ-KR infection followed by amber LED illumination were examined in human glioblastoma (GBM) and malignant meningioma (MM) models in vitro. G47Δ-KR infection of tumor cells mediated KR expression that was activated by amber LED and produced reactive oxygen species, leading to cell death that was more robust than G47Δ-KR without light. In vivo, we tested G47Δ-KR-based PD-OV therapy employing intratumoral injection of G47Δ-KR followed by amber laser (wavelength: 585 nm) tumor irradiation in GBM and MM xenografts. PD-OV therapy was feasible in GBM and MM xenografts and resulted in potent anti-tumor effects that were superior to G47Δ-KR alone (without laser) or laser alone. RNA sequencing analysis of post-treatment tumor samples revealed PD-OV therapy-induced increases in TME infiltration of variable immune cell types. This study thus demonstrated the proof-of-concept that G47Δ-KR enables PD-OV therapy for neuro-oncological malignancies and warrants further research to advance potential clinical translation.

Photodynamic augmentation of oncolytic virus therapy for central nervous system malignancies

Oncolytic viruses (OVs) have emerged as a clinical therapeutic modality potentially effective for cancers that evade conventional therapies, including central nervous system malignancies. Rationally designed combinatorial strategies can augment the efficacy of OVs by boosting tumor-selective cytotoxicity and modulating the tumor microenvironment (TME). Photodynamic therapy (PDT) of cancer not only mediates direct neoplastic cell death but also primes the TME to sensitize the tumor to secondary therapies, allowing for the combination of two potentially synergistic therapies with broader targets. Here, we created G47Δ-KR, clinical oncolytic herpes simplex virus G47Δ that expresses photosensitizer protein KillerRed (KR). Optical properties and cytotoxic effects of G47Δ-KR infection followed by amber LED illumination (peak wavelength: 585–595 nm) were examined in human glioblastoma (GBM) and malignant meningioma (MM) models in vitro. G47Δ-KR infection of tumor cells mediated KR expression that was activated by LED and produced reactive oxygen species, leading to cell death that was more robust than G47Δ-KR without light. In vivo, we tested photodynamic-oncolytic virus (PD-OV) therapy employing intratumoral injection of G47Δ-KR followed by laser light tumor irradiation (wavelength: 585 nm) in GBM and MM xenografts. PD-OV therapy was feasible in these models and resulted in potent anti-tumor effects that were superior to G47Δ-KR alone (without laser light) or laser light alone. RNA sequencing analysis of post-treatment tumor samples revealed PD-OV therapy-induced increases in TME infiltration of variable immune cell types. This study thus demonstrated the proof-of-concept that G47Δ-KR enables PD-OV therapy for neuro-oncological malignancies and warrants further research to advance potential clinical translation.

Neoadjuvant Afatinib for stage III EGFR-mutant non-small cell lung cancer: a phase II study

Afatinib, an irreversible ErbB-family blocker, could improve the survival of advanced epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer patients (NSCLCm+). This phase II trial (NCT04201756) aimed to assess the feasibility of neoadjuvant Afatinib treatment for stage III NSCLCm+. Forty-seven patients received neoadjuvant Afatinib treatment (40 mg daily). The primary endpoint was objective response rate (ORR). Secondary endpoints included pathological complete response (pCR) rate, pathological downstaging rate, margin-free resection (R0) rate, event-free survival, disease-free survival, progression-free survival, overall survival, treatment-related adverse events (TRAEs). The ORR was 70.2% (95% CI: 56.5% to 84.0%), meeting the pre-specified endpoint. The major pathological response (MPR), pCR, pathological downstaging, and R0 rates were 9.1%, 3.0%, 57.6%, and 87.9%, respectively. The median survivals were not reached. The most common TRAEs were diarrhea (78.7%) and rash (78.7%). Only three patients experienced grade 3/4 TRAEs. Biomarker analysis and tumor microenvironment dynamics by bulk RNA sequencing were included as predefined exploratory endpoints. CISH expression was a promising marker for Afatinib response (AUC = 0.918). In responders, compared to baseline samples, increasing T-cell- and B-cell-related features were observed in post-treatment tumor and lymph-node samples, respectively. Neoadjuvant Afatinib is feasible for stage III NSCLC+ patients and leads to dynamic changes in the tumor microenvironment.

Abstract 2118: Development of RNAscope multiplex-based assay for exploratory pharmacodynamic biomarkers assessment in breast cancer patients from Phase I clinical trial of ORM-5029, a potent GSPT1 degrader

Abstract Breast cancer is the most common invasive cancer in women worldwide and remains a clinical challenge. Addressing the unmet need for better therapeutics, we used our Dual-Precision Targeted Protein Degradation (TPD² TM) approach that merges the power of protein degraders with precision of ADC technology to design ORM-5029. It delivers a highly potent and precise catalytic GSPT1 protein degrader molecule (SMol006) selectively to HER2-expressing tumor cells via conjugation to pertuzumab. GSPT1 is a protein associated with regulation of the translational termination complex, and its loss results in activation of the integrated stress response (ISR) which ultimately leads to apoptosis. Activation of ISR triggers overall reduction of protein synthesis and activation of stress response genes (e.g., ATF3 and DDIT3). ORM-5029 is currently being investigated for safety and tolerability in an open label, multicenter, phase 1, dose escalation and expansion study in patients with HER2-expressing advanced breast tumors (NCT05511844). Here, we evidence that the efficacy of ORM-5029 correlates with pharmacodynamic degradation of GSPT1 and activation of ATP3 and DDIT3 transcripts both in vitro and in tumor xenografts in mice. With the aim of evaluating GSPT1 degradation and consequent activation of ATF3 and DDIT3 in pre- and post-treatment tumor samples from HER2+ breast cancer patients, we are developing an RNAscope multiplex (Leica System) assay panel. This approach combines detection of GSPT1 protein with 20zz ISH probe-mediated detection of ATF3 and DDIT3 mRNA in tumor sections. As a part of method development, we treated BT474 cells with an efficacious concentration of GSPT1 degrader and optimized the immunofluorescence and immunohistochemistry protocol with our in-house generated GSPT1 antibody (MAb001). Data showed that GSPT1- MAb001 could be successfully used for both immunofluorescence and immunohistochemistry assays. The optimized protocol is currently being developed into RNAscope multiplex workflow using BT474 FFPE sections and tissue microarrays. In summary, RNAscope multiplex assay could be used as a method to determine pharmacodynamic response in HER2+ breast cancer patients treated with ORM-5029. Citation Format: Shikha Saini, Yeonjoon Kim, Wesley Wong, Tina Karunaratne, Olaf Christensen, Sujoy Dutta, Palash Bhar, Khuloud Takrouri, Nathan Fishkin, Dong-Ki Choi, Min-Soo Kim, Joanne Lim, Anna Skaletskaya, James Palacino, Peter Park. Development of RNAscope multiplex-based assay for exploratory pharmacodynamic biomarkers assessment in breast cancer patients from Phase I clinical trial of ORM-5029, a potent GSPT1 degrader [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2118.

Perioperative treatment and biomarker analysis of LP002, an anti-PD-L1 antibody, plus chemotherapy in resectable gastric and gastroesophageal junction cancer.

The addition of immune checkpoint inhibitors to perioperative chemotherapy in operable gastric or gastroesophageal junction (GEJ) cancer has become one of the research hotspots, while reliable biomarkers for efficacy are lacking. We conducted a phase 1 trial to assess the safety and efficacy of LP002, an anti-PD-L1 antibody, plus chemotherapy as perioperative treatment in patients with gastric or GEJ cancer. We enrolled patients with resectable and PD-L1 positive gastric or GEJ cancers. Eligible patients received three preoperative and six postoperative cycles of intravenous LP002 with cisplatin and 5-fluorouracil, repeated every 2 weeks. The primary endpoint was safety. Secondary endpoints included rate of margin-free (R0) resection and pathological complete response (pCR). We also characterized changes in the tumor immune microenvironment using multiplex immunofluorescence (MIF) staining and next-generation sequencing (NGS) with pre- and post-treatment tumor samples. Thirty patients were enrolled, of whom 28 had GEJ cancer. With a median follow-up of 7.9 months, all patients completed preoperative treatment, and 27 patients underwent surgery. Twenty-four patients underwent R0 resection. Six patients (20.0%) had Mandard tumor regression grade (TRG) 1-3, including one achieving pCR. Twenty-seven patients had treatment-related adverse events (TRAEs), while grade 3-4 TRAEs were observed in 11 patients. No treatment-related deaths occurred. MIF staining revealed that TRG 1-3 group was associated with a higher density of PD-L1+/CD68+ cells in the pre-treatment tumor parenchyma than TRG 4-5 group (p=0.048). NGS studies with paired pre- and post-treatment tumor samples revealed the disappearance of pre-existing mutations, the emergence of new mutations, and variations in the abundance of mutations after preoperative LP002 and chemotherapy. Meanwhile, tumor mutational burden decreased in patients with TRG 1-3 (p=0.0313). LP002 plus cisplatin and 5-fluorouracil are safe in patients with gastric or GEJ cancer, and patient selection via appropriate biomarkers is needed in the future.

CD8+ and FoxP3+ T-Cell Cellular Density and Spatial Distribution After Programmed Death-Ligand 1 Check Point Inhibition.

To analyze CD8+ and FoxP3+ T-cell cellular density (CD) and intercellular distances (ID) in head and neck squamous cell carcinoma (HNSCC) samples from a neoadjuvant trial of durvalumab +/- metformin. Paired pre- and post-treatment primary HNSCC tumor samples were stained for CD8+ and FoxP3+. Digital image analysis was used to determine estimated mean CD8+ and FoxP3+ CDs and CD8+-FoxP3+ IDs in the leading tumor edge (LTE) and tumor adjacent stroma (TAS) stratified by treatment arm, human papillomavirus (HPV) status, and pathologic treatment response. A subset of samples was characterized for T-cell related signatures using digital spatial genomic profiling. Post-treatment analysis revealed a significant decrease in FoxP3+ CD and an increase in CD8+ CDs in the TAS between patients receiving durvalumab and metformin versus durvlaumab alone. Both treatment arms demonstrated significant post-treatment increases in ID. Although HPV+ and HPV- had similar immune cell CDs in the tumor microenvironment, HPV+ pre-treatment samples had 1.60 times greater ID compared with HPV- samples, trending toward significance (p= 0.05). At baseline, pathologic responders demonstrated a 1.16-fold greater CD8+ CDs in the LTE (p= 0.045) and 2.28-fold greater ID (p= 0.001) than non-responders. Digital spatial profiling revealed upregulation of FoxP3+ and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) in the TAS (p= 0.006, p= 0.026) in samples from pathologic responders. Analysis of CD8+ and FoxP3+ detected population differences according to HPV status, pathologic response, and treatment. Greater CD8+-FoxP3+ ID was associated with pathologic response. CD8+ and FoxP3+ T-cell distributions may be predictive of response to immune checkpoint inhibition. gov (Identifier NCT03618654). 3 Laryngoscope, 133:1875-1884, 2023.

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Abstract 1237: Randomized phase II trial of pre-operative afatinib in non-metastatic head and neck squamous cell carcinoma patients: Identification of predictive biomarkers of response

Abstract Background: Epidermal growth factor receptor (EGFR) inhibitors, including monoclonal antibodies and tyrosine kinase inhibitors, have limited efficacy in head and neck squamous cell carcinoma (HNSCC) patients. In the randomized phase II PREDICTOR trial, we aimed at identifying predictive and pharmacodynamics biomarkers of 2-4 weeks afatinib (an irreversible pan-HER inhibitor) versus no treatment in the pre-operative setting (NCT01415674). We previously reported a 59% metabolic response rate on PET imaging in the afatinib arm. We report here the evaluation of predictive genomic and transcriptomic biomarkers of afatinib efficacy. Patients and Methods: All patients (41 in the afatinib arm and 20 in the no treatment arm) underwent a pre-treatment biopsy. We performed targeted DNA sequencing using an in-house NGS panel of 571 genes on baseline biopsies from 56 patients, and RNA-sequencing (RNAseq) in 54 patients. In the afatinib arm, 26 patients had paired pre- and post-treatment tumor samples. DNA and RNA alterations were correlated with metabolic response to afatinib using PET imaging, as well as overall survival (OS). Results: Most frequent molecular alterations, including known activating mutations and/or focal amplifications for oncogenes or homozygous deletions and inactivating mutations for tumor suppressor genes, involved genome integrity (TP53 [70%]), cell cycle (CCND1 [38%], CDKN2A [32%], CDKN2B [14%]), senescence (TERT [23%]), Wnt signaling (NOTCH1 [16%]), and the PI3K pathway (PIK3CA [14%]). In the afatinib arm, metabolic response was observed in 1 out of 7 patients (14%) and in 19 out of 28 patients (68%) in the Wnt altered and unaltered groups (p = 0.03, fisher exact test), respectively. In the whole cohort of patients, homozygous deletions of both CDKN2A and CDKN2B correlated with shorter OS, with 6-year survival of 22% in the CDKN2A/B altered group and 70% in the CDKN2A/B wild-type group (p = 0.004; log-rank test). In the afatinib treated patients, using a generalized linear mixed model with a patient as random effect and a quasi-binomial family, the ratio of B cells expression levels in the post-treated versus pre-treated samples was significantly higher in responder as compared to non-responder patients (p = 0.001). Conclusions: Wnt signaling pathway alterations and treatment-related dynamic changes in B cells proportions were identified as predictive and pharmacodynamics biomarkers of afatinib efficacy. CDKN2A/B homozygous deletions were associated with a poor prognosis in HNSCC patients treated with upfront surgery. Citation Format: Grégoire Marret, Maud Kamal, Jocelyn Gal, Stéphane Temam, Jerzy Klijianenko, Jean-Pierre Delord, Caroline Hoffmann, Gilles Dolivet, Olivier Malard, Jerôme Fayette, Olivier Capitain, Caroline Even, Sébastien Vergez, Lionel Geoffrois, Frédéric Rolland, Philippe Zrounba, Laurent Laccourreye, Joël Guigay, Nicolas Aide, Valérie Bénavent, Constance Lamy, Elodie Girard, Marta Jimenez, Ivan Bièche, Christophe Le Tourneau. Randomized phase II trial of pre-operative afatinib in non-metastatic head and neck squamous cell carcinoma patients: Identification of predictive biomarkers of response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1237.

Phase 2 study of trastuzumab deruxtecan in the neoadjuvant treatment for patients with HER2-positive gastric and gastroesophageal junction adenocarcinoma (EPOC2003).

TPS4161 Background: Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate consisting of an anti-HER2 antibody, a cleavable tetrapeptide-based linker, and a cytotoxic topoisomerase I inhibitor. DESTINY-Gastric01 study showed significantly higher objective response rate with T-DXd than physician's choice of chemotherapy (51.3%, vs. 14.3%, Shitara K, et al. NEJM 2020) as well as prolonged overall survival (median 12.5 vs. 8.4 months, hazard ratio 0.59). Considering that high response rate or major pathological response (MPR), which is defined as the proportion of subjects with < 10% residual tumor may be associated with favorable survival outcomes after preoperative chemotherapy, T-DXd could be a promising neoadjuvant treatment for patients with gastric and gastroesophageal junction (GEJ) cancer. However, to our knowledge, there is no currently ongoing study of T-DXd as a neoadjuvant setting for patients with gastric and GEJ cancers. Methods: This is an open-label, single-arm, multicenter, investigator initiated phase 2 trial to evaluate antitumor activity of T-DXd as the neoadjuvant treatment for patients with HER2 positive gastric and GEJ adenocarcinoma. Eligible patients should have previously untreated locally advanced gastric and GEJ adenocarcinoma with clinical stage of T2-4 and/or N+ without distant metastasis. The main cohort will enroll patients with HER2 overexpression defined as immunohistochemistry （IHC）3+ or IHC2+/ in situ hybridization（ISH）+ by local assessment. According to the results of exploratory biomarker analysis of DESTINY-Gastric01 study (Shitara K, et al. ESMO-GI2021), patients with HER2-low expression (IHC1+ or 2+ with ISH negative) with HER2-extra cellular domein (ECD) higher than 11.6 ng/ml also will be enrolled into the exploratory cohort. Patients will receive 3 cycles of T-DXd (6.4mg/kg) by intravenous infusion every 3 weeks followed by surgery at 3 to 8 weeks after last T-DXd infusion.The primary endpoint is MPR rate. The threshold in the main cohort is defined as 20%, and the expected MPR rate was set at 45%. The minimum sample size of this trial is 23 patients in the main cohort (α- and β-error probabilities, 0.1 and 0.15, respectively). Total number of patients will be increased up to 27 patients depending on the enrollment. In addition, 10 patients will be recruited into the exploratory cohort. Secondary endpoints are pathologic complete response (pCR) rate, curative resection rate, and adverse event rate. Biomarker analyses including whole exome sequencing, RNA sequencing, proteomics, and ctDNA changes will also be conducted using pre- and post-treatment tumor and blood samples. Enrollment is ongoing at six sites in Japan from November 2021. Clinical trial information: NCT05034887.

Fosciclopirox clinical proof of concept in patients with nonmuscle invasive and muscle-invasive bladder cancer.

e16557 Background: Fosciclopirox (F) is being developed for the treatment of non-muscle invasive (NMIBC) and muscle invasive (MIBC) bladder cancer. F is a prodrug which is rapidly and completely metabolized in blood to its active metabolite, ciclopirox (CPX). In preclinical models of bladder cancer, CPX acts in part as a γ-secretase inhibitor by binding to γ-secretase complex proteins Presenilin 1 and Nicastrin, resulting in Notch and Wnt inhibition. The F Recommended Phase 2 Dose (RP2D), 900 mg/m2 administered IV over 20 minutes, was identified in the Phase 1 dose escalation trial (NCT03348514) in advanced solid tumor patients. Methods: The F RP2D was investigated in two early phase NMIBC and MIBC clinical proof of concept trials. In NCT04608045, neoadjuvant F was administered as monotherapy in cisplatin-ineligible (C-I) MIBC patients and in combination with gemcitabine + cisplatin in chemotherapy-eligible (C-E) MIBC patients. Clinical stage was assessed in pre-treatment (TURBT/CT) and post-treatment pathological state determined at radical cystectomy, (RC). The steady-state plasma and urine pharmacokinetics of F were also characterized. In NCT04525131, F was administered once daily for five days prior to TURBT. Pre- and post-treatment (at TURBT) bladder tumor samples underwent single cell sequencing to identify treatment effects on gene expression. Plasma, urine, and bladder tumor concentrations of F and its metabolites were determined in samples collected at TURBT. Results: Five C-E and 4 C-I MIBC patients received neoadjuvant F prior to RC. Twelve NMIBC patients received F prior to TURBT. There were no treatment-related serious adverse events observed in either study. Each patient experienced at least one treatment-emergent adverse event (TEAE), none of which resulted in study discontinuation. The most common TEAEs were nausea, fatigue, and constipation. Pathologic downstaging (< ypT2) of bladder tumors was observed in 3 C-E MIBC patients with 2 CRs (ypT0). Two of 4 C-I patients had evident clinical response by CT scan with only microscopic residual ypT2 disease. Treatment-related changes in expression of Notch 1, Notch 2, Hes 1, Hey-1, c-Myc, ß-catenin and survivin were observed in the majority of NMIBC patients. F disappeared from plasma within 2 hours of administration. The mean CPX elimination half-life of CPX, apparent systemic clearance, and volume of distribution values were 8.8 hours, 46 L/hr and 549 L, respectively. Mean plasma, tumor and urine concentrations of CPX at TURBT were approximately 27, 9 and 100 µM, respectively. Conclusions: To date, fosciclopirox is well tolerated and achieves sufficient systemic, tumor, and urine CPX exposure at the RP2D. Evidence of target inhibition was demonstrated in NMIBC tumors and preliminary signs of clinical activity observed in MIBC patients. Safety and efficacy trials are planned to confirm and expand findings in NMIBC and MIBC patients. Clinical trial information: NCT04608045; NCT04525131.

105P CDK4/6 inhibitors in hormone receptor-positive, HER2-negative, locally advanced breast cancer (LABC): Biological and clinical activity, and post-surgical approaches

Endocrine therapy (ET) combined with cyclin-dependent kinases 4 and 6 inhibitors (CDK4/6i) represents an alternative to chemotherapy (CT) in patients (pts) with hormone receptor-positive, HER2-negative (HR+/HER2-) LABC. Limited data exist on the real world and effectiveness of CDK4/6i and the post-surgical treatment patterns in this setting. Clinical and pathological data of consecutive pts with HR+/HER2- LABC treated with CDK4/6i + letrozole (+ ovarian suppression, if premenopausal) at the University “Federico II” and the National Cancer Institute “G. Pascale” (Naples, Italy) between July 2018 and July 2021 were collected. Study objectives included evaluation of clinical and pathologic responses, HR, HER2 and Ki67 expression in pre- and post-treatment tumor samples, post-surgical loco regional and systemic treatments. Fifty-nine pts (56% postmenopausal) were included in this study. Among these, 25 (42%), 29 (49%), and 5 (9%) pts received palbociclib, ribociclib, and abemaciclib, respectively. After a median duration of 7.5 months (range 1-16), 48 (81%) pts achieved an objective response and underwent surgery, however, no pathological complete response was detected. Three pts experienced disease progression during the treatment, while 8 pts were still under therapy at the time of this analysis. The mean expression of progesterone receptor and Ki67 significantly decreased in residual tumors compared to baseline (paired t-test P <0.001). After surgery, 24% of pts continued ET + CDK4/6i, 48% received anthracycline and/or taxane-based CT followed by ET, and 29% were treated with ET alone. Age <65 years, presence of residual disease >2 cm in breast and/or lymph node metastases after CDK4/6i were associated with CT use (chi-square t-test <0.05). CDK4/6i + ET showed biological and clinical activity in HR+/HER2- LABC. Treatment of physician's choice in pts who achieved tumor down staging and conversion to surgery was not uniform, probably due to the lack of evidence-based recommendations. Further clinical investigations are needed to define the optimal treatment approach for HR+/HER2- LABC.

Immune-checkpoint inhibitors for glioblastoma: what have we learned?

Glioblastoma, the most common malignant primary brain tumor, remains a lethal disease with few therapeutic options. Immunotherapies, particularly immune checkpoint inhibitors (ICPi), have revolutionized cancer treatment, but their role in glioblastoma is uncertain. To review the state of immunotherapies in glioblastoma, with an emphasis on recently published ICPi clinical trials. In this editorial/opinion article, we critically review results of the first generation of trials of ipilimumab, nivolumab and pembrolizumab in glioblastoma, as well as future directions. Expression of PD-L1 is frequent in glioblastoma, ranging from 60-70% of patients. Phase 1 studies of nivolumab with and without ipilimumab, as well as pembrolizumab, showed no new safety concerns in brain tumors, and no neurotoxicity. However, randomized phase 3 trials of nivolumab showed no survival improvements over bevacizumab in recurrent glioblastoma; no role in newly diagnosed disease as a replacement for temozolomide in unmethylated MGMT promoter tumors; and no benefit as an addition to temozolomide in methylated MGMT tumors. However, studies examining post treatment tumor samples have shown signs of increased immunologic response, and occasional long lasting radiographic responses have been seen. A small study of pembrolizumab suggested a potential role as a "neoadjuvant" treatment in resectable recurrent glioblastoma, while other studies are investigating selection of patients with higher mutational burden and novel agents and combinatorial strategies. Despite initial negative trials, immunotherapy remains of high interest in glioblastoma, and many trials are still ongoing. Improving our mechanistic understanding of the immunosuppression and T cell dysfunction induced by both tumor and the CNS microenvironment remains however crucial for the development of successful immunotherapeutic approaches in this disease.

HLA Class I Downregulation in Progressing Metastases of Melanoma Patients Treated With Ipilimumab.

Characterization of the molecular mechanisms underlying antitumor immune responses and immune escape mechanisms has resulted in the development of more effective immunotherapeutic strategies, including immune checkpoint inhibitor (ICI) therapy. ICIs can induce durable responses in patients with advanced cancer in a wide range of cancer types, however, the majority of the patients fail to respond to this therapy or develop resistance in the course of the treatment. Information about the molecular mechanisms underlying primary and acquired resistance is limited. Although HLA class I molecules are crucial in the recognition of tumor antigens by cytotoxic T lymphocytes, only a few studies have investigated the role of their expression level on malignant cells in ICI resistance. To address this topic, utilizing immunohistochemical staining with monoclonal antibodies (mAbs) we analyzed HLA class I expression level in pre-treatment and post-treatment tumor samples from melanoma patients treated with ipilimumab. Twenty-nine metastases removed from six patients were available for the study, including 18 pre-treatment and 11 post-treatment lesions. Compared to metastases excised before ipilimumab therapy, post-treatment lesions displayed a significantly lower HLA class I expression level on melanoma cells; HLA class I downregulation was most marked in progressing metastases from nonresponding patients. We also evaluated the level of infiltration by CD8+ T cells and NK cells but did not find consistent changes between pre- and post-treatment samples. Our results indicate the potential role of HLA class I downregulation as a mechanism of ICI resistance.

Comprehensive molecular characterization of muscle-invasive bladder cancer (MIBC) treated with durvalumab plus olaparib in the neoadjuvant setting: Neodurvarib trial.

546 Background: Immune checkpoint inhibitors have been incorporated to early-stage bladder carcinoma treatment recently. Durvalumab is a PD-L1 blocking antibody active in advance urothelial tumors and under evaluation in other settings of the disease. PARP inhibitors have shown activity in a variety of tumors with Homologous Recombination Deficiencies (HRD). The combination of Durvalumab plus Olaparib could present a synergistic effect, but its efficacy and potential biomarkers are under exploration. NEODURVARIB is a phase II clinical trial assessing the combination of Durvalumab plus Olaparib in MIBC (NCT03534492; SOGUG-2017-AIEC(VEJ)-2). Clinical activity and safety have been previously communicated by our group. Here we present the basal molecular profiles and their evolution under treatment with this combination. Methods: cT2-T4a MIBC aimed for cystectomy were treated during 6-8 weeks precystectomy. Pre- and post-treatment tumor and blood samples from 26 patients were collected. Pattern of immune infiltration was determined by IHQ. Genomics (mutational pattern, HRD and Tumor Mutation Burden [TMB]) and transcriptomics (differentially expressed loci, functional enrichment, molecular clustering and MIBC molecular subtyping) analysis were performed. Circulating immune populations were assessed using flow cytometry. Results: In basal (TURBT) samples, the frequency of mutations in genes commonly altered in MIBC ( TP53, MLL2, ARID1A, FGFR3, among others), HRD and TMB were similar to previous reports in MIBC and did not differ between responders and non-responders. Additionally, mutational patterns remained stable between baseline (TURBT) and post-treatment (cystectomy) samples. Regarding transcriptomics, GSEA showed enrichment of Epithelial Mesenchymal Transition (EMT), TGFβ and inflammatory/infection related classes in resistant tumors. Interestingly, differentially expressed genes in responders vs. non-responders were significantly regulated by epigenetic factors (EZH2/Suz12/PRC2 network). Transcriptomic-based estimations of the stromal/immune infiltration and MIBC molecular subtyping also showed a switch of the tumor microenvironment due to the treatment (luminal to basal/squamous transitions), reinforced by significant changes in the expression of immune markers (higher PDL1 and FAP scores in cystectomies). Lastly, circulating senescent T-cells were correlated with pathological complete response. Conclusions: Genetic alterations remained unchanged in bladder cancers treated with Durvalumab plus Olaparib. However, an enrichment of EMT signatures and a switch towards basal/squamous phenotypes were observed in resistant tumors. These findings underscore the relevance of modifications in gene expression as potential mechanisms of resistance to this combination. Clinical trial information: NCT03534492.

An open-label phase 2 study of lenvatinib plus pembrolizumab in the neoadjuvant and adjuvant treatment for patients with gastric and gastroesophageal junction adenocarcinoma (EPOC2001).

TPS372 Background: Lenvatinib, a multikinase inhibitor of VEGF receptors and other receptor tyrosine kinases, substantially decreased the tumor-associated macrophages and increased infiltration of CD8-positive T cells and enhanced anti-tumor activity of PD-1 inhibitors in vivo model. A phase 2 study demonstrated remarkable clinical activity of lenvatinib plus pembrolizumab for advanced gastric cancer with objective response rate of 69% (Kawazoe A, et al. Lancet Oncol. 2020), leading to the global phase 3 LEAP-015 trial at first-line (NCT04662710). The objective of this study is to evaluate anti-tumor activity of lenvatinib with pembrolizumab as neoadjuvant and adjuvant treatment for patients with gastric and gastroesophageal junction adenocarcinoma. Methods: The study is an open-label, single-arm, single-center, phase 2 clinical trial. Eligible patients are with previously untreated gastric and gastroesophageal junction adenocarcinoma as defined by cT2-4 and/or cN+ without evidence of metastatic disease. Patients will receive 3 cycles of 20 mg oral lenvatinib daily plus 200 mg intravenous pembrolizumab every 3 weeks as the neoadjuvant treatment followed by surgery, and then 3 cycles of lenvatinib plus pembrolizumab followed by 11 cycles of pembrolizumab monotherapy as the adjuvant treatment. The primary endpoint is major pathological response rate, and the secondary endpoints are pathological complete response rate, tumor shrinkage effect on primary lesions, radical resection rate, treatment completion rate until surgery or adjuvant treatment, event-free survival, overall survival, and incidence of adverse events. We also investigate several biomarkers using pre- and post-treatment tumor and blood samples. First patient will be enrolled in November 2021. Clinical trial information: NCT04745988.