Tumor Biopsies Research Articles

The combination of tumor mutational burden and T-cell receptor repertoire predicts the response to immunotherapy in patients with advanced non-small cell lung cancer.

Tumor mutational burden (TMB) and T-cell receptor (TCR) might predict the response to immunotherapy in patients with non-small cell lung cancer (NSCLC). However, the predictive value of the combination of TMB and TCR was not clear. Targeted DNA and TCR sequencing were performed on tumor biopsy specimens. We combined TMB and TCR diversity into a TMB-and-TCR (TMR) score using logistic regression. In total, 38 patients with advanced NSCLC were divided into a discovery set (n=17) and validation set (n=21). A higher TMR score was associated with better response and longer progression-free survival to immunotherapy in both the discovery set and validation set. The performance of TMR score was confirmed in the two external validation cohorts of 225 NSCLC patients and 306 NSCLC patients. Tumors with higher TMR scores were more likely to combine with LRP1B gene mutation (p=0.027) and top 1% CDR3 sequences (p=0.001). Furthermore, LRP1B allele frequency was negatively correlated with the top 1% CDR3 sequences (r=-0.55, p=0.033) and positively correlated with tumor shrinkage (r=0.68, p=0.007). The TMR score could serve as a potential predictive biomarker for the response to immunotherapy in advanced NSCLC.

OBX-115, an interleukin 2 (IL2)-sparing engineered tumor-infiltrating lymphocyte (TIL) cell therapy, in patients (pts) with immune checkpoint inhibitor (ICI)-resistant unresectable or metastatic melanoma.

9515 Background: Investigational unengineered TIL cell therapy has shown promising activity in ICI-resistant metastatic melanoma but requires systemic high-dose IL2, which has well-described high-grade toxicity frequently requiring specialized management and limiting pt eligibility. OBX-115 autologous engineered TIL cell therapy does not require co-administration of IL2 due to its regulatable expression of membrane-bound IL15 (mbIL15) using the FDA-approved small-molecule drug acetazolamide (ACZ) to provide cytokine support for TIL expansion and persistence. Methods: This Phase 1 study (NCT05470283) assesses the OBX-115 regimen in pts with ICI-resistant unresectable/metastatic melanoma. OBX-115 is manufactured from the pt’s tumor tissue (surgical excision or core needle biopsy [CNB]). After lymphodepletion (cyclophosphamide, fludarabine), pts receive OBX-115 followed by ACZ once daily for up to 7 days; ACZ redosing (up to 7 days) is permitted at Wk 6 for non-responders. No systemic IL2 is administered. Peripheral blood and tumor tissue is collected for longitudinal immune profiling. Results: As of 02 Jan 2024, 9 pts (median age, 50 y) with ICI-resistant metastatic melanoma were infused with OBX-115 (median study follow up, 17 wks; range, 2–58 wks). Median lines of prior therapy was 3 (range, 1–6). OBX-115 was successfully manufactured for all pts, including from CNB tumor tissue (n = 5). The infusion product had a high proportion of CD8+ cells (≥96%) and stem-like cells (CD8+CD39-CD69-; median 76%) with very low PD-1 expression in the CD8+ population ( < 1%). Post-infusion safety included no DLTs, 3 Gr 3 nonhematologic TEAEs in 2 pts (abdominal pain, ALT elevation, syncope), and no Gr 4 nonhematologic TEAEs. ACZ was redosed and well-tolerated in 4 of 5 eligible pts. Among patients with minimum 12-wk follow-up (n = 6), ORR per RECIST v1.1 was 50% (2 CR, 1 PR, 3 SD); all responses occurred between Wk 6–18 and were ongoing, with longest response sustained > 12 mo. One pt developed new metastatic disease (liver) and progressed at Wk 24, despite continued target lesion reduction; no pt developed brain metastasis. Post-infusion ctDNA was not detectable in any of the responders at Day 14 or 42. Though OBX-115 had minimal NK cells ( < 1%), post-infusion peripheral blood and tumor biopsies showed NK cell expansion, consistent with trans-presentation of mbIL15 to circulating NK cells. Updated efficacy data on all infused pts will be presented. Conclusions: OBX-115regulatableengineeredTIL cell therapy was well-tolerated and produced consistently deepening and durable responses, indicating that OBX-115 may mediate CRs and durable clinical benefit in ICI-resistant metastatic melanoma without high-dose IL2. OBX-115 investigation continues in this and an ongoing Phase 1/2 multicenter study (NCT06060613). Clinical trial information: NCT05470283 .

A window-of-opportunity (WOO) trial of giredestrant +/- LHRHa vs anastrozole+LHRHa in premenopausal women with ER+/HER2- early breast cancer (EBC; IBCSG 67-22; PREcoopERA).

TPS628 Background: Adjuvant endocrine therapy (ET) for premenopausal women with ER+/HER2- EBC often includes ovarian function suppression (OFS) via a GnRH or LHRH agonist (LHRHa) plus an aromatase inhibitor (AI) or tamoxifen. Younger age is associated with lower rates of ET adherence indicating side effects are less acceptable to these women. The development of drug regimens without OFS are clearly an unmet medical need in this setting. New oral selective ER degraders (SERDs) are emerging as potentially useful ETs in the (neo)adjuvant settings. Giredestrant is an efficient and potent SERD and a full antagonist, which translates into better anti-proliferation activity than known SERDs. PREcoopERA is a WOO trial aiming to determine, among premenopausal women with ER+/HER2- EBC: if giredestrant+LHRHa provides greater anti-proliferative activity than anastrozole+LHRHa; and if giredestrant without LHRHa provides similar (non-inferior) anti-proliferative activity to giredestrant+LHRHa. Results would provide rationale for subsequent (neo)adjuvant clinical trials. Methods: PREcoopERA (NCT05896566) is a randomized, open-label, 3-arm, WOO trial evaluating the anti-proliferative activity and safety of the oral SERD giredestrant +/- LHRHa triptorelin, as compared to the AI anastrozole + triptorelin. Eligible are premenopausal women with untreated ER+/HER2- operable stage I-III (excl. T4) EBC, with Ki-67 ≥10%. 220 women will be randomized in 2:2:1 ratio to giredestrant (30 mg/d po for 4 weeks until rebiopsy) + triptorelin (3.75 mg IM on D1); giredestrant alone; or anastrozole (1 mg/d po) + triptorelin. Rebiopsy is planned for D29, either during primary surgery or standalone procedures. The primary endpoint is change in Ki-67 labeling index between pre-treatment diagnostic tumor biopsy and post-treatment rebiopsy, as assessed by the IBCSG Pathology Office. Sample size of 80:80:40 randomized women are planned to test superiority of 4 weeks giredestrant+triptorelin vs anastrozole+triptorelin to reduce Ki-67 (hypothesized percentage changes -80% vs -65%) and to test non-inferiority of 4 weeks giredestrant vs giredesrant+triptorelin (within 10% as non-inferiority margin). The trial was activated on 9 October 2023. The first woman was enrolled on 25 January 2024. Clinical trial information: NCT05896566 .

A phase 2 trial of IO102-IO103 and nivolumab-relatlimab fixed-dose combination in previously untreated, unresectable melanoma.

TPS9605 Background: One emerging strategy to augment immune checkpoint inhibitor efficacy is to eliminate immunosuppressive cell populations in the tumor microenvironment (TME) through vaccination. IO102-IO103 is an investigational cancer vaccine that targets both tumor and immune-suppressive cells in the TME. IO102-IO103 treatment promotes inflammation and potentiates anti-tumor activity via activation and expansion of T cells against IDO1 and/or PD-L1 positive cells. IO102-IO103 plus nivolumab demonstrated an impressive objective response rate (ORR) of 80% in the phase 1/2 MM1636 trial of 30 patients with previously untreated, unresectable melanoma, and a randomized phase 3 trial of pembrolizumab with or without IO102-IO103 (NCT05155254) is ongoing. No prior studies have evaluated IO102-IO103 in combination with dual PD-1 and LAG-3 blockade with nivolumab-relatlimab (nivo-rela), and to our knowledge there are no existing clinical data for other investigational agents in combination with nivo-rela. Methods: NCT05912244 is a single-arm, investigator-initiated trial testing IO102-IO103 in combination with nivo-rela in patients with previously untreated, unresectable melanoma. Twelve of forty-three planned patients have been enrolled. All patients will be treated with nivo-rela every four weeks for up to two years. IO102-IO103 will be administered subcutaneously every two weeks for the initial eight weeks, and then every four weeks for up to two years. An interim efficacy analysis for futility will be performed after response data are available for 21 patients. Eligible patients must have histologically confirmed, unresectable stage III or stage IV non-uveal melanoma, measurable disease by RECIST v1.1, an ECOG performance status of 0 or 1, and definitive treatment of any brain metastases. Prior systemic treatment in the neoadjuvant or adjuvant setting is allowed if completed at least six months prior to trial enrollment. Available pre-treatment melanoma tissue is required, and all patients will undergo an on-treatment study biopsy between the second and third dose of nivo-rela. Peripheral blood mononuclear cells will be collected prior to the first three doses of nivo-rela. The primary endpoint is ORR by RECIST v1.1, weighted by PD-L1 expression. Secondary endpoints include safety assessed by Common Terminology Criteria for Adverse Events v 5.0, progression-free survival (PFS) by RECIST v1.1, duration of disease response, and disease control rate. Exploratory correlative endpoints include assessment of changes in PD-L1 and IDO-expressing cells in the TME on paired tumor biopsies, identification of peripheral PD-L1 and IDO-reactive cells in the peripheral blood by ELISpot, and assessment of peripheral pre-treatment and on-treatment T cell clonality by TCR sequencing. Clinical trial information: NCT05912244 .

Long-term survival follow-up for tebentafusp in previously treated metastatic uveal melanoma

BackgroundTebentafusp, a bispecific (gp100×CD3) ImmTAC, significantly improved overall survival (OS) outcomes for HLA-A*02:01+ adult patients with untreated metastatic uveal melanoma (mUM) and showed promising survival in previously treated mUM with...

Epigenomic signatures of sarcomatoid differentiation to guide the treatment of renal cell carcinoma

Renal cell carcinoma with sarcomatoid differentiation (sRCC) is associated with poor survival and a heightened response to immune checkpoint inhibitors (ICIs). Two major barriers to improving outcomes for sRCC are the limited understanding of its gene regulatory programs and the low diagnostic yield of tumor biopsies due to spatial heterogeneity. Herein, we characterized the epigenomic landscape of sRCC by profiling 107 epigenomic libraries from tissue and plasma samples from 50 patients with RCC and healthy volunteers. By profiling histone modifications and DNA methylation, we identified highly recurrent epigenomic reprogramming enriched in sRCC. Furthermore, CRISPRa experiments implicated the transcription factor FOSL1 in activating sRCC-associated gene regulatory programs, and FOSL1 expression was associated with the response to ICIs in RCC in two randomized clinical trials. Finally, we established a blood-based diagnostic approach using detectable sRCC epigenomic signatures in patient plasma, providing a framework for discovering epigenomic correlates of tumor histology via liquid biopsy.

Discriminating subtypes in advanced breast cancer with ctDNA methylation profiling.

1013 Background: Immunohistochemical subtyping of advanced breast cancer is critical to direct appropriate therapy. Yet, biopsy of recurrent cancer may not be technically possible, single-site tumour biopsies sample only a single metastasis and subtype may change through therapy. We developed a non-invasive blood test to subtype tumours based on circulating tumor DNA (ctDNA) methylation profiling. Methods: We interrogated TCGA-BRCA (level 3) methylation data from 783 tumours to identify regions with aberrant CpG methylation that differed between breast cancers and normal tissues, and between different breast cancer subtypes. A capture based enzymatic ctDNA based methylation profile was developed targeting 1257 differentially methylated regions (TWIST Methyl custom panel, TWIST Bioscience, panel size 0.4Mb). Plasma samples from patients with breast cancer, and healthy donors, were sequenced with the capture assay. A machine learning (ML) classifier was built to detect the presence of breast cancer, and a second classifier was built to discriminate ER positive v ER negative, and HER2 positive v HER2 negative from ctDNA methylation profiles. A 10-fold stratified nested cross-validation was used to assess classifier characteristics. Results: Plasma samples from 427 people (126 health donor, 27 early breast cancer patients and 291 metastatic breast cancer patients), along with 133 tumor tissue samples, were sequenced and a ML classifier built to detect the presence of breast cancer. In cross-validation, a random forest classifier yielded a median AUC of 0.996 (SD 0.020), with sensitivity of 94.9% at 99% specificity. A total of 231 ER positive and 63 ER negative plasma samples from patients were used for IHC ER status differentiation. In cross-validation a LGBM classifier yielded a median AUC of 0.928 (SD 0.051). Median sensitivity was 76.7% at specificity 86-99%. A total of 11% (25/231) ER positive and 8% (5/63) ER negative samples were misclassified. A total of 41 HER2 positive and 253 HER2 negative plasma samples were used for IHC HER2 status differentiation. In cross-validation a LGBM classifier yielded a median AUC of 0.934 (SD 0.075). Median sensitivity was 77.5% at specificity 85-92%, A total of 7% (3/41) HER-2 positive and 13% (33/253) HER2 negative samples were misclassified. In both IHC classifiers, subtype misclassifications were likely due to multiple concurrent BC tumours of differing IHC subtypes, biopsy proven subtype transitions and low purity. Effects of tumor purity on IHC-based classifiers will also be presented. Conclusions: Non-invasivemethylation based ctDNA subtyping can detect the presence of breast cancer, and discriminate IHC subtypes, with high accuracy. Further assessment is warranted to assess whether methylation-based subtyping has the potential to report IHC subtypes when repeat biopsy is not possible, detect subtype transitions and direct advanced breast cancer treatment.

A first-in-human study to evaluate a personalized neoantigen-based mRNA-loaded dendritic cell vaccine, in combination with ablation, in patients with hepatocellular carcinoma.

e14513 Background: Local recurrence of Hepatocellular Carcinoma (HCC) following potentially curative resection or ablation is an inevitable risk. T cells recognizing neoantigens are present in most cancers including HCC and offer a specific and highly immunogenic target for the personalized vaccination as an adjuvant therapy. The purpose of this study is to evaluate the safety and T cell immune response of a personalized neoantigen-based mRNA loaded dendritic cell (DC) vaccine (LK101) combined with regular ablation in patients with HCC (NCT03674073). Methods: Each patient’s fresh tumor biopsy and matched peripheral blood sample were subjected to whole-exome sequencing and RNA-sequencing. Following determination of somatic mutations and gene expression, up to 30 in silico–predicted potential neoantigens were selected with bioinformatic tool. DCs were generated from the monocytes enriched by leukapheresis, followed by transfection with neoantigen-encoding mRNA through electroporation. In total, 7 doses of LK101 were administered at 1.5×107 cells/injection intradermally with 4-dose priming weekly and 3 boosters in 4-week intervals. The primary endpoint of the clinical study was safety, and key secondary endpoints were immunogenicity, overall survival and recurrence. 24 HCC patients at HKLC stage IIa were enrolled 1:1 to ablation control arm and ablation combined with DC (LK101) arm. Results: The study was completed, patients were followed up for a median of 48.4 months and 38.8 months in vaccination arm and control arm. All of adverse events were Grade 1-2. Most frequent treatment-related adverse events are injection site reactions, inclusive of local pain (63.6%), itch (27.3%) and others (27.3%), such as redness and edema. 3 patients in ablation control arm died, whereas all vaccinated patients survived. The 1-year and 2-year recurrence rates in vaccination arm compared with control arm are 18.2%, respectively. LK101 induced neoantigen-specific T cell responses were investigated by ELISpot analysis. Overall, all patients elicited at least one measurable response. Immune responses to 60-90% of neoantigen peptides were seen post vaccination across patients. Both preexisting immune response and De Novo immune response that targeted neoantigens were observed in vaccinated patients. Moreover, both CD4 and CD8 positive effector memory T cells showed the trend of increase, and PD-1 positive T cells reduced in peripheral blood of patients after vaccination. Conclusions: Combination of LK101 with ablation therapy has a manageable safety profile, showing the evidence of immune activation and potential of prolonged survival. In combination with regular ablation, neoantigen based mRNA loaded DC vaccine is a promising immunotherapy for the treatment of HCC. Clinical trial information: NCT03674073 .

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]

Monocyte/lymphocyte ratio to predict immunoradiotherapy response in patients with advanced pretreated sarcoma.

e23523 Background: Immuno-radiotherapy may improve outcomes for patients with advanced solid tumors, although optimized combination modalities remain unclear. Here, we report the sarcoma cohort analysis from the SABR-PDL1 trial that evaluated the PD-L1 inhibitor atezolizumab in combination with stereotactic body radiation therapy (SBRT) in patients with advanced cancer. Methods: Eligible patients received atezolizumab 1200 mg every 3 weeks until progression or unmanageable toxicity, together with ablative SBRT delivered concurrently with the 2nd cycle (recommended dose of 45 Gy in 3 fractions, adapted upon normal tissue tolerance constraint). SBRT was delivered to at least one tumor site, with at least one additional measurable lesion being kept from the radiation field. The primary efficacy endpoint was one-PFS rate from the start of atezolizumab. Sequential tumor biopsies were collected for deep multi-feature immune profiling and monocyte/lymphocyte ratio (sum of cells derived from the monocytic lineage including monocytes, macrophages and myeloid dendritic cells, divided by sum of lymphocytes) were computed both on tumor and blood. Results: 61 highly pretreated (median of 5 prior lines) patients with advanced sarcomas (leiomyosarcoma: 28% and other types; 32 men [53%]; median age, 47 years [range, 37-55 years]) were enrolled in two centers (France; Spain) from 03/2020 to 04/2021. All but one received atezolizumab and 56/61 (92%) received SBRT. The most frequently irradiated site was lung (n = 36/72 lesions; 50%). Treatment-related G3 (no G4-5) toxicity was observed in one (2%) patient. Median OS and PFS were respectively 12.2 [95%CI:7-18.4] and 2.5 months [95%CI:1.2-2.6], including five (8.2%) patients with PFS > 1 year. Best overall responses consisted of PR (n = 3;5%) and SD (n = 36; 60%). Altogether, 182 (n = 86 paraffin; n = 96 freshly frozen) sequential tumor biopsies from 35 patients were successfully analyzed. None (0%) of the biopsies harbored tertiary-lymphoid structures. Tumor RNA sequencing analysis using independent immune deconvolution technics including CIBERSORTabs, EPIC and xCELL revealed an increased tumor infiltration of monocytic lineage-derived cells in patients who rapidly progressed. Differential gene expression analysis showed a significant upregulation of genes implicated in immunosuppression in unressponsive tumors. Unresponsive tumors were enriched with immune chemoattractants. We found higher monocyte/lymphocyte ratio in unresponsive patients compared to patients who had stable disease or tumor response both in the tumor and in the blood. Matched blood and tumor monocyte/lymphocyte ratios were significantly correlated in this population. Conclusions: This study provides new data on the feasibility, efficacy, and immune context of tumors that may help identifying patients with advanced sarcomas most likely to respond to immuno-radiotherapy. Clinical trial information: NCT02992912 .

Biomarker discordance after neoadjuvant systemic therapy and associated patterns of recurrence.

3149 Background: Understanding and addressing intratumor heterogeneity in breast cancer (BC) is crucial for advancing research and improving patient outcomes. The identification of biomarkers for targeted therapies is complex when dealing with heterogeneous tumors, and this heterogeneity complicates cancer outcomes since it affects treatment response, and the development of resistance. Methods: A single institution retrospective review of patients with BC who received neoadjuvant systemic therapy (NAST) followed by definitive surgical resection from 2008 to 2022 was performed. Initial tumor biopsies were tested for hormone receptors (HR) [estrogen receptor (ER) and progesterone receptor (PR)], and HER2 status and repeated on the residual tumor tissue after NAST. HER2 status was categorized into HER2-negative, HER2-low, and HER2-positive and HR status was categorized into ER and/or PR positive or negative based on the 2023 ASCO-CAP guidelines. HER2 and HR concordance and discordance rates were obtained and patients who had disease recurrence were identified and analyzed using descriptive statistics and Fisher’s exact test. Results: 480 patients with BC received NAST, and 143 patients with residual disease had pre and post treatment biomarker data available for analysis with a median post-surgery follow up of 15.2 months. Discordance in either HER2 or HR status was identified in 50% (72/143) of samples. Out of 143 patients, 44 patients had disease recurrence in which 50% (22/44) of tumors exhibited biomarker discordance. Among the changes in biomarker status, a shift in PR expression occurred most frequently in 27% of tumors (12/44). A change from HER2-low to HER2-negative status, as well as changes in ER expression occurred in 11% of tumors (5/44). 14% (6/44) of tumors had changes in both HER2 and HR. In patients who did not have recurrence, 51% (50/99) of tumors exhibited biomarker discordance. A HER2-low to HER2-negative status occurred most frequently in 18% (18/99). A change in ER expression occurred second most frequently, in 14% (14/99) of tumors. There was no statistical significance of discordance rates in the recurrence group and non-recurrence group (p-value=0.96). Conclusions: Tumor heterogeneity plays a role in shaping the distinct tumor biology of each patient. While the rates of biomarker discordance were comparable between samples with and without recurrence, the importance of HR discordance in this group requires extensive long-term monitoring. Our research highlights the importance of biomarker changes in a curative setting. Considering the implications for treatment, further investigation into additional biological and tumor-related factors associated with recurrence is warranted. [Table: see text]

Apatinib and camrelizumab plus hepatic arterial infusion with oxaliplatin and 5-fluorouracil vs. apatinib and camrelizumab as the first-line treatment for hepatocellular carcinoma with portal vein tumor thrombus: A randomized multi-center clinical trial.

TPS4194 Background: The combination of immune checkpoint inhibitors and antiangiogenic drugs has been approved as the first-line treatment for advanced hepatocellular carcinoma (HCC). However, the survival benefit is still limited, especially in patients with high-risk features (Vp4, and/or bile duct invasion and/or tumor occupancy of ≥50% of the liver). Hepatic arterial infusion chemotherapy (HAIC) plus apatinib (an antiangiogenic drug) and camrelizumab (a PD-1 antibody) showed encouraging antitumor activity for these patients in a previous phase 2 study. Herein, we investigated the efficacy and safety of apatinib and camrelizumab plus HAIC compared with apatinib and camrelizumab as the first-line treatment for HCC with portal vein tumor thrombus (PVTT). Methods: In this randomized, open-label, phase 3 trial, eligible patients are ≥18 years with HCC confirmed by radiology, histology, or cytology. Other eligibility criteria were as follows: with PVTT, ECOG PS 0/1, no history of systemic therapy, not amenable to curative treatment approach, Child-Pugh A class liver score, and ≥1 measurable lesion by RECIST v1.1. A total of 214 patients (anticipated) will be randomized (1:1) to receive apatinib (250mg orally once daily) and camrelizumab (200 mg IV Q2W) plus HAIC with FOLFOX (oxaliplatin 85 mg/m2, leucovorin 400 mg/m2, 5-fluorouracil bolus 400 mg/m2 on day 1, and 5-fluorouracil infusion 2400 mg/m2 for 20 hours via hepatic artery Q4W) or apatinib (250mg orally once daily) and camrelizumab (200 mg IV Q2W) until disease progression or intolerable toxicity. Stratification will be by patients with or without high-risk features. The primary endpoint was overall survival. Secondary endpoints were progression-free survival, objective response rate per RECIST 1.1 and mRECIST, disease control rate per RECIST 1.1 and mRECIST, time to progression, duration of response, time to response, and safety. Whole-Exome Sequencing and RNA Sequencing of tumor biopsy samples were performed for predictive biomarker exploration. Clinical trial information: NCT05198609 .

Mutational analysis of cfDNA to identify predictive biomarkers in previously untreated patients with metastatic pancreatic cancer receiving the GSK-3 inhibitor elraglusib (9-ING-41) in combination with gemcitabine/nab-paclitaxel in the 1801 phase 2 study.

e16306 Background: Glycogen Synthase Kinase-3beta has been well-credentialed as a therapeutic target in patients with advanced cancer including pancreatic cancer. Elraglusib (9-ING-41), a novel best-in-class GSK-3 inhibitor, is being developed in combination with gemcitabine/nab-paclitaxel (GnP) as first-line therapy for patients with metastatic pancreatic cancer (mPDAC). Using liquid biopsy samples from an ongoing randomized phase 2 trial (1801 Part 3B; NCT03678883), we examined whether gene mutations in cfDNA correlated with clinical outcomes in mPDAC patients treated with GnP or elraglusib/GnP. Methods: Pre-dose plasma samples were obtained from 37 (GnP arm) and 89 (elraglusib/GnP arm) mPDAC patients previously untreated for advanced disease. Mutational analysis of cfDNA extracted from plasma samples was performed by Tempus (Chicago, IL) using the xF+ platform. The Tempus xF+ assay is an NGS cfDNA liquid biopsy tumor profiling assay for identifying genomic alterations in 523 genes. Tempus xF+ reports were used for analysis of gene mutations. Results: In 126 pancreatic cancer patients, frequently mutated genes ( > 10%) consisted of KRAS (93/126, 74%), TP53 (79/126, 63%), CDKN2A (26/126, 21%), DNMT3A (25/126, 20%), NOTCH1/2 (25/126, 20%), and MLL3 (23/126, 18%). Gain-of-function mutations in KRAS, and loss-of-function mutations of TP53, and CDKN2A genes were associated with worse overall survival (OS) in patients treated with elraglusib/GnP (P < 0.05) but not in GnP-treated patients. Multivariate analysis of frequently mutated genes showed that patients with double (KRAS+TP53) and triple (KRAS+TP53+CDKN2A) mutations had significantly worse OS only in the elraglusib/GnP arm. The presence of DNMT3A, NOTCH1/2 or MLL3 gene mutations did not correlate with any clinical outcomes. Conclusions: Our preliminary analysis of gene mutations in liquid biopsy samples obtained from patients in the ongoing 1801 Part 3B phase 2 trial identified potential predictive biomarkers of clinical outcome in mPDAC patients treated with combination of elraglusib/GnP. Clinical trial information: NCT03678883 .

Hormone Receptor Expression and Activity for Different Tumour Locations in Patients with Advanced and Recurrent Endometrial Carcinoma.

Response to hormonal therapy in advanced and recurrent endometrial cancer (EC) can be predicted by oestrogen and progesterone receptor immunohistochemical (ER/PR-IHC) expression, with response rates of 60% in PR-IHC > 50% cases. ER/PR-IHC can vary by tumour location and is frequently lost with tumour progression. Therefore, we explored the relationship between ER/PR-IHC expression and tumour location in EC. Pre-treatment tumour biopsies from 6 different sites of 80 cases treated with hormonal therapy were analysed for ER/PR-IHC expression and classified into categories 0-10%, 10-50%, and >50%. The ER pathway activity score (ERPAS) was determined based on mRNA levels of ER-related target genes, reflecting the actual activity of the ER receptor. There was a trend towards lower PR-IHC (33% had PR > 50%) and ERPAS (27% had ERPAS > 15) in lymphogenic metastases compared to other locations (p = 0.074). Hematogenous and intra-abdominal metastases appeared to have high ER/PR-IHC and ERPAS (85% and 89% ER-IHC > 50%; 64% and 78% PR-IHC > 50%; 60% and 71% ERPAS > 15, not significant). Tumour grade and previous radiotherapy did not affect ER/PR-IHC or ERPAS. A trend towards lower PR-IHC and ERPAS was observed in lymphogenic sites. Verification in larger cohorts is needed to confirm these findings, which may have implications for the use of hormonal therapy in the future.

A pediatric case of Burkitt’s lymphoma with bile duct obstruction requiring surgical biliary reconstruction

BackgroundBiliary obstruction due to compression by a B-cell solid tumor occurs rarely. A few reports have described biliary reconstruction surgery for obstructive jaundice caused by Burkitt’s lymphoma. However, there are no detailed reports on pediatric cases. We report a pediatric case of obstructive jaundice due to malignant lymphoma treated with biliary reconstruction surgery.Case presentationA 5-year-old girl presented to our hospital with a massive abdominal tumor that caused biliary stricture. Chemotherapy was initiated after an open tumor biopsy. However, endoscopic biliary stent placement was performed owing to elevated bilirubin levels. We treated the patient with chemotherapy for 9 months while endoscopically replacing the biliary stent every few months. She achieved complete tumor remission. However, sclerotic lymph nodes were persistent on the dorsal side of the cholecystic duct junction, and biliary stricture at the same site had changed to stent-dependent biliary obstruction. Therefore, we performed choledochojejunostomy and retrocolic Roux-en-Y reconstruction 15 months after initial admission. There were no postoperative complications or tumor recurrences, and the bilirubin level remained low. Histopathologically, the resected bile duct wall was fibrotic and thick, and the bile duct lumen narrowed.ConclusionsBiliary reconstruction is effective to achieve long-term biliary patency in pediatric patients with stent-dependent biliary obstruction due to malignant lymphoma. However, the decision on when to stop biliary stent replacement and proceed to biliary reconstruction surgery is a matter of debate. Further case studies are required to address this issue.

Rapid detection of IDH mutations in gliomas by intraoperative mass spectrometry

The development and performance of two mass spectrometry (MS) workflows for the intraoperative diagnosis of isocitrate dehydrogenase (IDH) mutations in glioma is implemented by independent teams at Mayo Clinic, Jacksonville, and Huashan Hospital, Shanghai. The infiltrative nature of gliomas makes rapid diagnosis necessary to guide the extent of surgical resection of central nervous system (CNS) tumors. The combination of tissue biopsy and MS analysis used here satisfies this requirement. The key feature of both described methods is the use of tandem MS to measure the oncometabolite 2-hydroxyglutarate (2HG) relative to endogenous glutamate (Glu) to characterize the presence of mutant tumor. The experiments i) provide IDH mutation status for individual patients and ii) demonstrate a strong correlation of 2HG signals with tumor infiltration. The measured ratio of 2HG to Glu correlates with IDH-mutant (IDH-mut) glioma (P < 0.0001) in the tumor core data of both teams. Despite using different ionization methods and different mass spectrometers, comparable performance in determining IDH mutations from core tumor biopsies was achieved with sensitivities, specificities, and accuracies all at 100%. None of the 31 patients at Mayo Clinic or the 74 patients at Huashan Hospital were misclassified when analyzing tumor core biopsies. Robustness of the methodology was evaluated by postoperative re-examination of samples. Both teams noted the presence of high concentrations of 2HG at surgical margins, supporting future use of intraoperative MS to monitor for clean surgical margins. The power of MS diagnostics is shown in resolving contradictory clinical features, e.g., in distinguishing gliosis from IDH-mut glioma.

Ganglioside SSEA-4 in Ewing sarcoma marks a tumor cell population with aggressive features and is a potential cell-surface immune target

Carbohydrate markers of immature cells during prenatal human development can be aberrantly expressed in cancers and deserve evaluation as immune targets. A candidate target in Ewing sarcoma is the globo-series ganglioside stage-specific embryonic antigen-4 (SSEA-4). We detected SSEA-4 expression on the cell surface of all of 14 EwS cell lines and in 21 of 31 (68%) primary EwS tumor biopsies. Among paired subpopulations of tumor cells with low versus high SSEA-4 expression, SSEA-4high expression was significantly and consistently associated with functional characteristics of tumor aggressiveness, including higher cell proliferation, colony formation, chemoresistance and propensity to migrate. SSEA-4low versus SSEA-4high expression was not related to expression levels of the EWSR1-FLI1 fusion transcript or markers of epithelial/mesenchymal plasticity. SSEA-4low cells selected from bulk populations regained higher SSEA-4 expression in vitro and during in vivo tumor growth in a murine xenograft model. T cells engineered to express SSEA-4-specific chimeric antigen receptors (CARs) specifically interacted with SSEA-4 positive EwS cells and exerted effective antigen-specific tumor cell lysis in vitro. In conclusion, with its stable expression and functional significance in EwS, SSEA-4 is an attractive therapeutic immune target in this cancer that deserves further evaluation for clinical translation.

In silico analysis of the wild-type and mutant-type of BRCA2 gene

BackgroundThe aim of this study was to conduct an in silico analysis of a novel compound heterozygous variant in breast cancer susceptibility gene 2 (BRCA2) to clarify its structure–function relationship and elucidate the molecular mechanisms underlying triple-negative breast cancer (TNBC).MethodsA tumor biopsy sample was obtained from a 42-year-old Chinese woman during surgery, and a maxBRCA™ test was conducted using the patient’s whole blood. We obtained an experimentally determined 3D structure (1mje.pdb) of the BRCA2 protein from the Protein Data Bank (PDB) as a relatively reliable reference. Subsequently, the wild-type and mutant structures were predicted using SWISS-MODEL and AlphaFold, and the accuracy of these predictions was assessed through the SAVES online server. Furthermore, we utilized a high ambiguity-driven protein–protein docking (HADDOCK) algorithm and protein–ligand interaction profiler (PLIP) to predict the pathogenicity of the mutations and elucidate pathogenic mechanisms that potentially underlies TNBC.ResultsHistological examination revealed that the tumor biopsy sample exhibited classical pathological characteristics of TNBC. Furthermore, the maxBRCA™ test revealed two compound heterozygous BRCA2 gene mutations (c.7670 C > T.pA2557V and c.8356G > A.pA2786T). Through performing in silico structural analyses and constructing of 3D models of the mutants, we established that the mutant amino acids valine and threonine were located in the helical domain and oligonucleotide binding 1 (OB1), regions that interact with DSS1.ConclusionOur analysis revealed that substituting valine and threonine in the helical domain region alters the structure and function of BRCA2 proteins. This mutation potentially affects the binding of proteins and DNA fragments and disrupts interactions between the helical domain region and OB1 with DSS1, potentially leading to the development of TNBC. Our findings suggest that the identified compound heterozygous mutation contributes to the clinical presentation of TNBC, providing new insights into the pathogenesis of TNBC and the influence of compound heterozygous mutations in BRCA2.

Administration of intratumoral GD2-directed interleukin-2 immunocytokine and local radiation therapy to activate immune rejection of spontaneous canine melanoma.

Canine malignant melanoma provides a clinically relevant, large animal parallel patient population to study the GD2-reactive hu14.18-IL-2 immunocytokine as it is similar to human melanoma and expresses GD2. The objectives of this study were to evaluate safety, radiation fractionation, and identify informative biomarkers of an in-situ tumor vaccine involving local radiation therapy plus intratumoral-immunocytokine in melanoma tumor-bearing dogs. Twelve dogs (six dogs/arm) with locally advanced or metastatic melanoma were randomized to receive a single 8 Gy fraction (arm A) or three 8 Gy fractions over 1 week (arm B) to the primary site and regional lymph nodes (when clinically involved) with the single or last fraction 5 days before intratumoral-immunocytokine at 12 mg/m 2 on 3 consecutive days. Serial tumor biopsies were obtained. All 12 dogs completed protocol treatment, and none experienced significant or unexpected adverse events. Evidence of antitumor activity includes one dog with a complete response at day 60, one dog with a partial response at day 60, and four dogs with mixed responses. Histology of serial biopsies shows a variably timed increase in intratumoral lymphocytic inflammation in some dogs. Canine NanoString analyses of serial biopsies identified changes in gene signatures of innate and adaptive cell types versus baseline. There were no significant differences in NanoString results between arm A and arm B. We conclude that intratumoral-immunocytokine in combination with local radiation therapy in canine melanoma is well tolerated and has antitumor activity with the potential to inform clinical development in melanoma patients.

Abstract PR011: Imaging mass cytometry captures patient heterogeneity enabling BCG response stratification in non-muscle invasive bladder cancer

Abstract BCG immunotherapy is the standard of care for high-risk non-muscle invasive bladder cancer (NMIBC). The mechanisms of response to BCG are not fully understood. Patient response to BCG is highly variable with ~30-40% of individuals experiencing recurring or progressing disease after treatment within 24 months. Prognostic markers of response to treatment are not fully understood and remain insufficient for patient stratification. In order to develop an understanding of BCG response by comprehensive assessment of the tumor context. We performed single cell spatial proteomic profiling via imaging mass cytometry (IMC) on tissue microarrays of tumor biopsies from 53 bladder cancer patients (36 responding and 17 non-responding). We then validated our finding on an external cohort of whole pathology slides from 10 patients (4 responding and 6 non-responding). The IMC panel was developed based on scRNAseq profiles of bladder tumor samples to provide an unbiased cellular context of NMIBC. Additionally, IMC data is accompanied by hematoxylin and eosin (H&amp;E) images of adjacent cuts and annotated by a clinical pathologist to distinguish tumor, immune, and stromal regions. We first developed a convolutional neural network (CNN) to reproduce pathologist annotations of tumor, stroma, and immune regions from H&amp;E data with high accuracy (AUC&amp;gt;93%). We then registered IMC and annotated H&amp;E images to train a random forest model to predict pathologist annotations using IMC data (AUC&amp;gt;95%). To determine the cellular composition of TMA spots, we used sparse non-negative matrix factorization (sNMF) to encode and cluster IMC data within tumor, stroma, and immune regions. NMF analysis revealed that a component defined primarily by KRT5, CD11b, and CD14, specifically within tumor-annotated regions adjoining COL1A1+ stromal regions, is a prognostic marker of poor response (p-value&amp;lt;0.05). This is consistent with previous findings that tumor-associated myeloid cells indicate poor prognosis. Additional NMF components of the immune annotated region showed weak associations with response. Immune regions defined by CD68, CD11b, CD31, and PDPN corresponded with poor BCG response, indicating myeloid-stromal-vascular interactions outside the tumor influence outcome, while vascular immune regions defined by high aSMA presence correspond with better response. Collectively, combining NMF components to generate a single response score for responding and non-responding patients (p-value&amp;lt;0.05) proved more effective at patient stratification than any single NMF component alone. The external cohort validated our random forest model for separating tumor, stroma, and immune regions, our sNMF model for clustering IMC images, and our response score (p-value&amp;lt;0.1). Citation Format: Ali Foroughi Pour, Dylan Baker, Santhosh Sivajothi, Bonnie Choy, Khyati Meghani, Yanni Yu, Leigh A. Fall, Benjamin Ristau, Joshua J. Meeks, Paul Robson, Jeffrey H. Chuang. Imaging mass cytometry captures patient heterogeneity enabling BCG response stratification in non-muscle invasive bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr PR011.