Kahila, Mohamed M.

Abstract Recent advances in the field of immuno‐oncology have brought transformative changes in the management of cancer patients. The immune profile of tumours has been found to have key value in predicting disease prognosis and treatment response in various cancers. Multiplex immunohistochemistry and immunofluorescence have emerged as potent tools for the simultaneous detection of multiple protein biomarkers in a single tissue section, thereby expanding opportunities for molecular and immune profiling while preserving tissue samples. By establishing the phenotype of individual tumour cells when distributed within a mixed cell population, the identification of clinically relevant biomarkers with high‐throughput multiplex immunophenotyping of tumour samples has great potential to guide appropriate treatment choices. Moreover, the emergence of novel multi‐marker imaging approaches can now provide unprecedented insights into the tumour microenvironment, including the potential interplay between various cell types. However, there are significant challenges to widespread integration of these technologies in daily research and clinical practice. This review addresses the challenges and potential solutions within a structured framework of action from a regulatory and clinical trial perspective. New developments within the field of immunophenotyping using multiplexed tissue imaging platforms and associated digital pathology are also described, with a specific focus on translational implications across different subtypes of cancer. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Abstract Modern histologic imaging platforms coupled with machine learning methods have provided new opportunities to map the spatial distribution of immune cells in the tumor microenvironment. However, there exists no standardized method for describing or analyzing spatial immune cell data, and most reported spatial analyses are rudimentary. In this review, we provide an overview of two approaches for reporting and analyzing spatial data (raster versus vector‐based). We then provide a compendium of spatial immune cell metrics that have been reported in the literature, summarizing prognostic associations in the context of a variety of cancers. We conclude by discussing two well‐described clinical biomarkers, the breast cancer stromal tumor infiltrating lymphocytes score and the colon cancer Immunoscore, and describe investigative opportunities to improve clinical utility of these spatial biomarkers. © 2023 The Pathological Society of Great Britain and Ireland.

AbstractThe clinical significance of the tumor‐immune interaction in breast cancer is now established, and tumor‐infiltrating lymphocytes (TILs) have emerged as predictive and prognostic biomarkers for patients with triple‐negative (estrogen receptor, progesterone receptor, and HER2‐negative) breast cancer and HER2‐positive breast cancer. How computational assessments of TILs might complement manual TIL assessment in trial and daily practices is currently debated. Recent efforts to use machine learning (ML) to automatically evaluate TILs have shown promising results. We review state‐of‐the‐art approaches and identify pitfalls and challenges of automated TIL evaluation by studying the root cause of ML discordances in comparison to manual TIL quantification. We categorize our findings into four main topics: (1) technical slide issues, (2) ML and image analysis aspects, (3) data challenges, and (4) validation issues. The main reason for discordant assessments is the inclusion of false‐positive areas or cells identified by performance on certain tissue patterns or design choices in the computational implementation. To aid the adoption of ML for TIL assessment, we provide an in‐depth discussion of ML and image analysis, including validation issues that need to be considered before reliable computational reporting of TILs can be incorporated into the trial and routine clinical management of patients with triple‐negative breast cancer. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Abstract Triple-negative breast cancer (TNBC) is characterized by elevated locoregional recurrence risk despite aggressive local therapies. New tumor-specific radiosensitizers are needed. We hypothesized that the ATR inhibitor, VX-970 (now known as M6620), would preferentially radiosensitize TNBC. Noncancerous breast epithelial and TNBC cell lines were investigated in clonogenic survival, cell cycle, and DNA damage signaling and repair assays. In addition, patient-derived xenograft (PDX) models generated prospectively as part of a neoadjuvant chemotherapy study from either baseline tumor biopsies or surgical specimens with chemoresistant residual disease were assessed for sensitivity to fractionated radiotherapy, VX-970, or the combination. To explore potential response biomarkers, exome sequencing was assessed for germline and/or somatic alterations in homologous recombination (HR) genes and other alterations associated with ATR inhibitor sensitivity. VX-970 preferentially inhibited ATR-Chk1-CDC25a signaling, abrogated the radiotherapy-induced G2–M checkpoint, delayed resolution of DNA double-strand breaks, and reduced colony formation after radiotherapy in TNBC cells relative to normal-like breast epithelial cells. In vivo, VX-970 did not exhibit significant single-agent activity at the dose administered even in the context of genomic alterations predictive of ATR inhibitor responsiveness, but significantly sensitized TNBC PDXs to radiotherapy. Exome sequencing and functional testing demonstrated that combination therapy was effective in both HR-proficient and -deficient models. PDXs established from patients with chemoresistant TNBC were also highly radiosensitized. In conclusion, VX-970 is a tumor-specific radiosensitizer for TNBC. Patients with residual TNBC after neoadjuvant chemotherapy, a subset at particularly high risk of relapse, may be ideally suited for this treatment intensification strategy. Mol Cancer Ther; 17(11); 2462–72. ©2018 AACR.

Abstract Purpose: TNBC represents an aggressive subtype characterized by earlier relapse and worse survival compared to non-TNBC. Patients with residual TNBC after neoadjuvant chemotherapy (NAC) have a high risk of locoregional recurrence despite aggressive subsequent local therapies, including RT. Thus, new radiosensitizers that address chemoresistant TNBC (i.e residual disease after NAC) are needed. ATR plays a key role in the cellular response to replication stress and DNA damage. TNBC is characterized by G1 checkpoint loss, homologous recombination deficiency (HRD), and other features which increase dependence on ATR pathway signaling. We hypothesized that VX-970, a selective inhibitor of ATR, would effectively radiosensitize TNBC. Methods: Cell lines representing varying subtypes of TNBC (MDA-MB-231, HCC1806, and BT-549) and the normal breast epithelial cell line MCF10a were investigated in clonogenic survival, cell cycle, and DNA damage signaling and repair assays. In addition, Mayo Clinic (MC)TNBC1 and MCTNBC2, patient derived xenograft models (PDXs) generated prospectively from baseline and chemoresistant residual disease surgical specimens after NAC, respectively, of unique patients in the Breast Cancer Genome Guided Therapy Study were analyzed. Tumors were injected into hind-legs of athymic nude mice and animals were randomized to: 1) Vehicle 2) RT 3) VX-970 4) RT + VX-970. RT was delivered in 5 daily fractions of 2 Gy. VX-970 was administered daily 1 hour prior to RT at a dose of 60 mg/kg. Exome sequencing was assessed for germ-line and/or somatic alterations in HR genes and an ex-vivo RAD51 foci formation assay was applied to confirm the functional status of HR in each PDX model. Results: In vitro, VX-970 preferentially inhibited ATR-Chk1-CDC25a signaling, abrogated the RT-induced G2/M checkpoint, delayed resolution of γH2AX and 53BP1 foci and reduced colony formation after RT in MDA-MB-231, HCC1806, and BT-549 relative to MCF10a. In vivo, VX-970 did not exhibit single agent activity at the dose administered but markedly sensitized both MCTNBC1 and MCTNBC2 to RT, suggesting that VX-970 could overcome chemoresistant TNBC biology. For MCTNBC1 and MCTNBC2, the median time to tumor tripling was 26 vs 49 days (p=0.002) and 25 vs 43 days (p=0.006) for the RT and RT + VX-970 groups, respectively. Ex-vivo RAD51 foci formation was robust in MCTNBC1, a PDX without HR gene alterations. In contrast, there was no RT-induced RAD51 foci formation in MCTNBC2, suggesting a BRCA1 mutation in the corresponding human tumor resulted in loss of HR function in that model. Conclusion: VX-970 + RT effectively radiosensitized TNBC PDXs established from both pre-treatment biopsy specimens and chemoresistant residual tumor samples and in both HR deficient and HR proficient settings. ATR inhibition should be investigated in the clinic in combination with RT for patients with residual TNBC after NAC. Citation Format: Robert W. Mutter, Mohamed M. Kahila, Xinyi Tu, Qin Zhou, Jia Yu, Krishna R. Kalari, Liewei Wang, Judy C. Boughey, Matthew P. Goetz, Jann N. Sarkaria, Zhenkun Lou. ATR inhibition is a promising radiosensitizing strategy for chemotherapy-resistant triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 840.