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.

The immunomodulatory drugs (IMiDs) lenalidomide (Len) and pomalidomide (Pom) are standard of care for multiple myeloma (MM). Cereblon (CRBN), a component of the CRL4CRBN E3 ligase, is required for IMiD's anti-myeloma activity. Binding of IMiD to CRBN promotes the recruitment of transcription factors IKZF1 and IKZF3 to CRL4CRBN for ubiquitination and degradation, leading to loss of IRF4, an IKZF1/3 target essential for survival of myeloma cells. MEIS2 was identified by protein array as an endogenous substrate that competes with IMiD for binding to the same domain on CRBN. However, whether MEIS2 functions as an inhibitor of CRBN and its relevance in IMiD therapy has not been investigated. To address this, first we discovered that high MEIS2 RNA expression in myeloma cells before therapy was tightly associated with poorer overall survival (OS) (p-value = 0.0001) in the CoMMpass Bor-Lenalidomide-Dex clinical trial (N=272). Conversely, high CRBN RNA expression correlated with improved OS (p-value =0.0077). Next, we demonstrated that MEIS2 protein was differentially expressed in primary MM cells but absent in normal plasma cells (PC)s, B cells and lymphoma cells. In contrast, the expression of CRBN protein varied in PCs, and was significantly greater in treatment-naïve MM cells than in IMiD refractory MM cells. This resulted in a ~3-fold increase in the MEIS2 to CRBN ratio in IMiD refractory patients, implicating a role for MEIS2 in inhibiting CRBN and promoting clinical resistance to IMiD therapy. In addition, high expression of core genes that promote cell cycle progression through early G1 (CDK4 and CDK6) and late G1 (E2F1, CDK2, CCNA2) also tightly correlated with poorer OS. This is in line with dysregulation of G1-S progression in relapsed-refractory MM we previously reported (Ely et al, 2005) and suggests that inhibition of CDK4/6 may reprogram MEIS2 for enhanced IMiD killing. Indeed, induction of prolonged early G1 arrest by CDK4/6 inhibition with palbociclib 1) enhanced Len and Pom killing in model MM cell lines; 2) reduced the MEIS2 protein by transcriptional repression and increased the CRBN protein by post-transcriptional regulation in cooperation with Len; 3) this led a > 5 fold reduction in the MEIS2/CRBN protein ratio, exacerbated loss of IKZF1/3 and IRF4, and enhanced apoptosis (caspase- cleavage). Importantly. these findings were validated in freshly isolated primary bone marrow myeloma cells (BMMCs) (n=27) in a stromal co-culture, and the IMiD response ex vivo mimicked the clinical response to the first post-biopsy Len or Pom therapy in patients independent of prior therapy. Thus, induction of early G1 arrest by CDK4/6 inhibition reprograms primary MM cells for IMiD killing by reducing the MEIS2 to CRBN protein ratio through opposing regulation of MEIS2 and CRBN. Loss and gain of function studies demonstrated that MEIS2 inhibits IMiD killing by attenuating the induction of interferon downstream of the IKZF1/3-IRF4 axis, more prominently in early G1 arrest induced by CDK4/6 inhibition in MM cell lines as well as primary BMMCs. Further biochemical analyses revealed that CDK4/6 inhibition rapidly accelerated the displacement of MEIS2 from CRBN within one hour of Len treatment, concurrent with enhanced ubiquitination and degradation of IKZF1 and IKZF3 in both MM cell lines and primary BMMCs. In summary, we have demonstrated for the first time that MEIS2, an endogenous CRBN substrate, is a negative regulator of IMiD therapy in MM: 1) High expression of MEIS2 is a prognostic indicator for poor OS in IMiD therapy, in opposition to improved OS with high CRBN expression; 2) MEIS2 inhibits IMiD killing of MM cells by attenuating the induction of interferon; 3) inhibition of CDK4/6 enhances IMiD killing by rapidly displacing MEIS2 from CRBN through enhanced Len recruitment of IKZF1/3 and by time-dependent opposing regulation of MEIS2 and CRBN synthesis. Selective expression of MEIS2 in MM cells but not in normal PCs, B cells or lymphoma cells further suggest MEIS2 as a druggable target in MM. In this study, we provide the first evidence that CDK4/6 inhibition reverses MEIS2 inhibition of CRBN that has a potential to enhance the clinical response to IMiD. The prospect that through cell cycle control CDK4/6 inhibition may also enhance the anti-MM activity of CELMoDs, the newly developed IMiDs such as iberdomide, in relapsed-refractory setting is exciting and warrants future investigation.

PDF file - 53K, Validation of gene expression data using QPCR compared to microarray values (data reported as fold change).

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.

The 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.

PDF file - 60K, Kaplan-Meier estimates of (A)PFS and (B)OS in patients in the ITT population (N-38).

PDF file - 72K, Most common all-grade and grade ≥3 AEs during DoVeD induction and bortezomib-based stem cell mobilization.

PDF file - 59K, Responses rates after DoVeD induction, bortezomib- or non-bortezomib-based mobilization, and SCT.

Detection of BTK C481S mutation in MCL patients at relapse from a durable but not transient ibrutinib response or no response.

Supplementary Methods and Supplementary Figure Legends from Cell-Cycle Reprogramming for PI3K Inhibition Overrides a Relapse-Specific C481S BTK Mutation Revealed by Longitudinal Functional Genomics in Mantle Cell Lymphoma

Significant non-synonymous damaging SNVs detected in CDS by WTS in Pt 1 serial samples.

NIK and p52 of the alternative NF-kappaB pathway are expressed in JEKO-1 MCL cells and not modulated by targeting of CDK4 with PD 0332991 in combination with ibrutinib.

Supplementary Figures 1-2, Table 1 from A Novel Orally Active Small Molecule Potently Induces G1 Arrest in Primary Myeloma Cells and Prevents Tumor Growth by Specific Inhibition of Cyclin-Dependent Kinase 4/6

PDF file - 58K, Study design and patient flow by intent to treat (ITT).

Background: Prior studies in breast cancer (BC) have demonstrated variability in the immune microenvironment of BCs across subtypes, with higher levels of immune infiltration observed in triple-negative (TN) BC. Clinical trials of immunotherapy in BC have largely targeted patients with TNBC, however many of these patients do not respond to treatment, suggesting heterogeneity across TN tumors. Further, immune infiltrated hormone receptor–positive, HER2-negative (HR+/HER2-) tumors have been observed. Patients with these tumors may benefit from immune checkpoint blockade (ICB). Identification of patients with immunogenic tumors would be valuable for future patient selection and stratification in clinical trials. In this study, we conducted comprehensive molecular profiling of a set of commercially procured BC samples to characterize the tumor microenvironment (TME) of HR+/HER2- and TN tumors and to identify patient or tumor features associated with immune infiltration. Methods: 163 surgically resected BC samples were used for this study (93 HR+/HER2-, 70 TN). Clinically validated immunohistochemistry (IHC) assays were used to evaluate the expression of two immune checkpoint (IC) molecules, PD-L1 (by VENTANA PD-L1 (SP142) Assay and Agilent PD-L1 IHC 28-8 pharmDx) and LAG3 (antibody clone 17B4). The presence of CD8+ cells was measured by IHC (Dako/Agilent clone C8/144B) and used to derive the spatial location and density (topology) of CD8+ cells within the TME. RNA sequencing (RNA-seq) was used to construct gene expression signatures representative of immune cell types. Furthermore, RNAseq data was used to identify expressed somatic mutations and to calculate the total expressed tumor mutational burden (‘eTMB’). Results: As previously reported, the prevalence of PD-L1 + tumors was higher in TN vs HR+/HER2− BC, however close to half (44.1% SP142 IC> 1%) of HR+/HER2- tumors showed some degree of PD-L1 expression. Similar patterns were observed for LAG3 IHC expression (32.3% and 64.7% of HR+ and TN tumors respectively with >1% expression). Concordant expression of the two markers (SP142 IC> 1% and LAG3 >=1% or < 1% and < 1%) was observed in approximately 80% of tumors in both subtypes. Less than 10% of tumors in both subtypes had high LAG3 (>1%) and low PD-L1(< 1%) expression. Most (82.4%) HR+/HER2- tumors in the dataset were deficient in CD8+ cells in both stroma and tumor parenchyma, however a subset (12.9%) were identified as having an ‘excluded’ CD8 pattern, and four (4.7%) were classified as ‘inflamed’. The frequency of both the excluded and inflamed states was slightly higher among the TN tumors (20.6% and 15.9% respectively). Expression of PD-L1+ occurred more frequently among tumors with higher CD8 expression. Among both TN and HR+ tumors, most ‘inflamed’ tumors were grade 3. We also examined a range of gene expression signatures representative of various immune cell subpopulations, their distribution within each subtype, and relationships with other molecular data. For example, expression of a CXCL13+ T cell signature, a cell type that has been shown to be predictive of response to anti-PD-L1 + chemo combo therapy in TNBC, was significantly higher in TN vs HR+ tumors, however, a large subset of HR+ tumors had levels of expression of the signature above the median observed in TN tumors. In addition, we noted that eTMB, while low overall, was positively correlated with certain immune-related expression signatures across HR+/HER2− samples, including signatures previously associated with improved response to ICB, as well as PD-L1 positivity. Conclusions: We observed heterogeneity in the immune profile of the real-world HR+ and TN tumors in this cohort. A subset of tumors in both subtypes expressed markers or signatures previously reported to be associated with response to ICB. (1) Cancer Cell. 2021 Dec 13;39(12):1578-1593.e8. Citation Format: Aparna Chhibber, Lloye M. Dillon, John B. Wojcik, Vishantie Dostal, George Lee, Fayaz Seifuddin, Scott Ely, Mark D. Stern, Charlie Benson-Garnett, Mustimbo Roberts, Jenny Wu. Characterization of Immune Contexture in HR+/HER2- and Triple Negative Breast Cancer in a Real-world Cohort [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-09-07.

Integrative WTS and WES analysis of MCL cells from serial samples of Pt 1.

Figure S1. MCL cell lines are differentially sensitive to killing and cell cycle control by ibrutinib.

Supplementary Figures 1-2, Table 1 from A Novel Orally Active Small Molecule Potently Induces G₁ Arrest in Primary Myeloma Cells and Prevents Tumor Growth by Specific Inhibition of Cyclin-Dependent Kinase 4/6