Immune-active microenvironment in small cell carcinoma of the ovary, hypercalcemic type : rationale for immune checkpoint blockade

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT), is a highly aggressive monogenic cancer driven by SMARCA4 mutations. Here, we report responses to anti-PD1 immunotherapy in four patients and characterize the immune landscape of SCCOHT tumors using quantitative immunofluorescence and gene expression profiling. Unexpectedly for a low mutation burden cancer, the majority of the tumors (eight of 11 cases) demonstrated PD-L1 expression with strong associated T-cell infiltration (R2 = 0.60–0.95). PD-L1 expression was detected in both tumor and stromal cells, with macrophages being the most abundant PD-L1-positive cells in some tumors (three of 11 cases). Transcriptional profiling revealed increased expression of genes related to Th1 and cytotoxic cell function in PD-L1-high tumors, suggesting that PD-L1 acts as a pathway of adaptive immune resistance in SCCOHT. These findings suggest that although SCCOHT are low–mutational burden tumors, their immunogenic microenvironment resembles the landscape of tumors that respond well to treatment with PD-1/PD-L1 blockade

The Testis-Specific Factor CTCFL Cooperates with the Protein Methyltransferase PRMT7 in H19 Imprinting Control Region Methylation

Expression of imprinted genes is restricted to a single parental allele as a result of epigenetic regulation—DNA methylation and histone modifications. Igf2/H19 is a reciprocally imprinted locus exhibiting paternal Igf2 and maternal H19 expression. Their expression is regulated by a paternally methylated imprinting control region (ICR) located between the two genes. Although the de novo DNA methyltransferases have been shown to be necessary for the establishment of ICR methylation, the mechanism by which they are targeted to the region remains unknown. We demonstrate that CTCFL/BORIS, a paralog of CTCF, is an ICR-binding protein expressed during embryonic male germ cell development, coinciding with the timing of ICR methylation. PRMT7, a protein arginine methyltransferase with which CTCFL interacts, is also expressed during embryonic testis development. Symmetrical dimethyl arginine 3 of histone H4, a modification catalyzed by PRMT7, accumulates in germ cells during this developmental period. This modified histone is also found enriched in both H19 ICR and Gtl2 differentially methylated region (DMR) chromatin of testis by chromatin immunoprecipitation (ChIP) analysis. In vitro studies demonstrate that CTCFL stimulates the histone-methyltransferase activity of PRMT7 via interactions with both histones and PRMT7. Finally, H19 ICR methylation is demonstrated by nuclear co-injection of expression vectors encoding CTCFL, PRMT7, and the de novo DNA methyltransferases, Dnmt3a, -b and -L, in Xenopus oocytes. These results suggest that CTCFL and PRMT7 may play a role in male germline imprinted gene methylation

Report on computational assessment of Tumor Infiltrating Lymphocytes from the International Immuno-Oncology Biomarker Working Group

Funder: U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)Funder: National Center for Research Resources under award number 1 C06 RR12463-01, VA Merit Review Award IBX004121A from the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service, the DOD Prostate Cancer Idea Development Award (W81XWH-15-1-0558), the DOD Lung Cancer Investigator-Initiated Translational Research Award (W81XWH-18-1-0440), the DOD Peer Reviewed Cancer Research Program (W81XWH-16-1-0329), the Ohio Third Frontier Technology Validation Fund, the Wallace H. Coulter Foundation Program in the Department of Biomedical Engineering and the Clinical and Translational Science Award Program (CTSA) at Case Western Reserve University.Funder: Susan G Komen Foundation (CCR CCR18547966) and a Young Investigator Grant from the Breast Cancer Alliance.Funder: The Canadian Cancer SocietyFunder: Breast Cancer Research Foundation (BCRF), Grant No. 17-194Abstract: Assessment of tumor-infiltrating lymphocytes (TILs) is increasingly recognized as an integral part of the prognostic workflow in triple-negative (TNBC) and HER2-positive breast cancer, as well as many other solid tumors. This recognition has come about thanks to standardized visual reporting guidelines, which helped to reduce inter-reader variability. Now, there are ripe opportunities to employ computational methods that extract spatio-morphologic predictive features, enabling computer-aided diagnostics. We detail the benefits of computational TILs assessment, the readiness of TILs scoring for computational assessment, and outline considerations for overcoming key barriers to clinical translation in this arena. Specifically, we discuss: 1. ensuring computational workflows closely capture visual guidelines and standards; 2. challenges and thoughts standards for assessment of algorithms including training, preanalytical, analytical, and clinical validation; 3. perspectives on how to realize the potential of machine learning models and to overcome the perceptual and practical limits of visual scoring

Application of a risk-management framework for integration of stromal tumor-infiltrating lymphocytes in clinical trials

Funder: Breast Cancer Research Foundation (BCRF); doi: https://doi.org/10.13039/100001006Abstract: Stromal tumor-infiltrating lymphocytes (sTILs) are a potential predictive biomarker for immunotherapy response in metastatic triple-negative breast cancer (TNBC). To incorporate sTILs into clinical trials and diagnostics, reliable assessment is essential. In this review, we propose a new concept, namely the implementation of a risk-management framework that enables the use of sTILs as a stratification factor in clinical trials. We present the design of a biomarker risk-mitigation workflow that can be applied to any biomarker incorporation in clinical trials. We demonstrate the implementation of this concept using sTILs as an integral biomarker in a single-center phase II immunotherapy trial for metastatic TNBC (TONIC trial, NCT02499367), using this workflow to mitigate risks of suboptimal inclusion of sTILs in this specific trial. In this review, we demonstrate that a web-based scoring platform can mitigate potential risk factors when including sTILs in clinical trials, and we argue that this framework can be applied for any future biomarker-driven clinical trial setting

ChIP Analysis of CTCFL Association with the <i>H19</i> ICR and <i>Gtl2</i> DMR

<div><p>(A) Diagrams illustrate the regions within <i>H19</i> ICR and <i>Gtl2</i> DMR analyzed by ChIP.</p> <p>(B) Adult testis analysis. Upper panels show agarose gels of real-time PCR products for <i>H19</i> ICR and standard PCR products for <i>Gtl2</i> DMR. A no–DNA control (−)is indicated. Input (In) represents 10% of total amount of samples used. Pre-immune (PI) was used as a control for the enrichment of CTCFL immunoprecipitated with α-CTCFL (α-CL) antibody. For <i>Gtl2</i> DMR, normal rabbit serum (NRS) was used. Lower panel shows real-time PCR analysis of <i>H19</i> ICR. Control represents genomic region lacking CTCF binding consensus sequence.</p> <p>(C) The 15.5-dpc testis analysis. Figure is organized as for (B). In this case, control is included in the upper panel.</p></div

Immunohistochemistry of CTCFL Expression in Developing Testis

<div><p>(A) Characterization of α-CTCFL antibody. Myc-tagged CTCFL expressed in 293T cells was used to assess antibody titer and specificity. First (left) panel shows different anti-sera (dilution 1:1,000); pre-immune; 1: first bleed; 2: second bleed; 3: final bleed. Second panel: verification of myc-tagged CTCFL by α-myc antibody. Third panel: determination of α-CTCFL antibody specificity. The α-CTCFL antibody (dilution 1:10,000) was pre-incubated with GST-N-terminal CTCFL (GA) and GST (G). The antibody, pre-incubated with specific antigen, was neutralized, whereas α-CTCFL pre-incubated with GST alone retained its reactivity. Fourth panel: α-CTCFL reacted with a Western blot of adult testis extract. The black arrow indicates the position of myc epitope-tagged CTCFL, whereas the grey arrow indicates the position of endogenous CTCFL.</p> <p>(B–G) CTCFL expression in developing and adult testis. The pre-immune and adjacent α-CTCFL panels are at the same magnification (400×). A 5× magnification of the α-CTCFL image is given in the rightmost panel.</p> <p>(B) CTCFL is not detected at 13.5 dpc.</p> <p>(C) Mitotically arrested gonocytes (marked with white arrows) exhibit CTCFL staining at 14.5 dpc.</p> <p>(D) A few centrally localized gonocytes and cells at the periphery of seminiferous tubules express CTCFL at 17.5 dpc.</p> <p>(E) CTCFL is localized in gonocytes in newborn mice.</p> <p>(F) At 15 d after birth, nuclei of spermatogonia expressed CTCFL, as did their counterparts in adult testis (G).</p> <p>(H) Immunohistochemical detection of CTCF expression in adult testis. Normal rabbit serum (NRS) (left panel) and α-CTCF are presented as for (B–G). CTCF is expressed uniquely in Sertoli cells (marked with arrows).</p></div

N-terminal CTCFL Interacts with PRMT7

<div><p>(A) Diagrams show the domain structure of the PRMT7 and CTCFL proteins. MTase I and II represent the methyltransferase regions within PRMT7. The position of the recovered yeast two-hybrid clone of PRMT7 is indicated.</p> <p>(B) Reciprocal GST pull-downs of CTCFL and PRMT7. Scheme of the tested interactions is given in diagrams. GST and GST fusion proteins (CMV-GST vector) were co-expressed in 293T cells with myc- or V5-tagged preys. N-ter., N-terminal.</p> <p>(C) Co-immunoprecipitation of CTCFL and PRMT7. The 293T extracts containing over-expressed CTCFL-Myc or CTCFL-Myc&PRMT7-V5 were immunoprecipitated with α-V5 antibody or normal mouse serum (NMS). CTCFL was detected with α-Myc antibody. N-ter., N-terminal.</p> <p>(D) No interaction between CTCF and PRMT7 is detectable by GST pull-downs.</p></div

Model of <i>H19</i> ICR Methylation

<div><p>(A) An initial complex of CTCFL and PRMT7 localizes to the <i>H19</i> ICR, resulting in symmetrical dimethyl modification of arginine residues in histones H2A and H4 composing the adjacent nucleosomes. The localization to ICR is assured by the zinc finger portion of CTCFL, whereas the interactions with histones and PRMT7 are taking place via the N-terminal portion of the protein.</p> <p>(B) Following disengagement of the CTCFL complex, the de novo DNA methyltransferases Dnmt3a and Dnmt3b are recruited by either their PWWP motifs or through a bridging protein to the methylated histones. Dnmt3L is recruited by direct interaction with Dnmt3a and Dnmt3b. Subsequent to their recruitment, the de novo DNA methyltransferases methylate adjacent CpGs, resulting in ICR methylation.</p></div

CTCFL Interacts with Histones H1, H2A, and H3

<div><p>(A) Farwestern analysis. Western blots containing 1-μg and 0.25-μg histones were reacted with transfected 293T cell lysates expressing myc-tagged CTCFL. The presence of CTCFL was detected by α-Myc and α-CTCFL antibodies. Individual histones are indicated. The lower panel shows the ponceau red–stained Western blot. The migration of the individual histones are indicated to the left.</p> <p>(B) N-terminal and full-length CTCFL interacts with histones H1, H2A, and H3. Bacterial-produced GST histone fusion proteins were reacted with lysates from 293T cells transfected with either full-length or N-terminal CTCFL expression vectors. No detectable interaction of PRMT7 with histones is observed.</p></div