Combined Tumor-Based BRCA1/2 and TP53 Mutation Testing in Ovarian Cancer.

Somatic/germline BRCA1/2 mutations (m)/(likely) pathogenic variants (PV) (s/gBRCAm) remain the best predictive biomarker for PARP inhibitor efficacy. As >95% of high-grade serous ovarian cancers (HGSOC) have a somatic TP53m, combined tumor-based BRCA1/2 (tBRCA) and TP53 mutation testing (tBRCA/TP53m) may improve the quality of results in somatic BRCAm identification and interpretation of the 'second hit' event, i.e., loss of heterozygosity (LOH). A total of 237 patients with HGSOC underwent tBRCA/TP53m testing. The ratio of allelic fractions (AFs) for tBRCA/TP53m was calculated to estimate the proportion of cells carrying BRCAm and to infer LOH. Among the 142/237 gBRCA results, 16.2% demonstrated a pathogenic/deleterious variant (DEL) gBRCA1/2m. Among the 195 contributive tumor samples, 43 DEL of tBRCAm (22.1%) were identified (23 gBRCAm and 20 sBRCAm) with LOH identified in 37/41 conclusive samples. The median AF of TP53m was 0.52 (0.01-0.93), confirming huge variability in tumor cellularity. Initially, three samples were considered as wild type with <10% cellularity. However, additional testing detected a very low AF (<0.05) in both BRCA1/2m and TP53m, thus reidentifying them as sBRCA1/2m. Combined tBRCA/TP53m testing is rapid, sensitive, and identifies somatic and germline BRCA1/2m. AF TP53m is essential for interpreting sBRCA1/2m in low-cellularity samples and provides indirect evidence for LOH as the 'second hit' of BRCA1/2-related tumorigenesis.

Clinical utility of SMARCA4 testing by immunohistochemistry in rare ovarian tumours.

BACKGROUND: Ovarian small cell carcinoma, hypercalcaemic type (SCCOHT) is a rare and lethal disease affecting young women. As histological diagnosis is challenging and urgent, there is a clear need for a robust diagnostic test. While mutations in the chromatin-remodelling gene, SMARCA4, appear to be typical, it may not be feasible routinely to be clinically relevant. METHODS: Previous studies have described the value of SMARCA4 IHC to differentiate SCCOHT from ovarian neoplasms (ON), with similar histologic appearances. We aimed to evaluate its clinical utility among a cohort of 44 SCCOHT and 94 rare ON frequently misdiagnosed as SCCOHT. RESULTS: Forty-three percent (16/36) of SCCOHT had been classified locally as non-SCCOHT confirming the diagnosis challenge. Sensitivity and specificity of SMARCA4 IHC were excellent at 88% and 94%, respectively. In a community setting with a much lower prevalence of the disease, estimated PPV is 40% while NPV remained high at 99%. Finally, among the 16 SCCOHT misclassified locally, SMARCA4 IHC testing would have resulted in corrected diagnosis in 88% of cases. CONCLUSIONS: SMARCA4 IHC is a highly sensitive, and specific test for the diagnosis of SCCOHT and is of huge clinical utility in providing a timely and accurate diagnosis of this challenging disease.

Impact of neoadjuvant chemotherapy on the immune microenvironment in advanced epithelial ovarian cancer: Prognostic and therapeutic implications.

Over the last decade, increasing evidence highlights the role of the host immune system in the control of tumor growth and the prognostic implications of tumor infiltrating lymphocytes (TILs) in ovarian cancer. Most data support a better prognosis with accumulation of CD3+ and CD8 + TILs and a poor outcome associated with increased regulatory T cells. However, only a small number of studies have focused on the effect of neoadjuvant chemotherapy (NACT) on the tumor immune microenvironment. This review will provide an update on the prognostic value of TIL subpopulations at diagnosis and a comprehensive overview of the recent studies evaluating the impact of neoadjuvant chemotherapy on TILs and their relationship to clinical outcome in advanced ovarian cancer. This information could help in future investigations of immunotherapy as maintenance following primary treatment.

Refinement of high-risk endometrial cancer classification using DNA damage response biomarkers: a TransPORTEC initiative.

The TransPORTEC consortium previouslclassified high-risk endometrial cancer including poor-risk histologies such as clear cells, into four molecular subtypes "POLE mutated," "microsatellite unstable," "TP53 mutated," and "no specific molecular profile." We evaluated whether DNA damage response biomarkers could further refine this high-risk tumors classification, in particular the heterogeneous "no specific molecular profile" and "TP53 mutated" subsets recently qualified as poor prognosis in high-risk endometrial cancer. DNA damage response biomarkers including proteins involved in DNA damage (δ-H2AX), homologous recombination (RAD51), regulators of error-prone Non Homologous End-Joining (DNA-pk, FANCD2), and PARP-1 were evaluated in 116 high-risk tumors by immunohistochemistry. CD8 and PD-1 expression by immunochemistry and mutation analyses were performed previously. Survival outcome were calculated using Kaplan-Meier and Log-rank test. None of the DNA damage response biomarkers alone were prognostic. However markers were informative within molecular subsets. Among the "no specific molecular profile" subset, δ-H2AX+ was significantly predictive of poor disease free survival (Hazard Ratio = 2.56; p = 0.026), and among "TP53 mutated," a DNA-pk+/FANCD2- profile (favouring error-prone Non Homologous End-Joining) predicted worst disease free survival (Hazard Ratio = 4.95; p = 0.009) resulting in five distinct prognostic subgroups from best to worst prognosis: group1 "POLE mutated/Microsatellite unstable" > group2 "no specific molecular profile with no DNA damage" > group3 "TP53 mutated/Non Homologous End-Joining negative" > group4 "no specific molecular profile with high DNA damage" > group5 "TP53 mutated/Non Homologous End-Joining positive"; p = 0.0002). Actionable targets were also different among subsets. Group3 had significantly higher infiltration of PD-1+ immune cells (p = 0.003), segregating with group1. Group2 had frequent PI3K pathway mutations and ER positivity. While group5, with the worst prognosis, had high DNA damage and PARP-1 expression providing a rationale for PARP inhibition. Our findings have refined the TransPORTEC prognostic classification of high-risk endometrial cancer into five distinct subgroups by integrating DNA damage response biomarkers and identified molecular subtype-specific therapeutic strategies.

Molecular analyses of juvenile granulosa cell tumors bearing AKT1 mutations provide insights into tumor biology and therapeutic leads.

Juvenile granulosa cell tumors (JGCTs) of the ovary are pediatric neoplasms representing 5% of all granulosa cell tumors (GCTs). Most GCTs are of adult type (AGCTs) and bear a mutation in the FOXL2 gene. The molecular basis of JGCTs is poorly understood, although mutations in the GNAS gene have been reported. We have detected in-frame duplications within the oncogene AKT1 in >60% of the JGCTs studied. Here, to evaluate the functional impact of these duplications and the existence of potential co-driver alterations, we have sequenced the transcriptome of four JGCTs and compared them with control transcriptomes. A search for gene variants detected only private alterations probably unrelated with tumorigenesis, suggesting that tandem duplications are the best candidates to underlie tumor formation in the absence of GNAS alterations. We previously showed that the duplications were specific to JGCTs. However, the screening of eight AGCTs samples without FOXL2 mutation showed the existence of an AKT1 duplication in one case, also having a stromal luteoma. The analysis of RNA-Seq data pinpointed a series of differentially expressed genes, involved in cytokine and hormone signaling and cell division-related processes. Further analyses pointed to the existence of a possible dedifferentiation process and suggested that most of the transcriptomic dysregulation might be mediated by a limited set of transcription factors perturbed by AKT1 activation. Finally, we show that commercially available AKT inhibitors can modulate the in vitro activity of various mutated forms. These results shed light on the pathogenesis of JGCTs and provide therapeutic leads for a targeted treatment.

DNA damage response as a therapeutic target in gynecological cancers.

PURPOSE OF REVIEW: The proven activity of poly ADP ribose polymerase (PARP) inhibitors in BRCA-mutated homologous recombination deficient (HRD) ovarian cancer has led to the availability to patients with ovarian cancer of the first targeted therapy with an associated predictive biomarker. Our focus has recently turned towards expanding the clinical utility of PARP inhibitors beyond BRCA mutated ovarian cancer, and to a search for novel targets within DNA damage response (DDR). RECENT FINDINGS: Early trials in unselected patients with ovarian cancer showed responses to PARP inhibition in BRCA-wildtype ovarian cancer, and recent genomic studies have demonstrated that germline or somatic aberrations in other homologous recombination genes are present in a significant proportion of ovarian cancers. In addition, PARP inhibition may be of value in molecularly defined subsets of endometrial or cervical cancers. Novel DDR inhibitors such as ATR, ATM, WEE1 or DNA-PK inhibitors are also being tested in patients. Finally, combinatorial strategies of DDR inhibitors with antiangiogenic agents, phosphoinositide 3-kinase inhibitors or immunotherapies may further increase therapeutic efficacy. SUMMARY: In the future, patients with gynaecological malignancies may be rationally selected for PARP inhibition on the basis of comprehensive evaluation of homologous recombination genomic alterations, or HRD assays. Furthermore, novel DDR inhibitors have the potential to expand the repertoire of therapeutic options available to these patients.

WNT4 and RSPO1 together are required for cell proliferation in the early mouse gonad.

The gonad arises from the thickening of the coelomic epithelium and then commits into the sex determination process. Testis differentiation is activated by the expression of the Y-linked gene Sry, which promotes cell proliferation and differentiation of Sertoli cells, the supporting cells of the testis. In absence of Sry (XX individuals), activation of WNT/CTNNB1 signalling, via the upregulation of Rspo1 and Wnt4, promotes ovarian differentiation. However, Rspo1 and Wnt4 are expressed in the early undifferentiated gonad of both sexes, and Axin2-lacZ, a reporter of canonical WNT/CTNNB1 signalling, is expressed in the coelomic region of the E11.5 gonadal primordium, suggesting a role of these factors in early gonadal development. Here, we show that simultaneous ablation of Rspo1 and Wnt4 impairs proliferation of the cells of the coelomic epithelium, reducing the number of progenitors of Sertoli cells in XY mutant gonads. As a consequence, in XY Wnt4(-/-); Rspo1(-/-) foetuses, this leads to the differentiation of a reduced number of Sertoli cells and the formation of a hypoplastic testis exhibiting few seminiferous tubules. Hence, this study identifies Rspo1 and Wnt4 as two new regulators of cell proliferation in the early gonad regardless of its sex, in addition to the specific role of these genes in ovarian differentiation.

The PI3K/Akt/mTOR pathway in ovarian cancer: therapeutic opportunities and challenges.

The phosphatidylinositol 3 kinase (PI3K) pathway is frequently altered in cancer, including ovarian cancer (OC). Unfortunately, despite a sound biological rationale and encouraging activity in preclinical models, trials of first-generation inhibitors of mammalian target of rapamycin (mTOR) in OC have demonstrated negative results. The lack of patient selection as well as resistance to selective mTOR complex-1 (mTORC1) inhibitors could explain the disappointing results thus far. Nonetheless, a number of novel agents are being investigated, including dual mTORC1/mTORC2, Akt, and PI3K inhibitors. Although it is likely that inhibition of the PI3K/Akt/mTOR pathway may have little effect in unselected OC patients, certain histological types, such as clear cell or endometrioid OC with frequent phosphatidylinositol-4,5-biphosphate 3-kinase, catalytic subunit alpha (PIK3CA) and/or phosphatase and tensin homolog (PTEN) alterations, may be particularly suited to this approach. Given the complexity and redundancy of the PI3K signaling network, PI3K pathway inhibition may be most useful in combination with either chemotherapy or other targeted therapies, such as MEK inhibitors, anti-angiogenic therapy, and hormonal therapy, in appropriately selected OC patients. Here, we discuss the relevance of the PI3K pathway in OC and provide an up-to-date review of clinical trials of novel PI3K inhibitors alone or in combination with cytotoxics and novel therapies in OC. In addition, the challenges of drug resistance and predictive biomarkers are addressed.

FOXL2 is regulated during the bovine estrous cycle and its expression in the endometrium is independent of conceptus-derived interferon tau.

FOXL2, a winged-helix/forkhead domain transcription factor, is a key gene involved in the differentiation and biological functions of the ovary. In a recent transcriptomic analysis, we found that FOXL2 expression in bovine caruncular endometrium was different from that in intercaruncular endometrium. In order to gain new insights into FOXL2 in this tissue, we determined the expression of this transcription factor during the estrous cycle and the establishment of pregnancy in cattle. The endometrial expression of FOXL2 did not vary during maternal recognition of pregnancy (Days 16-20). Using an in vivo bovine model and primary cell cultures, we showed that FOXL2 was not an interferon-tau target gene. Both FOXL2 transcript and protein were expressed from Day 5 to Day 20 of the estrous cycle, and their levels showed a significant increase during the luteolytic phase. A 2-day progesterone supplementation in heifers led to a clear down-regulation of FOXL2 protein levels, suggesting the negative impact of progesterone on FOXL2 expression. Immunohistochemistry data revealed the localization of FOXL2 in endometrial stromal and glandular cells. FOXL2 subcellular distribution was shown to be nuclear in endometrial samples collected during the luteolytic period, while it was not detected in nuclei during the luteal phase and at implantation. Collectively, our findings provide the first evidence that FOXL2 is involved in the regulation of endometrial tissue physiology.

A Novel B7-H6-Targeted IgG-Like T Cell-Engaging Antibody for the Treatment of Gastrointestinal Tumors.

PURPOSE: Advanced-stage gastrointestinal cancers represent a high unmet need requiring new effective therapies. We investigated the antitumor activity of a novel T cell-engaging antibody (B7-H6/CD3 ITE) targeting B7-H6, a tumor-associated antigen that is expressed in gastrointestinal tumors. EXPERIMENTAL DESIGN: Membrane proteomics and IHC analysis identified B7-H6 as a tumor-associated antigen in gastrointestinal tumor tissues with no to very little expression in normal tissues. The antitumor activity and mode of action of B7-H6/CD3 ITE was evaluated in in vitro coculture assays, in humanized mouse tumor models, and in colorectal cancer precision cut tumor slice cultures. RESULTS: B7-H6 expression was detected in 98% of colorectal cancer, 77% of gastric cancer, and 63% of pancreatic cancer tissue samples. B7-H6/CD3 ITE-mediated redirection of T cells toward B7-H6-positive tumor cells resulted in B7-H6-dependent lysis of tumor cells, activation and proliferation of T cells, and cytokine secretion in in vitro coculture assays, and infiltration of T cells into tumor tissues associated with tumor regression in in vivo colorectal cancer models. In primary patient-derived colorectal cancer precision-cut tumor slice cultures, treatment with B7-H6/CD3 ITE elicited cytokine secretion by endogenous tumor-infiltrating immune cells. Combination with anti-PD-1 further enhanced the activity of the B7-H6/CD3 ITE. CONCLUSION: These data highlight the potential of the B7-H6/CD3 ITE to induce T cell-redirected lysis of tumor cells and recruitment of T cells into noninflamed tumor tissues, leading to antitumor activity in in vitro, in vivo, and human tumor slice cultures, which supports further evaluation in a clinical study.

A study of goat SRY protein expression suggests putative new roles for this gene in the developing testis of a species with long-lasting SRY expression.

The testis-determining gene SRY is not well-conserved among mammals, and particularly between mouse and other mammals. To evaluate SRY function in a nonrodent species, we produced an antibody against goat SRY and used it to investigate the expression pattern of SRY throughout goat testicular development. By contrast with the mouse, SRY is primarily expressed in most cells of XY genital-ridges and not solely in pre-Sertoli cells. Between cord formation and prepuberty, SRY remains expressed in both Sertoli and germinal cells. During adulthood, SRY expression declines and then disappears from meiotic germ cells, only remaining present at low levels in some spermatogonia. Unlike the germinal lineage, SRY continues to be highly expressed in adult Sertoli cells with a typical nuclear staining. Our data indicate that in goat, the role of SRY may not be limited to testis determination and could have other functions in testicular maintenance and hence male fertility.

Histological classification of mucinous ovarian tumors: inter-observer reproducibility, clinical relevance, and role of genetic biomarkers.

The morphological distinction between the various types of mucinous ovarian tumors has major prognostic implications but may be challenging. The aims of our study were to describe inter-observer reproducibility in the morphological diagnosis of mucinous ovarian tumors, to evaluate the clinical relevance of possible diagnostic discrepancies, and to identify molecular abnormalities correlated with the histological type. Seventy-nine ovarian mucinous borderline tumors (MOB) and either expansile or infiltrative carcinomas (MOC) were independently reviewed by two gynecological pathologists. Molecular analysis was performed in 32 cases. Concordance between the two pathologists was reached in 67 cases (k: 0.78). The main discrepancies (8/12) were the evaluation of nuclear grade 3 or that of microfoci (< 5 mm) of infiltrative-type carcinoma in an otherwise typical expansile MOC. Our follow-up analysis showed that infiltrative MOC had a lower overall survival (OS) (p < 0.0024) and progression-free survival (PFS) (p = 0.0060) as compared with MOB and expansile MOC. The presence of nuclear grade 3 or microfoci (< 5 mm) of infiltrative-type pattern of invasion in an otherwise typical expansile MOC did not alter the prognosis as compared with expansile MOC without these features, in terms of OS (p < 0.0028) and PFS (p = 0.0074). KRAS mutations were more frequent in MOB (71%), than in expansile (50%) and infiltrative MOC (14%). In contrast, the prevalence of TP53 mutation was lower in MOB (43%), than in expansile (58%) and infiltrative MOC (71%). Our results confirm that in MOC, the expansile pattern of invasion is associated with a better prognosis than extensive (> 5 mm) infiltrative-type pattern of invasion. No specific or sensitive molecular profile might help in the differential diagnosis of mucinous ovarian tumors.

A Novel Antagonistic CD73 Antibody for Inhibition of the Immunosuppressive Adenosine Pathway.

Despite some impressive clinical results with immune checkpoint inhibitors, the majority of patients with cancer do not respond to these agents, in part due to immunosuppressive mechanisms in the tumor microenvironment. High levels of adenosine in tumors can suppress immune cell function, and strategies to target the pathway involved in its production have emerged. CD73 is a key enzyme involved in adenosine production. This led us to identify a novel humanized antagonistic CD73 antibody, mAb19, with distinct binding properties. mAb19 potently inhibits the enzymatic activity of CD73 in vitro, resulting in an inhibition of adenosine formation and enhanced T-cell activation. We then investigated the therapeutic potential of combining CD73 antagonism with other immune modulatory and chemotherapeutic agents. Combination of mAb19 with a PD-1 inhibitor increased T-cell activation in vitro Interestingly, this effect could be further enhanced with an agonist of the adenosine receptor ADORA3. Adenosine levels were found to be elevated upon doxorubicin treatment in vivo, which could be blocked by CD73 inhibition. Combining CD73 antagonism with doxorubicin resulted in superior responses in vivo Furthermore, a retrospective analysis of rectal cancer patient samples demonstrated an upregulation of the adenosine pathway upon chemoradiation, providing further rationale for combining CD73 inhibition with chemotherapeutic agents.This study demonstrates the ability of a novel CD73 antibody to enhance T-cell function through the potent suppression of adenosine levels. In addition, the data highlight combination opportunities with standard of care therapies as well as with an ADORA3 receptor agonist to treat patients with solid tumors.

Changes in DNA Damage Response Markers with Treatment in Advanced Ovarian Cancer.

Ovarian cancer (OC) is sensitive to upfront chemotherapy, which is likely attributable to defects in DNA damage repair (DDR). Unfortunately, patients relapse and the evolution of DDR competency are poorly described. We examined the expression of proposed effectors in homologous recombination (HR: RAD51, ATM, FANCD2), error-prone non-homologous end-joining (NHEJ: 53BP1), and base excision repair pathways (BER: PAR and PARP1) in a cohort of sequential OC samples obtained at diagnosis, after neoadjuvant chemotherapy (NACT), and/or at relapse from a total of 147 patients. Immunohistochemical (IHC) expression was quantified using the H-score (0-300), where H ≤ 10 defined negativity. Before NACT, a significant number of cases lacked the expression of some effectors: 60%, 60%, and 24% were PAR-, FANCD2-, or RAD51-negative, with a reassuringly similar proportion of negative biomarkers after NACT. In multivariate analysis, there was a poorer progression-free survival (PFS) and overall survival (OS) for cases with competent HR at diagnosis (PRE-NACT 53BP1-/RAD51+, hazard ratio (HR) 3.13, p = 0.009 and HR 2.78, p = 0.024) and after NACT (POST-NACT FANCD2+/RAD51+ HR 1.89, p = 0.05 and HR 2.38, p = 0.02; POST-NACT PARP-1+/RAD51+ HR 1.79, p = 0.038 and HR 2.04, p = 0.034), reflecting proficient DNA repair. Overall, HR-competent tumors appeared to have a dismal prognosis in comparison with tumors utilizing NHEJ, as assessed either at baseline or post-NACT. Accurate knowledge of the HR status during treatment is clinically important for the efficient timing of platinum-based and targeted therapies with poly(ADP-ribose) polymerase inhibitors (PARPi).

Somatic and Germline BRCA 1 and 2 Mutations in Advanced NSCLC From the SAFIR02-Lung Trial.

INTRODUCTION: Molecular profiling is considered a standard of care in advanced NSCLC. A comprehensive next-generation sequencing panel can discover somatic or germline BRCA1/2 mutations that are new druggable molecular alterations. However, the phenotypic and potential therapeutic relevance of BRCA1/2 mutation in NSCLC remains poorly defined. METHODS: From April 2014 to March 2017, 600 newly diagnosed, EGFR/ALK negative patients with advanced NSCLC were enrolled in the SAFIR02-Lung trial. Molecular profiling was done at study entry on archival tissue or frozen tissue collected from a new biopsy specimen before the third cycle of platinum-based chemotherapy. The prevalence of BRCA1/2 variants and its biological relevance were assessed. A homologous recombinant deficiency (HRD) score was based on the copy number variation data, and the germline status was determined by blood analysis. The BRCA Share database and the French CGG consortium were the references for the variant classification. RESULTS: Of 379 patients with a molecular profile discussed in a tumor molecular board, BRCA1/2 variants were identified in 20 patients (5.3%), including eight patients (2.1%) with a confirmed pathogenic BRCA mutation. Two patients (0.5%) harbored a germline BRCA2 mutation, and for six others, a somatic BRCA mutation was identified (1.6%). All were men and mainly smokers (88%). The overall response rate to chemotherapy was 13%. BRCA variants of unknown significance were detected in 12 patients (3.2%), achieving an 8.3% overall response rate with chemotherapy. One-third of tumors carrying pathogenic BRCA mutations or variants of unknown significance had biallelic inactivation and high HRD score. Overall survival of this cohort was 12.8 months. CONCLUSIONS: Pathogenic BRCA1/2 mutations occur in 2.1% of patients with advanced NSCLC. The predictive role of BRCA mutation for making treatment decisions in NSCLC seems limited based on clinical response (low platinum sensitivity) and molecular features (discrepancy between biallelic inactivation and high HRD score).

Small Cell Carcinoma of the Ovary, Hypercalcemic Type (SCCOHT) beyond SMARCA4 Mutations: A Comprehensive Genomic Analysis.

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is an aggressive malignancy that occurs in young women, is characterized by recurrent loss-of-function mutations in the SMARCA4 gene, and for which effective treatments options are lacking. The aim of this study was to broaden the knowledge on this rare malignancy by reporting a comprehensive molecular analysis of an independent cohort of SCCOHT cases. We conducted Whole Exome Sequencing in six SCCOHT, and RNA-sequencing and array comparative genomic hybridization in eight SCCOHT. Additional immunohistochemical, Sanger sequencing and functional data are also provided. SCCOHTs showed remarkable genomic stability, with diploid profiles and low mutation load (mean, 5.43 mutations/Mb), including in the three chemotherapy-exposed tumors. All but one SCCOHT cases exhibited 19p13.2-3 copy-neutral LOH. SMARCA4 deleterious mutations were recurrent and accompanied by loss of expression of the SMARCA2 paralog. Variants in a few other genes located in 19p13.2-3 (e.g., PLK5) were detected. Putative therapeutic targets, including MAGEA4, AURKB and CLDN6, were found to be overexpressed in SCCOHT by RNA-seq as compared to benign ovarian tissue. Lastly, we provide additional evidence for sensitivity of SCCOHT to HDAC, DNMT and EZH2 inhibitors. Despite their aggressive clinical course, SCCOHT show remarkable inter-tumor homogeneity and display genomic stability, low mutation burden and few somatic copy number alterations. These findings and preliminary functional data support further exploration of epigenetic therapies in this lethal disease.

Characterization of the tammar wallaby (Macropus eugenii) whey acidic protein gene: new insights into the function of the protein.

Whey acidic protein (WAP) belongs to a family of four disulfide core (4-DSC) proteins rich in cysteine residues and is the principal whey protein found in milk of a number of mammalian species. Eutherian WAPs have two 4-DSC domains, whereas marsupial WAPs are characterized by the presence of an additional domain at the amino terminus. Structural and expression differences between marsupial and eutherian WAPs have presented challenges to identifying physiological functions of the WAP protein. We have characterized the genomic structure of tammar WAP (tWAP) gene, identified its chromosomal localization and investigated the potential function of tWAP. We have demonstrated that tWAP and domain III (DIII) of the protein alone stimulate proliferation of a mouse mammary epithelial cell line (HC11) and primary cultures of tammar mammary epithelial cells (Wall-MEC), whereas deletion of DIII from tWAP abolishes this proliferative effect. However, tWAP does not induce proliferation of human embryonic kidney (HEK293) cells. DNA synthesis and expression of cyclin D1 and cyclin-dependent kinase-4 genes were significantly up-regulated when Wall-MEC and HC11 cells were grown in the presence of either tWAP or DIII. These data suggest that DIII is the functional domain of the tWAP protein and that evolutionary pressure has led to the loss of this domain in eutherians, most likely as a consequence of adopting a reproductive strategy that relies on greater investment in development of the newborn during pregnancy.

Goat RSPO1 over-expression rescues sex-reversal in Rspo1-knockout XX mice but does not perturb testis differentiation in XY or sex-reversed XX mice.

RSPO1 is a newly discovered gene involved in sex differentiation. Two goat BAC clones encompassing the RSPO1 gene (gRSPO1) were injected into mouse oocytes and several transgenic lines derived. Both clones induced gRSPO1 over-expression in various tissues, including male and female gonads, with no obvious phenotype and normal sex-ratios. Introgression of the gRSPO1 transgene into a mouse RSPO1 knockout genotype resulted in the rescue of the fertility and the disappearance of the masculinized gonadic features of the females, demonstrating the functionality of the goat protein in a mouse context. On the contrary, over-expression of gRSPO1 within a mSRY or a gSRY-XX genotypes did not interfere with the SRY-induced male phenotype.

Frequent Homologous Recombination Deficiency in High-grade Endometrial Carcinomas.

PURPOSE: The elevated levels of somatic copy-number alterations (SCNAs) in a subset of high-risk endometrial cancers are suggestive of defects in pathways governing genome integrity. We sought to assess the prevalence of homologous recombination deficiency (HRD) in endometrial cancers and its association with histopathologic and molecular characteristics. EXPERIMENTAL DESIGN: Fresh tumor tissue was prospectively collected from 36 endometrial cancers, and functional HRD was examined by the ability of replicating tumor cells to accumulate RAD51 protein at DNA double-strand breaks (RAD51 foci) induced by ionizing radiation. Genomic alterations were determined by next-generation sequencing and array comparative genomic hybridization/SNP array. The prevalence of BRCA-associated genomic scars, a surrogate marker for HRD, was determined in the The Cancer Genome Atlas (TCGA) endometrial cancer cohort. RESULTS: Most endometrial cancers included in the final analysis (n = 25) were of non-endometrioid (52%), grade 3 (60%) histology, and FIGO stage I (72%). HRD was observed in 24% (n = 6) of cases and was restricted to non-endometrioid endometrial cancers (NEEC), with 46% of NEECs being HRD compared with none of the endometrioid endometrial cancers (EEC, P = 0.014). All but 1 of the HRD cases harbored either a pathogenic BRCA1 variant or high somatic copy-number (SCN) losses of HR genes. Analysis of TCGA cases supported these results, with BRCA-associated genomic scars present in up to 48% (63/132) of NEEC versus 12% (37/312) of EEC (P < 0.001). CONCLUSIONS: HRD occurs in endometrial cancers and is largely restricted to non-endometrioid, TP53-mutant endometrial cancers. Evaluation of HRD may help select patients that could benefit from treatments targeting this defect, including platinum compounds and PARP inhibitors.

CDK4/6 inhibitors target SMARCA4-determined cyclin D1 deficiency in hypercalcemic small cell carcinoma of the ovary.

Inactivating mutations in SMARCA4 (BRG1), a key SWI/SNF chromatin remodelling gene, underlie small cell carcinoma of the ovary, hypercalcemic type (SCCOHT). To reveal its druggable vulnerabilities, we perform kinase-focused RNAi screens and uncover that SMARCA4-deficient SCCOHT cells are highly sensitive to the inhibition of cyclin-dependent kinase 4/6 (CDK4/6). SMARCA4 loss causes profound downregulation of cyclin D1, which limits CDK4/6 kinase activity in SCCOHT cells and leads to in vitro and in vivo susceptibility to CDK4/6 inhibitors. SCCOHT patient tumors are deficient in cyclin D1 yet retain the retinoblastoma-proficient/p16INK4a-deficient profile associated with positive responses to CDK4/6 inhibitors. Thus, our findings indicate that CDK4/6 inhibitors, approved for a breast cancer subtype addicted to CDK4/6 activation, could be repurposed to treat SCCOHT. Moreover, our study suggests a novel paradigm whereby critically low oncogene levels, caused by loss of a driver tumor suppressor, may also be exploited therapeutically.