High-grade squamous intraepithelial lesion cervicovaginal paps with negative high-risk HPV testing, a prospective study with histological follow-up.

BACKGROUND: High-risk human papillomavirus (hrHPV) testing has become integral in the screening and treatment protocols for cervical neoplasia. Stand-alone HPV testing is advocated as a screening tool for cervical neoplasia. However, negative hrHPV tests with diagnosis of high-grade squamous intraepithelial lesion or worse (≥HSIL) have been reported. We report our experience with paps diagnosed as HSIL, negative hrHPV testing, and subsequent follow-up. METHODS: Of 303 women with HSIL diagnosed on ThinPrep pap between 2019 and 2023, 84 (28%) were tested for hrHPV by Roche Cobas. Repeat testing was performed at a referral center. Immunohistochemistry (IHC) for p16 was performed on follow-up biopsies and hrHPV in-situ hybridization. RESULTS: Of 84 HSIL cases, 8 were hrHPV negative. Follow-up biopsies available in 7 cases were ≥HSIL (1 with invasive squamous cell carcinoma, 1 endocervical adenocarcinoma in situ). Follow-up HSIL was found on additional cases of HPV negative atypical glandular cells favor neoplastic and atypical squamous cells favor HSIL. IHC for p16 was positive on all biopsies. hrHPV FISH was negative. CONCLUSIONS: Our experience with hrHPV testing by Roche Cobas demonstrates that some morphologically diagnostic HSIL paps are hrHPV negative: 8.3% of HSIL paps with subsequent histological HSIL were HPV negative. The index case caused concern among our clinical colleagues. Positive staining for p16 is highly suggestive of HPV induced disease. Possible reasons for negative hrHPV testing could include non-hrHPV types, low HPV DNA levels, or HPV types not included in the Cobas testing panel.

Tumor FAK orchestrates immunosuppression in ovarian cancer via the CD155/TIGIT axis.

High-grade serous ovarian cancer (HGSOC) is a lethal malignancy characterized by an immunosuppressive tumor microenvironment containing few tumor infiltrating lymphocytes (TILs) and an insensitivity to checkpoint inhibitor immunotherapies. Gains in the PTK2 gene encoding focal adhesion kinase (FAK) at Chr8 q24.3 occur in ∼70% of HGSOC tumors, and elevated FAK messenger RNA (mRNA) levels are associated with poor patient survival. Herein, we show that active FAK, phosphorylated at tyrosine-576 within catalytic domain, is significantly increased in late-stage HGSOC tumors. Active FAK costained with CD155, a checkpoint receptor ligand for TIGIT (T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domains), in HGSOC tumors and a selective association between FAK and TIGIT checkpoint ligands were supported by patient transcriptomic database analysis. HGSOC tumors with high FAK expression were associated with low CD3 mRNA levels. Accordingly, late-stage tumors showed elevated active FAK staining and significantly lower levels of CD3+ TILs. Using the KMF (Kras, Myc, FAK) syngeneic ovarian tumor model containing spontaneous PTK2 (FAK) gene gains, the effects of tumor intrinsic genetic or oral small molecule FAK inhibitior (FAKi; VS-4718) were evaluated in vivo. Blocking FAK activity decreased tumor burden, suppressed ascites KMF-associated CD155 levels, and increased peritoneal TILs. The combination of FAKi with blocking TIGIT antibody (1B4) maintained elevated TIL levels and reduced TIGIT+ T regulatory cell levels, prolonged host survival, increased CXCL13 levels, and led to the formation of omental tertiary lymphoid structures. Collectively, our studies support FAK and TIGIT targeting as a rationale immunotherapy combination for HGSOC.

FAK activity sustains intrinsic and acquired ovarian cancer resistance to platinum chemotherapy.

Gene copy number alterations, tumor cell stemness, and the development of platinum chemotherapy resistance contribute to high-grade serous ovarian cancer (HGSOC) recurrence. Stem phenotypes involving Wnt-ß-catenin, aldehyde dehydrogenase activities, intrinsic platinum resistance, and tumorsphere formation are here associated with spontaneous gains in Kras, Myc and FAK (KMF) genes in a new aggressive murine model of ovarian cancer. Adhesion-independent FAK signaling sustained KMF and human tumorsphere proliferation as well as resistance to cisplatin cytotoxicity. Platinum-resistant tumorspheres can acquire a dependence on FAK for growth. Accordingly, increased FAK tyrosine phosphorylation was observed within HGSOC patient tumors surviving neo-adjuvant chemotherapy. Combining a FAK inhibitor with platinum overcame chemoresistance and triggered cell apoptosis. FAK transcriptomic analyses across knockout and reconstituted cells identified 135 targets, elevated in HGSOC, that were regulated by FAK activity and ß-catenin including Myc, pluripotency and DNA repair genes. These studies reveal an oncogenic FAK signaling role supporting chemoresistance.

Rgnef promotes ovarian tumor progression and confers protection from oxidative stress.

Ovarian cancer is the fifth-leading cause of cancer death among women. The dissemination of ovarian tumors and growth as spheroids accompanies late-stage disease. In cell culture, ovarian tumor cell spheroids can exhibit elevated resistance to environmental stressors, such as reactive oxygen species. Homeostatic balance of the antioxidant response is a protective mechanism that prevents anoikis, a form of programmed cell death. Signaling pathways activated by integrin receptors suppress anoikis. Rgnef (ARHGEF28/p190RhoGEF) is a guanine nucleotide exchange factor that is activated downstream of integrins. We find that Rgnef protein levels are elevated in late-stage serous ovarian cancer, high Rgnef mRNA levels are associated with decreased progression-free and overall survival, and genomic ARHGEF28 loss is associated with increased patient survival. Using transgenic and transplantable Rgnef knockout mouse models, we find that Rgnef is essential for supporting three-dimensional ovarian spheroid formation in vitro and tumor growth in mice. Using RNA-sequencing and bioinformatic analyses, we identify a conserved Rgnef-supported anti-oxidant gene signature including Gpx4, Nqo1, and Gsta4; common targets of the NF-kB transcription factor. Antioxidant treatment enhanced growth of Rgnef-knockout spheroids and Rgnef re-expression facilitated NF-κB-dependent tumorsphere survival. These studies reveal a new role for Rgnef in ovarian cancer to facilitate NF-κB-mediated gene expression protecting cells from oxidative stress.

Dramatic response to Laetrile and cannabidiol (CBD) oil in a patient with metastatic low grade serous ovarian carcinoma.

•Complimentary alternative medicine use is common in women with gynecologic cancers.•Cannabinoid receptors are potential therapeutic targets in ovarian cancer.•Communication with patients is critical regarding use of alternative therapies.