HPV Cotesting of Unsatisfactory Papanicolaou Tests: Implications for Follow-up Intervals.

OBJECTIVES: The 2019 American Society of Colposcopy and Cervical Pathology management guidelines recommend that patients with an unsatisfactory Papanicolaou (Pap) test (UPT) and negative human papillomavirus (HPV) cotest undergo repeat age-based screening in 2 to 4 months. The rationale is that a negative HPV test in the setting of an UPT may reflect an inadequate sample and therefore should not be interpreted as truly "negative." For patients 25 years and older who are cotested, if HPV is positive for the 16 or 18 genotypes, direct referral for colposcopy is recommended. Our study aimed to determine if a negative HPV cotest result is predictive of the absence of a high-grade squamous intraepithelial lesion (HSIL) and whether these patients may be called back for repeat testing at an interval longer than 2 to 4 months. METHODS: Follow-up cervical cytology and biopsy results in women with UPT and HPV cotests from January 2017 to December 2021 were collected. Original UPT and HPV cotest results were correlated with the follow-up Pap and biopsy results. RESULTS: There were 1,496 (2.28%) UPT cases out of 65,641 total Pap tests. Among the 1,496 UPT cases, 1,010 (67.5%) had HPV cotesting; 676 (45.1%) were followed by repeat Pap or biopsy within 4 months and 850 (56.8%) within 12 months. The total follow-up rate was 81%, with a range of 3 days to 36 months. The HSIL rate in HPV-positive cases was 5.7% (3/53) vs 0.4% (2/539) (P = .006) in HPV-negative cases. In UPT, HPV cotesting showed negative predictive values for low-grade and high-grade squamous intraepithelial lesion detection of 98.5% and 99.6%, respectively, while positive predictive values were 19% and 5.7%. CONCLUSIONS: A negative HPV cotest in individuals with UPT predicted the lack of HSIL in our study. Compliance with the recommended follow-up time of 2 to 4 months for women with UPT was low (45.1%). Our study suggests that women with UPT and negative HPV cotest may be safely called back at an interval longer than 4 months.

The Milan System for Reporting Salivary Gland Cytopathology.

OBJECTIVES: Our study assesses whether the Milan System for Reporting Salivary Gland Cytopathology (MSRSGC) offers any benefit over the original cytology classification, and measures interobserver agreement. METHODS: Four cytopathologists retrospectively blindly classified preoperative cytology by MSRSGC from 101 resected salivary tumors. Consensus MSRSGC diagnoses were correlated with surgical pathology diagnoses and compared with the original cytology classification. Diagnostic parameters were calculated for both systems. Interobserver variability was assessed. RESULTS: The original cytology classification vs MSRSGC had sensitivity, specificity, positive predictive value, and negative predictive value of 75.0% vs 78.3%, 97.1% vs 98.0%, 91.2% vs 94.7%, and 90.1% vs 90.0%, respectively. The original cytology classification risk of neoplasm (RON) was 91.7% for "negative for malignancy" and 100.0% for other categories. The MSRSGC RON was 71.4% in category II (nonneoplastic) and 100.0% in all other categories. The original cytology classification risk of malignancy (ROM) ranged from 0.0% for "atypical" to 100.0% for "positive for malignancy." The MSRSGC ROM ranged from 0.0% in categories I (nondiagnostic) and III (nonneoplastic) to 100.0% in category VI (malignant). Weighted agreement using the MSRSGC was 92% (Gwet AC1, 0.84); unweighted agreement was 69% (Gwet AC1, 0.64). MSRSGC category IVA (benign neoplasm) was most likely to show interobserver agreement, with complete agreement in 67% of cases. CONCLUSIONS: The MSRSGC performs similarly to the original cytology classification and shows relatively high interobserver agreement.

The RNF8 and RNF168 Ubiquitin Ligases Regulate Pro- and Anti-Resection Activities at Broken DNA Ends During Non-Homologous End Joining.

The RING-type E3 ubiquitin ligases RNF8 and RNF168 recruit DNA damage response (DDR) factors to chromatin flanking DNA double strand breaks (DSBs) including 53BP1, which protects DNA ends from resection during DNA DSB repair by non-homologous end joining (NHEJ). Deficiency of RNF8 or RNF168 does not lead to demonstrable NHEJ defects, but like deficiency of 53BP1, the combined deficiency of XLF and RNF8 or RNF168 leads to diminished NHEJ in lymphocytes arrested in G0/G1 phase. The function of RNF8 in NHEJ depends on its E3 ubiquitin ligase activity. Loss of RNF8 or RNF168 in G0/G1-phase lymphocytes leads to the resection of broken DNA ends, demonstrating that RNF8 and RNF168 function to protect DNA ends from nucleases, pos sibly through the recruitment of 53BP1. However, the loss of 53BP1 leads to more severe resection than the loss of RNF8 or RNF168. Moreover, in 53BP1-deficient cells, the loss of RNF8 or RNF168 leads to diminished DNA end resection. We conclude that RNF8 and RNF168 regulate pathways that both prevent and promote DNA end resection in cells arrested in G0/G1 phase.

Feasibility and clinical utility of a pan-solid tumor targeted RNA fusion panel: A single center experience.

Gene fusions are caused by chromosomal rearrangements and encode fusion proteins that can act as oncogenic drivers in cancers. Traditional methods for detecting oncogenic fusion transcripts include fluorescence in situ hybridization (FISH), reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC). However, these methods are limited in scalability and pose significant technical and interpretational challenges. Next-generation sequencing (NGS) is a high-throughput method for detecting genetic abnormalities and providing prognostic and therapeutic information for cancer patients. We present our experience with the validation of a custom-designed Archer Anchored Multiplex PCR (AMP™) technology-based NGS technology, "NYU FUSION-SEQer" using RNA sequencing. We examine both analytical performance and clinical utility of the panel using 75 retrospective validation samples and 84 prospective clinical samples of solid tumors. Our panel showed robust sequencing performance with strong enrichment for target regions. The lower limit of detection was 12.5% tumor fraction at 125 ng of RNA input. The panel demonstrated excellent analytic accuracy, with 100% sensitivity, 100% specificity and 100% reproducibility on validation samples. Finally, in the prospective cohort, the panel detected fusions in 61% cases (n = 51), out of which 41% (n = 21) enabling diagnosis and 59% (n = 30) enabling treatment and prognosis. We demonstrate that the fusion panel can accurately, efficiently and cost-effectively detect the majority of known fusion genes, novel clinically relevant fusions and provides an excellent tool for discovery of new fusion genes in solid tumors.

XLF and H2AX function in series to promote replication fork stability.

XRCC4-like factor (XLF) is a non-homologous end joining (NHEJ) DNA double strand break repair protein. However, XLF deficiency leads to phenotypes in mice and humans that are not necessarily consistent with an isolated defect in NHEJ. Here we show that XLF functions during DNA replication. XLF undergoes cell division cycle 7-dependent phosphorylation; associates with the replication factor C complex, a critical component of the replisome; and is found at replication forks. XLF deficiency leads to defects in replication fork progression and an increase in fork reversal. The additional loss of H2AX, which protects DNA ends from resection, leads to a requirement for ATR to prevent an MRE11-dependent loss of newly synthesized DNA and activation of DNA damage response. Moreover, H2ax-/-:Xlf-/- cells exhibit a marked dependence on the ATR kinase for survival. We propose that XLF and H2AX function in series to prevent replication stress induced by the MRE11-dependent resection of regressed arms at reversed replication forks.

MRI Is a DNA Damage Response Adaptor during Classical Non-homologous End Joining.

The modulator of retrovirus infection (MRI or CYREN) is a 30-kDa protein with a conserved N-terminal Ku-binding motif (KBM) and a C-terminal XLF-like motif (XLM). We show that MRI is intrinsically disordered and interacts with many DNA damage response (DDR) proteins, including the kinases ataxia telangiectasia mutated (ATM) and DNA-PKcs and the classical non-homologous end joining (cNHEJ) factors Ku70, Ku80, XRCC4, XLF, PAXX, and XRCC4. MRI forms large multimeric complexes that depend on its N and C termini and localizes to DNA double-strand breaks (DSBs), where it promotes the retention of DDR factors. Mice deficient in MRI and XLF exhibit embryonic lethality at a stage similar to those deficient in the core cNHEJ factors XRCC4 or DNA ligase IV. Moreover, MRI is required for cNHEJ-mediated DSB repair in XLF-deficient lymphocytes. We propose that MRI is an adaptor that, through multivalent interactions, increases the avidity of DDR factors to DSB-associated chromatin to promote cNHEJ.