Rapid on-site evaluation of fine-needle aspiration specimens using cytotechnologist-performed telecytology: Insights and advantages.

With multiple health care centres expanding and implementing remotely controlled technologies, the role of cytopathology laboratories will become crucial in optimizing collection and assessment of small biopsy specimens for diagnosis and personalized treatment. Cytopathology services provide rapid assessments of samples obtained from fine-needle aspirations and occasionally core needle biopsies (analysed by touch imprints). Over the past few decades, the cost barrier for hospitals, especially small ones, to support a cytopathologist or cytotechnologist for rapid on-site evaluation (ROSE) has been reduced by the introduction of computerized microscopes that allow cytopathologists to perform ROSE without being present at the satellite laboratory (i.e. remotely). Utilization of the cytotechnologist's experience in ROSE alongside these technologies made it possible to perform ROSE with telecytology (ROSE-TC), which positively impacts diagnosis and treatment and also lowers the cost of care. The satellite laboratories that successfully implemented ROSE-TC operate with experienced cytotechnologists who, working independently between the cytopathology laboratory and the biopsy suite, prepare the smears and stain the slides on site for the distant cytopathologists, who can evaluate the slides remotely (usually from their office) simply using the facility intranet. This article aims to encourage big and small centres to use senior-level cytotechnologists for both ROSE and ROSE-TC. This article also defines different types of ROSE, explains our institution's approach to ROSE procedures, and outlines some experience-driven considerations for successful ROSE-TC and its future evolution.

Pre-analytical effects on whole transcriptome and targeted RNA sequencing analysis in cytology: The effects of prolonged time in storage of effusion specimens prior to preservation.

OBJECTIVES: To investigate the pre-analytics of the molecular testing of cytology specimens, we studied the effects of time in refrigerator storage (4°C) of malignant effusions on RNA sequencing (RNAseq) results. METHODS: Ten effusion specimens were stored in a refrigerator (4°C) for different durations (day 0, 1, 4, and 7). All specimens were prepared as cytospins fixed in either Carnoy's solution or 95% ethanol (EtOH) and in an RNA preservative for a fresh frozen (FF) high-quality reference. Whole transcriptome (wt) and targeted (t)RNAseq of two multigene expression signatures were performed. We then compared transcript expression levels (including mutant allele fraction) according to pre-analytical variables using a concordance correlation coefficient (CCC) and a mixed effect model. RESULTS: Sequencing results were mostly stable over increasing time in storage. Cytospins fixed in Carnoy's solution were more concordant with FF samples than cytospins fixed in 95% EtOH at all timepoints. This finding was consistent for both wtRNAseq (averages: day 0 CCC = 0.98 vs 0.91; day 7 CCC = 0.88 vs 0.78) and tRNAseq methods (averages: day 0 CCC = 0.98 vs 0.81; day 7 CCC = 0.98 vs 0.90). Cytospins fixed in Carnoy's solution did not show significant changes in expression over timepoints or between expression signatures, whereas 95% EtOH did. CONCLUSION: RNAseq can be accurately performed on effusion specimens after prolonged refrigerator storage. RNA extracted from scraped cytospin slides fixed in Carnoy's solution was marginally superior to 95% EtOH fixation, but either method had comparable analytic performance to high-quality FF RNA samples.

Efficacy of Cobas HPV testing for predicting grade 2+ cervical intraepithelial neoplasia in a cancer prevention center and a gynecologic oncology clinic: A single-institution experience.

BACKGROUND: To evaluate the efficacy of Cobas human papillomavirus (HPV) testing to predict cervical intraepithelial neoplasia of grade 2 or higher (CIN2+), Cobas HPV testing results were correlated with follow-up biopsy in patients from Cancer Prevention Center (CPC) and Gynecologic Oncology Clinic (GOC) of The University of Texas MD Anderson Cancer Center. METHODS: Institutional data for patients who underwent Cobas HPV and Papanicolaou (Pap) cytology cotesting from 2019 to 2020 were retrospectively reviewed. Surgical follow-up results were compared with Cobas HPV testing results in two populations. RESULTS: A total of 2226 patients, including 921 women (mean age, 55.2 years) seen at the CPC and 1305 women (mean age, 49.3 years) seen at the GOC, were included. Specimens from GOC patients had a significantly higher HPV positivity rate than did those from CPC patients (22.9% vs. 10.1%; p < .001). Cobas HPV testing was positive in all seven CPC patients with surgical follow-up results showing CIN2+. Among 36 GOC patients with CIN2+ lesions, five patients had HPV-/Pap+ testing results. Although only seven CPC patients had CIN2+, Cobas HPV testing showed 100% sensitivity for predicting CIN2+ in this group. Sensitivity for CIN2+ was 86.5% in the GOC group, whereas 13.9% of GOC patients with CIN2+ had negative HPV testing results. CONCLUSIONS: Cobas HPV testing was highly efficacious for predicting CIN2+ lesions in the low-risk CPC population, which supports HPV primary screening for cervical cancer in low-risk populations. For high-risk patients, especially those with a history of CIN2+/cervical cancer, HPV/Pap cotesting may still be necessary to maintain a high clinical sensitivity for CIN2+.

Molecular testing opportunities on cytology effusion specimens: the pre-analytic effects of various body fluid cytology preparation methods on RNA extraction quality and targeted sequencing.

INTRODUCTION: RNA sequencing (RNAseq) analysis is emerging as a clinical research or diagnostic approach for cytologic samples, but there is need for formal comparison of different sample preparation methods in the cytology laboratory to identify which pre-analytic methods could provide alternatives to formalin-fixed paraffin-embedded (FFPE) sections. MATERIALS AND METHODS: We prepared 13 malignant effusions (metastatic estrogen receptor-positive breast cancer) in the cytology laboratory using 6 routine cytologic methods: FFPE cell block, Carnoy's solution, 95% ethanol (EtOH), air-dried and Diff-Quik, ThinPrep, and SurePath preparations. Measurements of RNA quality, expression of 2 multigene expression signatures, molecular subtype, and 4 common activating mutation sites in each preparation were compared with fresh frozen (FF) cell pellet in RNA preservative using distribution of fragment length and concordance correlation coefficient (CCC). RESULTS: The fraction of RNA fragments measuring 200 bases or more (DV200) were 24% higher from cytospins fixed in Carnoy's solution or 95% EtOH than DV200 from FFPE cell blocks. SurePath samples failed RNAseq quality control. There was high concordance of gene expression measurements with FF samples using cytospins fixed in Carnoy's solution, 95% EtOH, Diff-Quik (CCC = 0.829, 0.812, 0.760, respectively), or ThinPrep (CCC = 0.736), but lower using FFPE cell block (CCC = 0.564). The proportion of mutant transcripts was concordant between FF and any cytologic preparation methods. CONCLUSIONS: Cytospin preparations fixed with Carnoy's or 95% ETOH then Papanicolaou stained produced RNAseq results that were equivalent to FF samples and superior to FFPE cell block sections.

Detection accuracy of the Cobas HPV assay for high-risk HPV in head and neck FNA biopsy specimens.

BACKGROUND: This study evaluated the detection accuracy of the Cobas human papillomavirus (HPV) assay for high-risk human papillomavirus (hrHPV) and HPV-16 in head and neck fine-needle aspiration (FNA) specimens with squamous cell carcinoma. METHODS: Head and neck FNA biopsy specimens from 2012 to 2020 were retrospectively collected. Cobas HPV testing was performed on 90 FNA specimens with valid Cervista HPV testing results. Results of Cobas HPV and Cervista HPV assays were compared. A Linear Array or SPF10-LiPA25 HPV genotyping assay resolved cases with discrepant results. The κ value and accuracy of Cobas HPV testing were calculated. The accuracy of the Cobas HPV assay was also determined in 42 FNA needle-rinse specimens. RESULTS: Cobas HPV was positive in 82% of the FNA specimens (74 of 90). The concordance between Cobas HPV and Cervista HPV test results was 88.9% (80 of 90) with substantial agreement (κ = 0.669; 95% CI, 0.481-0.856). With HPV genotyping confirmation in cases with discrepant results between the 2 HPV assays, Cobas HPV showed 100% sensitivity and specificity for hrHPV. HPV-16 was detected in 88% of HPV-positive cases (65 of 74). HPV genotyping confirmed 1 false-negative HPV-16 result and 1 false-positive HPV-16 result. Overall, the accuracy of Cobas HPV for HPV-16 was 97.8%. The accuracy of Cobas HPV in FNA needle-rinse specimens was 100%. CONCLUSIONS: The Cobas HPV assay is highly accurate for determining the HPV status in head and neck FNA specimens. FNA needle rinse is valid for Cobas HPV testing in patients with squamous cell carcinoma.

p130Cas is an essential transducer element in ErbB2 transformation.

The ErbB2 oncogene is often overexpressed in breast tumors and associated with poor clinical outcome. p130Cas represents a nodal scaffold protein regulating cell survival, migration, and proliferation in normal and pathological cells. The functional role of p130Cas in ErbB2-dependent breast tumorigenesis was assessed by its silencing in breast cancer cells derived from mouse mammary tumors overexpressing ErbB2 (N202-1A cells), and by its reexpression in ErbB2-transformed p130Cas-null mouse embryonic fibroblasts. We demonstrate that p130Cas is necessary for ErbB2-dependent foci formation, anchorage-independent growth, and in vivo growth of orthotopic N202-1A tumors. Moreover, intranipple injection of p130Cas-stabilized siRNAs in the mammary gland of Balbc-NeuT mice decreases the growth of spontaneous tumors. In ErbB2-transformed cells, p130Cas is a crucial component of a functional molecular complex consisting of ErbB2, c-Src, and Fak. In human mammary cells, MCF10A.B2, the concomitant activation of ErbB2, and p130Cas overexpression sustain and strengthen signaling, leading to Rac1 activation and MMP9 secretion, thus providing invasive properties. Consistently, p130Cas drives N202-1A cell in vivo lung metastases colonization. These results demonstrate that p130Cas is an essential transducer in ErbB2 transformation and highlight its potential use as a novel therapeutic target in ErbB2 positive human breast cancers.

Validation of cobas 4800 HPV assay in SurePath Papanicolaou specimens for cervical cancer screening.

INTRODUCTION: The cobas (Roche Diagnostics, Indianapolis, IN) HPV assay was approved by the US Food and Drug Administration for human papillomavirus (HPV) testing in SurePath (Becton Dickinson, Franklin Lakes, NJ) Papanicolaou specimens for cervical cancer prevention. To validate the cobas HPV assay in SurePath specimens in our institution, we compared its accuracy and clinical efficacy to that of the Cervista (Hologic, Marlborough, MA) HPV HR assay. METHODS: This study used 138 Papanicolaou (Pap) cytology specimens collected in SurePath preservative fluid at our institution in 2018. After Pap cytology testing, the residual specimens were split for testing with the cobas and Cervista assays. Polymerase chain reaction (PCR)-based HPV testing (GP5+/GP6+) was performed on specimens with discrepant results. Clinical follow-up data were reviewed. RESULTS: The cobas HPV and Cervista HPV HR assays showed good concordance (89.1%), with a kappa value of 0.78 (95% CI: 0.675-0.885). Fifteen specimens showed discrepant results between the 2 assays. Of 7 cases with cobas+/Cervista- results, 5 (71%) were confirmed positive by PCR. Of 8 cases with cobas-/Cervista+ results, 4 (50%) were confirmed positive by PCR. cobas HPV and Cervista HPV HR showed the same HPV-positive rate in cases of pathologically diagnosed ASC-H, LSIL, or HSIL. The sensitivities and specificities for detecting high-risk HPV of cobas HPV (93.7%, 97.3%) and Cervista HPV HR (92.1%, 94.7%) were comparable. The cobas HPV assay had false-negative results in 4 cases (5.2%) including 1 false-negative case that failed to predict CIN3. CONCLUSIONS: The cobas HPV assay is valid in SurePath Pap cytology specimens for cervical cancer screening but has limitations of false-negative results with clinical implications.

Integrins and signal transduction.

Integrin signaling has a critical function in organizing cells in tissues during both embryonic development and tissue repair. Following their binding to the extracellular ligands, the intracellular signaling pathways triggered by integrins are directed to two major functions: organization of the actin cytoskeleton and regulation of cell behaviour including survival, differentiation and growth. Basic research conducted in the past twelve years has lead to remarkable breakthroughs in this field. Integrins are catalytically inactive and translate positional cues into biochemical signals by direct and/or functional association with intracellular adaptors, cytosolic tyrosine kinases or growth factor and cytokine receptors. The purpose of this chapter is to highlight recent experimental and conceptual advances in integrin signaling with particular emphasis on the ability of integrins to regulate Fak/Src family kinases (SFKs) activation and the cross-talk with soluble growth factors receptors and cytokines.

p130Cas as a new regulator of mammary epithelial cell proliferation, survival, and HER2-neu oncogene-dependent breast tumorigenesis.

To investigate the mechanisms through which p130Cas adaptor protein is linked to tumorigenesis, we generated mouse mammary tumor virus (MMTV)-p130Cas mice overexpressing p130Cas in the mammary gland. MMTVp130Cas transgenic mice are characterized by extensive mammary epithelial hyperplasia during development and pregnancy and by delayed involution at the end of lactation. These phenotypes are associated with activation of Src kinase, extracellular signal-regulated kinase 1/2, mitogen-activated protein kinase, and Akt pathways, leading to an increased rate of proliferation and a decreased apoptosis. A double-transgenic line derived from crossing MMTV-p130Cas with MMTV-HER2-Neu mice expressing the activated form of the HER2-Neu oncogene develops multifocal mammary tumors with a significantly shorter latency than the HER2-Neu parental strain alone. Mammary epithelial cells isolated from tumors of double-transgenic mice display increased tyrosine phosphorylation, c-Src, and Akt activation compared with cells derived from HER2-Neu tumors. In addition, p130Cas down-regulation by RNA interference increases apoptosis in HER2-Neu-expressing cells, indicating that p130Cas regulates cell survival. Consistently with the double-transgenic mice model, p130Cas is overexpressed in a significant subset of human breast cancers and high levels of p130Cas in association with HER2 expression correlate with elevated proliferation. These findings provide evidences for a role of p130Cas as a positive regulator of both proliferation and survival in normal and transformed mammary epithelial cells. Its overexpression contributes to HER2-Neu-induced breast tumorigenesis, thus identifying this protein as a putative target for clinical therapy.

Architecture of epigenetic reprogramming following Twist1-mediated epithelial-mesenchymal transition.

BACKGROUND: Epithelial-mesenchymal transition (EMT) is known to impart metastasis and stemness characteristics in breast cancer. To characterize the epigenetic reprogramming following Twist1-induced EMT, we characterized the epigenetic and transcriptome landscapes using whole-genome transcriptome analysis by RNA-seq, DNA methylation by digital restriction enzyme analysis of methylation (DREAM) and histone modifications by CHIP-seq of H3K4me3 and H3K27me3 in immortalized human mammary epithelial cells relative to cells induced to undergo EMT by Twist1. RESULTS: EMT is accompanied by focal hypermethylation and widespread global DNA hypomethylation, predominantly within transcriptionally repressed gene bodies. At the chromatin level, the number of gene promoters marked by H3K4me3 increases by more than one fifth; H3K27me3 undergoes dynamic genomic redistribution characterized by loss at half of gene promoters and overall reduction of peak size by almost half. This is paralleled by increased phosphorylation of EZH2 at serine 21. Among genes with highly altered mRNA expression, 23.1% switch between H3K4me3 and H3K27me3 marks, and those point to the master EMT targets and regulators CDH1, PDGFRα and ESRP1. Strikingly, Twist1 increases the number of bivalent genes by more than two fold. Inhibition of the H3K27 methyltransferases EZH2 and EZH1, which form part of the Polycomb repressive complex 2 (PRC2), blocks EMT and stemness properties. CONCLUSIONS: Our findings demonstrate that the EMT program requires epigenetic remodeling by the Polycomb and Trithorax complexes leading to increased cellular plasticity. This suggests that inhibiting epigenetic remodeling and thus decrease plasticity will prevent EMT, and the associated breast cancer metastasis.

FOXC2 expression links epithelial-mesenchymal transition and stem cell properties in breast cancer.

Resistance to chemotherapy and metastases are the major causes of breast cancer-related mortality. Moreover, cancer stem cells (CSC) play critical roles in cancer progression and treatment resistance. Previously, it was found that CSC-like cells can be generated by aberrant activation of epithelial-mesenchymal transition (EMT), thereby making anti-EMT strategies a novel therapeutic option for treatment of aggressive breast cancers. Here, we report that the transcription factor FOXC2 induced in response to multiple EMT signaling pathways as well as elevated in stem cell-enriched factions is a critical determinant of mesenchymal and stem cell properties, in cells induced to undergo EMT- and CSC-enriched breast cancer cell lines. More specifically, attenuation of FOXC2 expression using lentiviral short hairpin RNA led to inhibition of the mesenchymal phenotype and associated invasive and stem cell properties, which included reduced mammosphere-forming ability and tumor initiation. Whereas, overexpression of FOXC2 was sufficient to induce CSC properties and spontaneous metastasis in transformed human mammary epithelial cells. Furthermore, a FOXC2-induced gene expression signature was enriched in the claudin-low/basal B breast tumor subtype that contains EMT and CSC features. Having identified PDGFR-ß to be regulated by FOXC2, we show that the U.S. Food and Drug Administration-approved PDGFR inhibitor, sunitinib, targets FOXC2-expressing tumor cells leading to reduced CSC and metastatic properties. Thus, FOXC2 or its associated gene expression program may provide an effective target for anti-EMT-based therapies for the treatment of claudin-low/basal B breast tumors or other EMT-/CSC-enriched tumors.