Point of Care Molecular Testing: Community-Based Rapid Next-Generation Sequencing to Support Cancer Care.

Purpose: Biomarker data are critical to the delivery of precision cancer care. The average turnaround of next-generation sequencing (NGS) reports is over 2 weeks, and in-house availability is typically limited to academic centers. Lengthy turnaround times for biomarkers can adversely affect outcomes. Traditional workflows involve moving specimens through multiple facilities. This study evaluates the feasibility of rapid comprehensive NGS using the Genexus integrated sequencer and a novel streamlined workflow in a community setting. Methods: A retrospective chart review was performed to assess the early experience and performance characteristics of a novel approach to biomarker testing at a large community center. This approach to NGS included an automated workflow utilizing the Genexus integrated sequencer, validated for clinical use. NGS testing was further integrated within a routine immunohistochemistry (IHC) service, utilizing histotechnologists to perform technical aspects of NGS, with results reported directly by anatomic pathologists. Results: Between October 2020 and October 2021, 578 solid tumor samples underwent genomic profiling. Median turnaround time for biomarker results was 3 business days (IQR: 2-5). Four hundred eighty-one (83%) of the cases were resulted in fewer than 5 business days, and 66 (11%) of the cases were resulted simultaneously with diagnosis. Tumor types included lung cancer (310), melanoma (97), and colorectal carcinoma (68), among others. NGS testing detected key driver alterations at expected prevalence rates: lung EGFR (16%), ALK (3%), RET (1%), melanoma BRAF (43%), colorectal RAS/RAF (67%), among others. Conclusion: This is the first study demonstrating clinical implementation of rapid NGS. This supports the feasibility of automated comprehensive NGS performed and interpreted in parallel with diagnostic histopathology and immunohistochemistry. This novel approach to biomarker testing offers considerable advantages to clinical cancer care.

Cytomorphologic Features of Gastric-Type Endocervical Adenocarcinoma in Liquid-Based Preparations.

OBJECTIVE: Gastric-type endocervical adenocarcinoma (GAS) is a recently described, uncommon, and aggressive tumor with distinct morphologic features and HPV-independent etiology. Data on GAS in liquid-based cytology (LBC) Papanicolaou (Pap) test preparations from a North American patient population are scant. We systematically assessed the cytomorphologic characteristics of GAS in LBC from patients in Ontario and examined if glandular cell nuclear area could represent a readily assessable feature which may aid in GAS detection. STUDY DESIGN: Pap test slides preceding the diagnosis of GAS were retrieved locally or requested from outside laboratories. A structured review of 15 cytomorphologic features was performed using the available LBC Pap test slides of GAS and a set of usual-type endocervical adenocarcinomas (UEA). Morphometry of the glandular cell nuclear area was performed, and normalized values were compared to UEA and benign endocervical cells. RESULTS: At least 1 Pap test (5 ThinPrep®, 11 SurePath®, and 1 direct smear) was available for 14 patients. Original LBC Pap test diagnoses were negative for intraepithelial lesion or malignancy (NILM) (7), adenocarcinoma/carcinoma (6), atypical glandular cells (2), and adenocarcinoma in situ (1). Review detected abnormal glandular cells in 6/7 NILM cases. Honeycomb-like sheets, nuclear enlargement, and microvesicular cytoplasm were the single most common architectural, nuclear, and cytoplasmic features, respectively. Microvesicular cytoplasm (100 vs. 17%), honeycomb-like sheets (87 vs. 8%), prominent nucleoli (93 vs. 25%), and anisonucleosis (93 vs. 50%) were most discriminatory for GAS versus UEA, respectively. Yellow mucin, intranuclear cytoplasmic pseudoinclusions, and goblet/Paneth-like cells were uncommon, but unique for GAS. Glandular cell nuclear area normalized to neutrophils was found to be significantly increased in GAS compared to benign endocervical cells. CONCLUSIONS: GAS is under-recognized and may mimic reactive endocervical cells. Awareness of the tumor type and its cytomorphology is critical for early detection. Identification of glandular cells with uniform nuclear enlargement in conjunction with any of the other cytologic features may help avoid false-negative Pap results. Neutrophils may serve as convenient size reference and visual aid.

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells.

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction of differentiation of two mouse breast epithelial cell lines, HC11 and EpH4, and their use to study complementary stages of mammary gland development and neoplastic transformation. The HC11 mouse breast epithelial cell line originated from the mammary gland of a pregnant Balb/c mouse. It differentiates when grown to confluence attached to a plastic Petri dish surface in medium containing fetal calf serum and Hydrocortisone, Insulin and Prolactin (HIP medium). Under these conditions, HC11 cells produce the milk proteins ß-casein and whey acidic protein (WAP), similar to lactating mammary epithelial cells, and form rudimentary mammary gland-like structures termed "domes". The EpH4 cell line was derived from spontaneously immortalized mouse mammary gland epithelial cells isolated from a pregnant Balb/c mouse. Unlike HC11, EpH4 cells can fully differentiate into spheroids (also called mammospheres) when cultured under three-dimensional (3D) growth conditions in HIP medium. Cells are trypsinized, suspended in a 20% matrix consisting of a mixture of extracellular matrix proteins produced by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, plated on top of a layer of concentrated matrix coating a plastic Petri dish or multiwell plate, and covered with a layer of 10% matrix-containing HIP medium. Under these conditions, EpH4 cells form hollow spheroids that exhibit apical-basal polarity, a hollow lumen, and produce ß-casein and WAP. Using these techniques, our results demonstrated that the intensity of the cadherin/Rac signal is critical for the differentiation of HC11 cells. While Rac1 is necessary for differentiation and low levels of activated RacV12 increase differentiation, high RacV12 levels block differentiation while inducing neoplasia. In contrast, EpH4 cells represent an earlier stage in mammary epithelial differentiation, which is inhibited by even low levels of RacV12.