Accurate genome-wide genotyping from archival tissue to explore the contribution of common genetic variants to pre-cancer outcomes.

PURPOSE: The contribution of common genetic variants to pre-cancer progression is understudied due to long follow-up time, rarity of poor outcomes and lack of available germline DNA collection. Alternatively, DNA from diagnostic archival tissue is available, but its somatic nature, limited quantity and suboptimal quality would require an accurate cost-effective genome-wide germline genotyping methodology. EXPERIMENTAL DESIGN: Blood and tissue DNA from 10 individuals were used to benchmark the accuracy of Single Nucleotide Polymorphisms (SNP) genotypes, Polygenic Risk Scores (PRS) or HLA haplotypes using low-coverage whole-genome sequencing (lc-WGS) and genotype imputation. Tissue-derived PRS were further evaluated for 36 breast cancer patients (11.7 years median follow-up time) diagnosed with DCIS and used to model the risk of Breast Cancer Subsequent Events (BCSE). RESULTS: Tissue-derived germline DNA profiling resulted in accurate genotypes at common SNPs (blood correlation r2 > 0.94) and across 22 disease-related polygenic risk scores (PRS, mean correlation r = 0.93). Imputed Class I and II HLA haplotypes were 96.7% and 82.5% concordant with clinical-grade blood HLA haplotypes, respectively. In DCIS patients, tissue-derived PRS was significantly associated with BCSE (HR = 2, 95% CI 1.2-3.8). The top and bottom decile patients had an estimated 28% and 5% chance of BCSE at 10 years, respectively. CONCLUSIONS: Archival tissue DNA germline profiling using lc-WGS and imputation, represents a cost and resource-effective alternative in the retrospective design of long-term disease genetic studies. Initial results in breast cancer suggest that common risk variants contribute to pre-cancer progression.

PTEN Loss and ARID1A Mutation in an HPV-positive Metastatic Adenocarcinoma Diagnosed Almost 18 yr After an Intact Cone Excision for Endocervical Adenocarcinoma In Situ.

There have been previous reports of neoplasms with the morphology of endocervical adenocarcinoma in situ (AIS) that secondarily involve the ovaries, presumably through transtubal spread, with a smaller subset metastasizing to distant sites. These ovarian metastases have been discovered up to 7 yr postexcision of the endocervical lesion, consistent with the known potential for overtly invasive cervical carcinomas to recur late after primary curative management. Herein, we present a case of a premenopausal woman with a pelvic mass classified as metastatic human papillomavirus (HPV)-associated endocervical adenocarcinoma (p16-block immunoreactive, high-risk HPV positive by in situ hybridization with PTEN loss, ARID1A, and PBRM1 mutations detected by qualitative next-generation sequencing), identified 17.7 yr (212 mo) after a fertility-sparing cone excision with negative margins for endocervical AIS [HPV-associated, p16-block immunoreactive; PTEN, and BAF250a (ARID1a) expression retained]. Our case highlights: (1) the potential for a subset of lesions with the morphology of AIS to metastasize, and the extraordinarily long timeframe (almost 18 y, the longest reported to date) during which metastases may still be identified; (2) alterations in PTEN and ARID1A may play a role in the progression of a subset of endocervical carcinomas; and (3) the need for studies to evaluate the utility of incorporating ovarian/pelvic imaging into surveillance protocols following fertility-sparing excisions or ovarian-preserving hysterectomies, during the management of endocervical adenocarcinomas, as well as the need to counsel patients about the small but real risk of delayed discovery of ovarian metastases following fertility-preserving surgeries for AIS.

Implementation of the 2018 American Society of Clinical Oncology/College of American Pathologists Guidelines on HER2/neu Assessment by FISH in breast cancers: predicted impact in a single institutional cohort.

The 2018 American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP) update modified the interpretation guidelines for human epidermal growth factor receptor 2 (HER2) testing by incorporating immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) results in a subset of cases. Importantly, the new guidelines eliminate "equivocal" results, as well as the use of alternative chromosome 17 probes as the primary strategy for resolving the indeterminate FISH results. Herein, we investigate the predicted impact of implementing the 2018 ASCO/CAP guidelines on HER2 assessment by FISH in breast cancers, using data from a single institution. We compared the HER2 status of 1542 consecutive cases of breast carcinoma, interpreted by 2013 and 2018 ASCO/CAP guidelines. In total, 10.7% (165/1542) of the cases had a different final interpretation by 2018 guidelines compared with 2013 guidelines, including 70 previously HER2-positive cases reclassified as negative, four previously negative cases reclassified as positive, and 91 previously equivocal cases reclassified as negative. Overall, the number of HER2-positive cancers was reduced by 66 cases (4.3% reduction in the HER2 positivity rate). The newly HER2-negative cases were mostly estrogen receptor positive (90%), progesterone receptor positive (80%), stage 1 (60.9%), and grade 1-2 (59.4%) cancers; 70% of them had been designated as HER2 positive only after the use of an alternative chromosome 17 FISH probe after an intially equivocal result from the standard CEP17 probe. Overall, implementing the revised 2018 HER2 guidelines is predicted to change the HER2 results of 10.7% of breast cancers, mainly by reclassifying previously equivocal to negative results.

Phenotypic alterations in breast cancer associated with neoadjuvant chemotherapy: A comparison with baseline rates of change.

Several studies have documented phenotypic alterations in breast cancer associated with neoadjuvant chemotherapy [NACT], but many of these studies are limited by the fact that they did not account for the baseline rate of expected phenotypic change between biopsies and resections in the absence of NACT. Herein, we assess whether the NACT-associated rate of phenotypic change is significantly different than would be expected in a control population of patients that did not receive NACT. From a pathologic database, we documented the estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2/neu) phenotypes of consecutive invasive breast carcinomas (n=826), as well as the subset in which at least one of these tests was assessed in both the biopsy and resection (n=340). We then compared the rates of phenotypic change in the patients that did (n=65) and did not (n=275) receive NACT. Respectively, 49.2% and 36% of the NACT and non-NACT groups showed a biopsy-to-resection change in status for at least one biomarker (p=0.0005). The NACT and non-NACT groups showed the following respective rates of a biopsy-to-resection change in phenotype: ER (9.2% vs 2.5%, p=0.02); PR (30.7% vs 8%, p=0.000006); Her2/neu-IHC (25% vs 22.3%, p=0.7), Her2/neu-FISH (7% vs 3%, p=0.6). The direction of change in the NACT group was positive in the biopsy to negative in the resection in >70% of cases for all markers. For ER and PR, there was no statistically significant difference between cases that showed a biopsy-to-excision change in phenotype and those that were more phenotypically stable regarding a wide array of clinicopathologic variables. The average percentage of ER/PR-immunoreactive tumor cells in the pre-NACT biopsies was significantly lower in the phenotypically altered cases as compared to the phenotypically stable cases. Our findings confirm that phenotypic alterations in breast cancer occur after NACT, and that these changes are more pronounced for hormone receptors (especially PR); Significant NACT-associated alterations were not apparent for HER2/neu. A distinct pathologic profile for cases displaying a phenotypic change within the NACT group was not demonstrable. The pre-NACT levels of ER and PR may affect the likelihood of a phenotypic change. These results highlight the need for repeat testing in residual tumors after NACT.

Malignant cell contamination may lead to false-positive findings at endosonographic fine needle aspiration for tumor staging.

BACKGROUND AND STUDY AIMS: Endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) of lymph nodes is used for staging of gastrointestinal malignancies. False-positive rates of 5 % - 7 % are not well understood. Elements of EUS examinations that contribute to false-positive cytological findings were investigated. PATIENTS AND METHODS: 13 patients undergoing EUS staging of gastrointestinal luminal malignancy were consecutively enrolled together with 3 patients with extraluminal tumors (pancreas, lung) defined as controls. After EUS, cellular debris and fluid were collected from the FNA needle catheter, instrument channel, and endoscope tip for cytologic and histologic investigation. RESULTS: 6 of 13 patients (46 %) had malignant cells contaminating the FNA needle catheter, instrument channel, or endoscope tip. Malignant cells were present in the instrument channel in 5 cases (38 %), exterior tip of echoendoscope in 4 (31 %) and needle catheter in 2 (15 %). CONCLUSIONS: Echoendoscopes used for FNA in patients with luminal tumors are at risk for malignant cell contamination of the instrument channel, FNA needle catheter, and echoendoscope tip. FNA needle contamination can contribute to false-positive findings.

Fumarate Hydratase-Deficient Leiomyoma of the Uterine Corpus: Comparative Morphologic Analysis of Protein-Deficient Tumors With and Without Pathogenic Germline Fumarate Hydratase Gene Mutations.

Deficiency of fumarate hydratase (FH) protein expression in uterine corpus leiomyomas may be attributable to either germline or somatic mutations of the FH gene, the former being definitional for the hereditary leiomyomatosis and renal cell cancer syndrome. The authors assess whether, using previously reported FH-associated morphologic features, FH protein-deficient uterine corpus leiomyomas associated with a pathogenic germline mutations of the FH gene (group 1) are distinguishable from FH protein-deficient uterine corpus leiomyomas without such mutations (and whose FH protein loss is presumed to be attributable to somatic/epigenetic inactivation or other unknown phenomena: group 2). Groups 1 and 2 were compared regarding a variety of clinicopathologic features, including 7 core "FH-associated" tumoral morphologic features: staghorn vasculature; alveolar-type edema; bizarre nuclei; chain-like tumor nuclei; hyaline cytoplasmic globules; prominent nucleoli, intranuclear inclusions, and perinucleolar halos; and prominent eosinophilic/fibrillary cytoplasm. Among 2418 patients diagnosed with uterine corpus leiomyoma during the study period, FH-associated morphologic features were reported in 1.5% (37 patients), and FH immunohistochemistry was performed in 29 (1.19%). Fourteen (48.27%) of the 29 patients showed FH protein deficiency by immunohistochemistry. Twelve patients underwent germline testing, of which 8 (66.7%) were classified as group 1 and 4 (33.3%) as group 2. FH protein-deficient tumors were larger (10.44 vs 4.08 cm, P = 0.01) and associated with younger patients (42.05 vs 47.97, P = 0.004) than 370 randomly selected uterine leiomyoma controls. Groups 1 and 2 showed no significant differences in patient age and tumor size. In group 1 tumors, the FH-associated morphologic features were generally present diffusely; all group 1 tumors displayed ≥5 FH-associated features, whereas all group 2 tumors displayed <5 FH-associated features (means 6.5 ± 0.53 vs 3.5 ± 1.00, P < 0.001). Notably, eosinophilic/fibrillary cytoplasm and alveolar-type edema were each significantly more prevalent in group 1 tumors than group 2 tumors (P = 0.018 for both). No single morphologic feature was found to be completely sensitive and specific in making the distinction between group 1 and 2 tumors. Our findings suggest that groups 1 and 2 are unlikely to be morphologically distinguishable by individual morphologic features. Whether there is a combination of features that can reliably make this distinction is unclear and will require additional studies with larger cohorts.

PI3Kγ inhibition circumvents inflammation and vascular leak in SARS-CoV-2 and other infections.

Virulent infectious agents such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and methicillin-resistant Staphylococcus aureus (MRSA) induce tissue damage that recruits neutrophils, monocyte, and macrophages, leading to T cell exhaustion, fibrosis, vascular leak, epithelial cell depletion, and fatal organ damage. Neutrophils, monocytes, and macrophages recruited to pathogen-infected lungs, including SARS-CoV-2-infected lungs, express phosphatidylinositol 3-kinase gamma (PI3Kγ), a signaling protein that coordinates both granulocyte and monocyte trafficking to diseased tissues and immune-suppressive, profibrotic transcription in myeloid cells. PI3Kγ deletion and inhibition with the clinical PI3Kγ inhibitor eganelisib promoted survival in models of infectious diseases, including SARS-CoV-2 and MRSA, by suppressing inflammation, vascular leak, organ damage, and cytokine storm. These results demonstrate essential roles for PI3Kγ in inflammatory lung disease and support the potential use of PI3Kγ inhibitors to suppress inflammation in severe infectious diseases.

Evaluation of ultra-deep targeted sequencing for personalized breast cancer care.

INTRODUCTION: The increasing number of targeted therapies, together with a deeper understanding of cancer genetics and drug response, have prompted major healthcare centers to implement personalized treatment approaches relying on high-throughput tumor DNA sequencing. However, the optimal way to implement this transformative methodology is not yet clear. Current assays may miss important clinical information such as the mutation allelic fraction, the presence of sub-clones or chromosomal rearrangements, or the distinction between inherited variants and somatic mutations. Here, we present the evaluation of ultra-deep targeted sequencing (UDT-Seq) to generate and interpret the molecular profile of 38 breast cancer patients from two academic medical centers. METHODS: We sequenced 47 genes in matched germline and tumor DNA samples from 38 breast cancer patients. The selected genes, or the pathways they belong to, can be targeted by drugs or are important in familial cancer risk or drug metabolism. RESULTS: Relying on the added value of sequencing matched tumor and germline DNA and using a dedicated analysis, UDT-Seq has a high sensitivity to identify mutations in tumors with low malignant cell content. Applying UDT-Seq to matched tumor and germline specimens from the 38 patients resulted in a proposal for at least one targeted therapy for 22 patients, the identification of tumor sub-clones in 3 patients, the suggestion of potential adverse drug effects in 3 patients and a recommendation for genetic counseling for 2 patients. CONCLUSION: Overall our study highlights the additional benefits of a sequencing strategy, which includes germline DNA and is optimized for heterogeneous tumor tissues.

B3 thymoma mimicking poorly differentiated thyroid carcinoma: Diagnostic pitfalls of anterior mediastinal mass fine needle aspiration.

B3 thymoma is a rare malignant type of thymic epithelial neoplasm found in the anterior mediastinum. Diagnosis of thymoma from fine needle aspiration (FNA) can be challenging due to the infrequency of sampling and its morphologic overlap with other entities such as squamous cell carcinoma, lymphoma or thyroid carcinoma. We report a case of B3 thymoma mimicking poorly differentiated thyroid carcinoma. We present its diagnostic pitfalls on cytology specimens, especially where it concerns identifying the correct location of the lesion, discuss the differential diagnosis, and correlation with the corresponding surgical resection specimen. A neck computed tomography angiogram (CTA) revealed a partially calcified 2.1 cm mass inferior to the left thyroid lobe in a 51 yr old woman being evaluated for stroke/TIA symptoms. She was referred for evaluation of the lesion. On the initial FNA and core needle biopsy, the lesion showed high-grade epithelioid cells with abundant lymphocytic infiltration and occasional necrosis, and was diagnosed as a high-grade carcinoma, favored to represent a poorly differentiated thyroid carcinoma considering the location on imaging. The patient subsequently underwent total thyroidectomy, central neck dissection, and thymectomy. Final surgical pathologic diagnosis indicated a type B3 thymoma. Due to the infrequency of sampling, thymoma poses a diagnostic challenge on preoperative FNA or core needle biopsy. Herein, we present a case of B3 thymoma with a preoperative cytologic specimen that consisted of hyperchromatic sheets of epithelioid tumor cells with a background of lymphocytes without definitive follicular cells or colloid. The core needle biopsy and cell block material showed abundant necrosis, intermixed lymphocytes and neoplastic epithelial cells with strong positive staining for pan-keratin and p40. The cytology and core needle biopsy material were interpreted as representing a probable thyroid neoplasm and raised a broad differential including anaplastic thyroid carcinoma, poorly differentiated thyroid carcinoma with squamous features, metastatic squamous carcinoma, and metastatic carcinoma to a lymph node. The final surgical resection specimen showed a B3 type-thymoma.

Using computer-assisted content analysis to advance anal dysplasia natural history research.

OBJECTIVE: Our study aim was to validate the use of computer-aided narrative content analysis in the extraction of standard diagnostic categories using an archived cytology database that included individually overread reference classification. DESIGN: A retrospective analysis of narrative anal cytology results collected on HIV-infected patients at the University of California, San Diego between January and December 2001. METHODS: We used computer-assisted content analysis extraction methodology using Wordstat 8.0 (Provalis Research) that operated using a classification dictionary that we developed for the following diagnostic categories: NAMC, ASCUS, LSIL, HSIL. We compared its accuracy to a physician overread manually extracted method: that classified each report into the most severe diagnostic category referenced in the narrative report. Agreement between content analysis mapped diagnostic categories and the reference category was evaluated using kappa agreement. RESULTS: During 2001, 901 patients underwent 997 anal cytological examinations as routine screening. By reference diagnostic category: 54 (5.4%) were unsatisfactory, 460 (46.1%) were NAMC, 291 (29.2%) were ASCUS, 131 (13.1%) were LSIL, and 61 (6.1%) were HSIL. Computer-aided content analysis extracted a single diagnosis from each report in 963 (96.2%) cases and two diagnoses in 38 (3.8%) cases. The Kappa agreement was 0.96 (0.019 s.e.). There were 29 cases classified ASCUS by reference category but LSIL by adjudicated content analysis. A focused review indicated that the over reader assigned reference category was in error. CONCLUSION: Computer-aided narrative content analysis of anal cytology results yielded accurate and time-efficient classification into meaningful diagnostic categories that can be used to evaluate screening programs and modeling natural history.

The breast pre-cancer atlas illustrates the molecular and micro-environmental diversity of ductal carcinoma in situ.

Microenvironmental and molecular factors mediating the progression of Breast Ductal Carcinoma In Situ (DCIS) are not well understood, impeding the development of prevention strategies and the safe testing of treatment de-escalation. We addressed methodological barriers and characterized the mutational, transcriptional, histological, and microenvironmental landscape across 85 multiple microdissected regions from 39 cases. Most somatic alterations, including whole-genome duplications, were clonal, but genetic divergence increased with physical distance. Phenotypic and subtype heterogeneity was frequently associated with underlying genetic heterogeneity and regions with low-risk features preceded those with high-risk features according to the inferred phylogeny. B- and T-lymphocytes spatial analysis identified three immune states, including an epithelial excluded state located preferentially at DCIS regions, and characterized by histological and molecular features of immune escape, independently from molecular subtypes. Such breast pre-cancer atlas with uniquely integrated observations will help scope future expansion studies and build finer models of outcomes and progression risk.

Quantitative comparison of surgical margin histology following excision with traditional electrosurgery and a low-thermal-injury dissection device.

BACKGROUND: This study is the first to examine in vivo the effect of thermal injury in breast conservation pathology in a direct comparison of traditional electrosurgery and an alternative low-thermal-injury device. METHODS: A prospective study of 20 consecutive subjects with biopsy-proven invasive ductal carcinoma (IDC) tumors 1 cm was conducted. Following excision, incisions were made into the tumor with the two devices. Thermal injury depth, margin distance, tissue type, and histological effect were compared on the same breast tissue cut with each excision instrument. A probability evaluation of close and positive margin cases for the true tumor margins was conducted. RESULTS: Compared to traditional electrosurgery, the low-thermal-injury instrument reduced collagen denaturation depth from 435 to 102 µm (77%), fused tissue depth from 262 to 87 µm (67%), and distortion depth from 1,132 to 774 µm (30%). CONCLUSIONS: Based on analysis of the close subset of the true margins, using the traditional electrosurgical device in place of the low-thermal-injury device would have resulted in 48% of the close margin samples being negatively converted to false-positive, and in 11% converting from close to false-negative. The methodology of this work may be readily applied to larger, more definitive studies.

Nonmass enhancement lesions of the breast on core needle biopsy: outcomes, frequency of malignancy, and pathologic findings.

Nonmass enhancement (NME) on breast magnetic resonance imaging (MRI) is defined as an area whose internal enhancement characteristics can be distinguished from the normal surrounding breast parenchyma, without an associated mass in the Breast Imaging Reporting and Data System lexicon. In this study, we evaluated the pathologic correlates of NME lesions of the breast identified on MRI at our institution, including the frequency of atypical or malignant lesions in the core needle biopsies (CNBs), performed after such a radiologic finding. A retrospective study was performed on all CNBs performed for NME on breast MRI between 2010 and 2019. A total of 443 biopsies from 411 patients were identified, comprising 5.5% of all CNBs over the study period. The pathologic diagnoses were benign in the majority of the biopsies (68.0%), whereas 11.5% and 20.5% of the cases were atypical and malignant lesions, respectively. Of the malignant cases, 69.2% were ductal carcinoma in situ (DCIS) and 30.8% were invasive carcinomas. The most common invasive cancer was invasive ductal carcinoma (50%), followed by invasive lobular carcinoma (39.3%). NME identified on breast MRI carried a significant (32%) risk of atypia and malignancy in our cohort, which confirms that biopsy evaluation of these lesions is warranted. DCIS was the most commonly identified malignancy. Notably, among invasive cancers, invasive lobular carcinoma was identified at a substantially higher frequency that would be expected for that histotype.

Rectal endometriosis mimicking primary rectal adenocarcinoma.

Endometriosis is a benign entity defined as the presence of endometrium tissue outside of uterine cavity. It is a common disease involving peritoneum, pelvic organs, gastrointestinal tract, and so on. Diagnosis based on cytology specimen can be challenge when we encounter increased cytological atypia in the glandular epithelium, with abundant inflammatory cells and debris in the background. We presented a case of deep rectal endometriosis mimicking rectal adenocarcinoma on cytology specimen and on MRI imaging studies. The combination of endometrial glands, cellular Mullerian stroma, hemorrhage, and hemosiderin-laden macrophages are the characteristic features on cytologic specimens.

Independent Validation of Tumor Budding Activity and Cell Nest Size as Determinants of Patient Outcome in Squamous Cell Carcinoma of the Uterine Cervix.

A novel 3-tiered grading system that combines tumor budding activity and cell nest size has been found to be highly prognostic in squamous cell carcinomas (SCCs) of various sites, including lung, oral cavity, larynx, hypopharynx, and esophagus. A similar grading system has recently been proposed for SCC of the uterine cervix. In this study, we appraise this grading system in an institutional cohort of cervical SCC to assess its prognostic value in an independent dataset. Our study cohort consisted of 94 consecutive, surgically excised, neoadjuvant therapy-naive cases of SCC of the uterine cervix, stage pT1b or higher. Tumor budding activity and cell nest size were scored on each case, the sum of which formed the basis for assigning a grade in the 3-tiered grading system hereafter referred to as the "tumor budding/nest size" (TBNS) system. As individual variables, both high tumor budding and small nest size were each associated with reduced overall survival (OS), disease-specific survival, and disease-free survival. The full TBNS system was associated with decreased OS, disease-specific survival, and disease-free survival independent of patient age, pathologic stage, and regional lymph node status. TBNS grades 1, 2, and 3 subgroups were clearly distinguishable on multivariate analyses (hazard ratio for OS of 2.06 [95% confidence interval: 0.5-8.42] for grade 2 and 4.58 [95% confidence interval: 1.24-16.87] for grade 3 tumors, relative to their grade 1 counterparts [P=0.035]). Higher grade tumors in the TBNS system were significantly correlated with advanced pathologic stage and lymph node metastasis (P=0.044 and 0.04, respectively). Among the other, potentially prognostic factors, higher pathologic stage, and lymph node metastasis were associated with decreased OS (P<0.001 and 0.004, respectively), whereas keratinization, nuclear size, mitotic count, and World Health Organization (WHO) grade were not. In conclusion, the proposed TBNS grading system is an excellent prognostic indicator that may potentially provide information that is useful in clinical decision-making. Our findings validate the previous study that proposed this system for prognostically stratifying cervical SCC patients. If further confirmed, consideration should be given to routinely adding a TBNS grade to pathologic descriptions of cervical SCC.

Mutational profiling of micro-dissected pre-malignant lesions from archived specimens.

BACKGROUND: Systematic cancer screening has led to the increased detection of pre-malignant lesions (PMLs). The absence of reliable prognostic markers has led mostly to over treatment resulting in potentially unnecessary stress, or insufficient treatment and avoidable progression. Importantly, most mutational profiling studies have relied on PML synchronous to invasive cancer, or performed in patients without outcome information, hence limiting their utility for biomarker discovery. The limitations in comprehensive mutational profiling of PMLs are in large part due to the significant technical and methodological challenges: most PML specimens are small, fixed in formalin and paraffin embedded (FFPE) and lack matching normal DNA. METHODS: Using test DNA from a highly degraded FFPE specimen, multiple targeted sequencing approaches were evaluated, varying DNA input amount (3-200 ng), library preparation strategy (BE: Blunt-End, SS: Single-Strand, AT: A-Tailing) and target size (whole exome vs. cancer gene panel). Variants in high-input DNA from FFPE and mirrored frozen specimens were used for PML-specific variant calling training and testing, respectively. The resulting approach was applied to profile and compare multiple regions micro-dissected (mean area 5 mm2) from 3 breast ductal carcinoma in situ (DCIS). RESULTS: Using low-input FFPE DNA, BE and SS libraries resulted in 4.9 and 3.7 increase over AT libraries in the fraction of whole exome covered at 20x (BE:87%, SS:63%, AT:17%). Compared to high-confidence somatic mutations from frozen specimens, PML-specific variant filtering increased recall (BE:85%, SS:80%, AT:75%) and precision (BE:93%, SS:91%, AT:84%) to levels expected from sampling variation. Copy number alterations were consistent across all tested approaches and only impacted by the design of the capture probe-set. Applied to DNA extracted from 9 micro-dissected regions (8 PML, 1 normal epithelium), the approach achieved comparable performance, illustrated the data adequacy to identify candidate driver events (GATA3 mutations, ERBB2 or FGFR1 gains, TP53 loss) and measure intra-lesion genetic heterogeneity. CONCLUSION: Alternate experimental and analytical strategies increased the accuracy of DNA sequencing from archived micro-dissected PML regions, supporting the deeper molecular characterization of early cancer lesions and achieving a critical milestone in the development of biology-informed prognostic markers and precision chemo-prevention strategies.

Normal human lymph node T follicular helper cells and germinal center B cells accessed via fine needle aspirations.

Germinal centers (GC) are critically important for maturation of the antibody response and generation of memory B cells, processes that form the basis for long-term protection from pathogens. GCs only occur in lymphoid tissue, such as lymph nodes, and are not present in blood. Therefore, GC B cells and GC T follicular helper (TFH) cells are not well-studied in humans under normal healthy conditions, due to the limited availability of healthy lymph node samples. We used a minimally invasive, routine clinical procedure, lymph node fine needle aspirations (LN FNAs), to obtain LN cells from healthy human subjects. This study of 73 LNs demonstrates that human LN FNAs are a safe and feasible technique for immunological research, and suggests benchmarks for human GC biology under noninflammatory conditions. The findings indicate that assessment of the GC response via LN FNAs will have application to the study of human vaccination, allergy, and autoimmune disease.

Estrogen Receptor, Progesterone Receptor, and Human Epidermal Growth Factor Receptor-2 Testing in Breast Cancer: Assessing the Value of Repeated Centralized Testing in Excision Specimens.

At some tertiary breast care centers, where many patients are referred from other institutions, it is routine to repeat testing for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2/neu) in excision specimens if these tests were performed on the preceding biopsy at the referring facility. The goal of this study is to assess the value of this practice. We documented results from ER, PR, and HER2 testing in 541 consecutive invasive breast cancers excised over a 2.5-year period and analyzed the subset (n=153) for which testing was performed on the excision specimen solely due to the fact that testing on the preceding biopsy was performed at an outside institution. The rates and directions of biopsy-to-excision change were as follows: ER [1.3% (2/153), 100% from (+) to (-)]; PR [4% (6/153), 83% from (+) to (-)]; HER2/neu assessed by immunohistochemistry [21% (29/137)]; HER2/neu assessed by fluorescence in situ hybridization [3.3% (2/61); 50% from amplified to nonamplified and 50% vice versa]. There were no ER(-) and PR(-) biopsy cases that became ER and/or PR(+) in the excision. By coordinate analysis for the hormone receptors [ie, ER and/or PR(+) being indicative of "hormone receptor" (HR) positivity], there were no cases that changed from HR(+) in the biopsy to HR(-) in the excision (or vice versa), which suggests that repeat testing for ER and PR in this setting is of limited value. In an analysis that incorporated both immunohistochemistry and in situ fluorescence hybridization results, there were 2 cases with a clinically significant biopsy-to-excision change in HER2/neu status in which that change was detected primarily because the excision was retested. These findings provide baseline data for formulating policies on whether repeat testing should routinely be performed in the described scenario.

Intranodal palisaded myofibroblastoma of para-esophageal lymph node: Case report with cytologic and histologic findings.

Intranodal palisaded myofibroblastoma is a rare lymph node mesenchymal neoplasm that most commonly arises in inguinal lymph nodes. There is limited data about cytologic features of this tumor and its diagnostic pitfalls. The combination of bland appearing spindle cells, fibrillary matrix, and hemosiderin pigments are the characteristic features of this neoplasm in cytologic specimens.

Breast cancers with a HER2/CEP17 ratio of 2.0 or greater and an average HER2 copy number of less than 4.0 per cell: frequency, immunohistochemical correlation, and clinicopathological features.

The 2013 American Society of Clinical Oncology and College of American Pathologists (ASCO/CAP) guidelines classified breast cancers with a fluorescence in situ hybridization dual-probe HER2/CEP17 ratio of 2 or greater as "amplified," inclusive of cases with a HER2 copy number less than 4. The 2018 ASCO/CAP update assigns HER2/neu status for the latter group in a fashion that is highly dependent on the associated immunohistochemical findings. Herein, the authors define the frequency, immunohistochemical correlates, and other clinicopathological features of breast cancers with HER2/CEP17 ratio of 2 or greater and HER2/neu copy number less than 4 (group A), based on an analysis of an institutional cohort assessed for HER2/neu status by both florescence in situ hybridization and immunohistochemistry and scored using 2013 ASCO/CAP criteria. Group A cases were compared with a group B of HER2/neu-amplified breast cancers with a HER2/neu copy number of 4 or greater regarding a variety of clinicopathological features. One hundred sixty-nine (14%) of 1201 cases were HER2/neu amplified, 18 (10.7%) in group A and 151 (89.3%) in group B. By immunohistochemistry, 61.1% of group A cases were HER2/neu negative, 7 (38.9%) were equivocal, and none were positive. In contrast, 66.9% of group B cases were HER2 positive (3+). We could not demonstrate statistically significant differences between the 2 groups regarding standard clinicopathological variables. In summary, our group A cases account for 1.5% of breast cancers, and 10.7% of all HER2/neu-amplified cancers classified as such based on 2013 ASCO/CAP criteria. They are predominantly HER2/neu negative by immunohistochemistry, which suggests that they are biologically different from classically HER2/neu-amplified cases and which validates the 2018 ASCO/CAP guideline against automatically classifying such cases as HER2/neu amplified.