Increased PI3K pathway activity is associated with recurrent breast cancer in patients with low and intermediate 21-gene recurrence score.

AIMS: We investigated key signalling pathways' activity and mutational status of early-stage breast carcinomas with low and intermediate 21-gene recurrence score (RS) to identify molecular features that may predict recurrence. METHODS: This is a retrospective case-control study of 18 patients with recurrent breast carcinoma with low and intermediate 21-gene RS (<25) and control group of 15 non-recurrent breast cancer patients. DNA and mRNA were extracted from tumour tissue. mRNA expression of genes involved in oestrogen receptor (ER), androgen receptor (AR), PI3K and MAPK signalling pathways was measured by real-time quantitative reverse transcription-qPCR (OncoSIGNal G4 test, InnoSIGN). Tumour mutational landscape was assessed by targeted DNA sequencing (Oncomine Precision Assay). RESULTS: There were no statistical differences between the groups' demographic and clinicopathological characteristics. PI3K pathway showed significantly higher activity in cases compared with controls (p=0.0014). Receiver operating characteristic curve analysis showed an area under the curve of 0.79 for PI3K pathway activity in the prediction of recurrent disease in low and intermediate 21-gene RS breast cancer. There was no difference in ER, AR and MAPK pathway activity. PIK3CA alterations were the most common driver mutations, but no difference was found between the groups (p=0.46) and no association with PI3K pathway activity (p=0.86). Higher Ki67 gene expression was associated with recurrences (p=0.042) CONCLUSION: Increased PI3K pathway activity, independent of PIK3CA mutations, may play a role in the recurrence of early-stage breast cancer with low and intermediate 21-gene RS. Pathway analysis can help to identify high-risk patients in this setting.

Automated and robust extraction of genomic DNA from various leftover blood samples.

With the development of genomic technologies, the isolation of genomic DNA (gDNA) from clinical samples is increasingly required for clinical diagnostics and research studies. In this study, we explored the potential of utilizing various leftover blood samples obtained from routine clinical tests as a viable source of gDNA. Using an automated method with optimized pre-treatments, we obtained gDNA from seven types of clinical leftover blood, with average yields of gDNA ranging from 3.11 ± 0.45 to 22.45 ± 4.83 µg. Additionally, we investigated the impact of storage conditions on gDNA recovery, resulting in yields of 8.62-68.08 µg when extracting gDNA from EDTA leftover blood samples stored at 4 °C for up to 13 weeks or -80 °C for up to 78 weeks. Furthermore, we successfully obtained sequenceable gDNA from both Serum Separator Tube and EDTA Tube using a 96-well format extraction, with yields ranging from 0.61 to 71.29 µg and 3.94-215.98 µg, respectively. Our findings demonstrate the feasibility of using automated high-throughput platforms for gDNA extraction from various clinical leftover blood samples with the proper pre-treatments.

DNA Methylation Identifies Epigenetic Subtypes of Triple-Negative Breast Cancers With Distinct Clinicopathologic and Molecular Features.

Triple-negative breast cancers (TNBC) include diverse carcinomas with heterogeneous clinical behavior. DNA methylation is a useful tool in classifying a variety of cancers. In this study, we analyzed TNBC using DNA methylation profiling and compared the results to those of mutational analysis. DNA methylation profiling (Infinium MethylationEPIC array, Illumina) and 50-gene panel-targeted DNA sequencing were performed in 44 treatment-naïve TNBC. We identified 3 distinct DNA methylation clusters with specific clinicopathologic and molecular features. Cluster 1 (phosphoinositide 3-kinase/protein kinase B-enriched cluster; n = 9) patients were significantly older (mean age, 71 years; P = .008) with tumors that were more likely to exhibit apocrine differentiation (78%; P < .001), a lower grade (44% were grade 2), a lower proliferation index (median Ki-67, 15%; P = .002), and lower tumor-infiltrating lymphocyte fractions (median, 15%; P = .0142). Tumors carried recurrent PIK3CA and AKT1 mutations and a higher percentage of low HER-2 expression (89%; P = .033). Cluster 3 (chromosomal instability cluster; n = 28) patients were significantly younger (median age, 57 years). Tumors were of higher grade (grade 3, 93%), had a higher proliferation index (median Ki-67, 75%), and were with a high fraction of tumor-infiltrating lymphocytes (median, 30%). Ninety-one percent of the germline BRCA1/2 mutation carriers were in cluster 3, and these tumors showed the highest level of copy number alterations. Cluster 2 represented cases with intermediate clinicopathologic characteristics and no specific molecular profile (no specific molecular profile cluster; n = 7). There were no differences in relation to stage, recurrence, and survival. In conclusion, DNA methylation profiling is a promising tool to classify patients with TNBC into biologically relevant groups, which may result in better disease characterization and reveal potential targets for emerging therapies.

Integrated Analysis of Ovarian Juvenile Granulosa Cell Tumors Reveals Distinct Epigenetic Signatures and Recurrent TERT Rearrangements.

PURPOSE: Adult granulosa cell tumor (AGCT) is characterized by the somatic FOXL2 p.C134W mutation, and recurrences have been associated with TERT promoter and KMT2D-truncating mutations. Conversely, the molecular underpinnings of the rare juvenile granulosa cell tumor (JGCT) have not been well elucidated. To this end, we applied a tumor-only integrated approach to investigate the genomic, transcriptomic, and epigenomic landscape of 31 JGCTs to identify putative oncogenic drivers. EXPERIMENTAL DESIGN: Multipronged analyses of 31 JGCTs were performed utilizing a clinically validated next-generation sequencing (NGS) panel targeting 580 cancer-related genes for genomic interrogation, in addition to targeted RNA NGS for transcriptomic exploration. Genome-wide DNA methylation profiling was conducted using an Infinium Methylation EPIC array targeting 866,562 CpG methylation sites. RESULTS: We identified frequent KMT2C-truncating mutations along with other mutated genes implicated in the switch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex, in addition to previously reported hotspot AKT1 and DICER1 mutations. Targeted transcriptome sequencing revealed recurrent TERT rearrangements (13%) involving partners CLPTM1L or DROSHA, and differential gene expression analysis showed FGFR1 upregulation in the TERT non-rearranged JGCTs under direct promoter control. Genome-wide DNA methylation rendered a clear delineation between AGCTs and JGCTs at the epigenomic level, further supporting its diagnostic utility in distinguishing among these tumors. CONCLUSIONS: This is the largest comprehensive molecular study of JGCTs, where we further expand our current understanding of JGCT pathogenesis and demonstrate putative oncogenic drivers and TERT rearrangements in a subset of tumors. Our findings further offer insights into possible targeted therapies in a rare entity.

Autoimmune anti-DNA and anti-phosphatidylserine antibodies predict development of severe COVID-19.

High levels of autoimmune antibodies are observed in COVID-19 patients but their specific contribution to disease severity and clinical manifestations remains poorly understood. We performed a retrospective study of 115 COVID-19 hospitalized patients with different degrees of severity to analyze the generation of autoimmune antibodies to common antigens: a lysate of erythrocytes, the lipid phosphatidylserine (PS) and DNA. High levels of IgG autoantibodies against erythrocyte lysates were observed in a large percentage (up to 36%) of patients. Anti-DNA and anti-PS antibodies determined upon hospital admission correlated strongly with later development of severe disease, showing a positive predictive value of 85.7% and 92.8%, respectively. Patients with positive values for at least one of the two autoantibodies accounted for 24% of total severe cases. Statistical analysis identified strong correlations between anti-DNA antibodies and markers of cell injury, coagulation, neutrophil levels and erythrocyte size. Anti-DNA and anti-PS autoantibodies may play an important role in the pathogenesis of COVID-19 and could be developed as predictive biomarkers for disease severity and specific clinical manifestations.

Platelets amplify endotheliopathy in COVID-19.

Given the evidence for a hyperactive platelet phenotype in COVID-19, we investigated effector cell properties of COVID-19 platelets on endothelial cells (ECs). Integration of EC and platelet RNA sequencing revealed that platelet-released factors in COVID-19 promote an inflammatory hypercoagulable endotheliopathy. We identified S100A8 and S100A9 as transcripts enriched in COVID-19 platelets and were induced by megakaryocyte infection with SARS-CoV-2. Consistent with increased gene expression, the heterodimer protein product of S100A8/A9, myeloid-related protein (MRP) 8/14, was released to a greater extent by platelets from COVID-19 patients relative to controls. We demonstrate that platelet-derived MRP8/14 activates ECs, promotes an inflammatory hypercoagulable phenotype, and is a significant contributor to poor clinical outcomes in COVID-19 patients. Last, we present evidence that targeting platelet P2Y12 represents a promising candidate to reduce proinflammatory platelet-endothelial interactions. Together, these findings demonstrate a previously unappreciated role for platelets and their activation-induced endotheliopathy in COVID-19.

Comparison of solid tissue sequencing and liquid biopsy accuracy in identification of clinically relevant gene mutations and rearrangements in lung adenocarcinomas.

Screening for therapeutic targets is standard of care in the management of advanced non-small cell lung cancer. However, most molecular assays utilize tumor tissue, which may not always be available. "Liquid biopsies" are plasma-based next generation sequencing (NGS) assays that use circulating tumor DNA to identify relevant targets. To compare the sensitivity, specificity, and accuracy of a plasma-based NGS assay to solid-tumor-based NGS we retrospectively analyzed sequencing results of 100 sequential patients with lung adenocarcinoma at our institution who had received concurrent testing with both a solid-tissue-based NGS assay and a commercially available plasma-based NGS assay. Patients represented both new diagnoses (79%) and disease progression on treatment (21%); the majority (83%) had stage IV disease. Tissue-NGS identified 74 clinically relevant mutations, including 52 therapeutic targets, a sensitivity of 94.8%, while plasma-NGS identified 41 clinically relevant mutations, a sensitivity of 52.6% (p < 0.001). Tissue-NGS showed significantly higher sensitivity and accuracy across multiple patient subgroups, both in newly diagnosed and treated patients, as well as in metastatic and nonmetastatic disease. Discrepant cases involved hotspot mutations and actionable fusions including those in EGFR, ALK, and NTRK1. In summary, tissue-NGS detects significantly more clinically relevant alterations and therapeutic targets compared to plasma-NGS, suggesting that tissue-NGS should be the preferred method for molecular testing of lung adenocarcinoma when tissue is available. Plasma-NGS can still play an important role when tissue testing is not possible. However, given its low sensitivity, a negative result should be confirmed with a tissue-based assay.

Genetic profile of primary mucinous cystadenocarcinoma of the breast-A case report.

Primary mucinous cystadenocarcinoma of the breast is a rare neoplasm with few reports in the literature. Here, we report for the first time a comprehensive genetic profile of a primary mucinous cystadenocarcinoma of the breast, using next-generation sequencing 580 cancer-associated gene panel. Mutations in TP53, RB1, and BAP1 were identified. The findings suggest that this tumor is driven mostly by abnormalities in tumor suppressor genes, primarily involved in cell cycle control and chromatin remodeling. Molecular characterization of additional primary mucinous cystadenocarcinomas of the breast is warranted and might provide information related to its biology and behavior.

Molecular analysis of encapsulated papillary carcinoma of the breast with and without invasion.

Encapsulated papillary carcinomas (EPCs) of the breast are a unique variant of papillary carcinoma confined to a cystic space with absent or attenuated myoepithelial cell layer. Although staged as an in situ lesion, it can be associated with invasive ductal carcinoma (IDC). We sought to compare the genomic characteristics of pure EPC and EPC with associated invasive carcinoma (EPCi) at the genomic level. All cases of EPCi harbored recurrent hotspot mutations in PIK3CA. PIK3CA, KMT2A, and CREBBP deleterious somatic events were found across both tumor groups, irrespective of invasion status. At the whole transcriptomic level, EPCi cases displayed remarkably similar mRNA profiles when compared to EPC. When EPCi cases were compared with their corresponding IDC, despite significant overlap, we identified differential gene expression in 39 genes with enrichment of multiple pathways including extracellular matrix regulation, cell adhesion, and collagen fibril organization. Despite morphologic, genotypic, and transcriptomic overlap between pure EPC and EPCi, the latter tumors are likely advanced lesions with PIK3CA activating mutations and enrichment of stromal-related genes implicated in the switch to IDC.

Primary cutaneous SMARCB1-deficient carcinoma.

BACKGROUND: SMARCB1-deficient malignancies can arise in various sites. We describe a novel primary SMARCB1-deficient carcinoma of skin (SDCS) and characterize SMARCB1 mutations in non-melanoma skin cancers (NMSC). METHODS: Cases underwent immunophenotyping and targeted exome sequencing (MSK-IMPACT) assay interrogating somatic mutations in 468 cancer-related genes. The MSK-IMPACT database from 2014 to 2020 encompassing 55, 000 cases was searched for NMSC with SMARCB1 mutations. RESULTS: SDCS arose on the scalp of an 18-year-old woman showing homozygous SMARCB1 deletion with a LATS2 G963E variant. Another case arose on the temple of a 76-year-old man harboring a SMARCB1 W206* mutation associated with loss of heterozygosity (LOH), 59 concurrent mutations, and a UV mutation signature (UV-MS). Both tumors exhibited INI1 loss, positive CK5/6, p40, p63, and claudin-4 with negative CD34. Of 378 NMSC cases, including 370 carcinomas, 7 SMARCB1-mutated tumors were identified: 3 squamous cell, 3 Merkel cell, and one basal cell carcinoma. Six showed UV-MS. Five INI1-interrogated cases retained protein expression suggesting they were SMARCB1-proficient. CONCLUSIONS: SDCS can be clinically aggressive, harbor SMARCB1 homozygous deletions or truncating SMARCB1 mutations associated with LOH, and can occur with or without UV-MS. Overall, SMARCB1 mutations in NMSC are rare with most being of undetermined significance and associated with retained INI1 and UV-MS.

Analytical performance of lateral flow immunoassay for SARS-CoV-2 exposure screening on venous and capillary blood samples.

OBJECTIVES: We validate the use of a lateral flow immunoassay (LFI) intended for rapid screening and qualitative detection of anti-SARS-CoV-2 IgM and IgG in serum, plasma, and whole blood, and compare results with ELISA. We also seek to establish the value of LFI testing on blood obtained from a capillary blood sample. METHODS: Samples collected by venous blood draw and finger stick were obtained from patients with SARS-CoV-2 detected by RT-qPCR and control patients. Samples were tested with Biolidics 2019-nCoV IgG/IgM Detection Kit lateral flow immunoassay, and antibody calls were compared with ELISA. RESULTS: Biolidics LFI showed clinical sensitivity of 92% with venous blood at 7 days after PCR diagnosis of SARS-CoV-2. Test specificity was 92% for IgM and 100% for IgG. There was no significant difference in detecting IgM and IgG with Biolidics LFI and ELISA at D0 and D7 (p = 1.00), except for detection of IgM at D7 (p = 0.04). Capillary blood of SARS-CoV-2 patients showed 93% sensitivity for antibody detection. CONCLUSIONS: Clinical performance of Biolidics 2019-nCoV IgG/IgM Detection Kit is comparable to ELISA and was consistent across sample types. This provides an opportunity for decentralized rapid testing and may allow point-of-care and longitudinal self-testing for the presence of anti-SARS-CoV-2 antibodies.

Upgrade rate of intraductal papilloma diagnosed on core needle biopsy in a single institution.

The management of intraductal papilloma (IDP) diagnosed on core needle biopsy (CNB) is controversial due to the variable upgrade rates to breast carcinoma (BC) on subsequent surgical excision reported in the literature. The purpose of our study was to investigate the upgrade rate of IDP diagnosed on CNB to BC in subsequent surgical excision and the impact of clinical, pathologic, and radiologic variables. This is a retrospective cohort of all women who had a diagnosis of IDP on a CNB between 2005 and 2018 in a tertiary academic center with subsequent surgical excision. Upgrade was defined as ductal carcinoma in situ (DCIS) and invasive carcinoma on surgical excision. Statistical analyses included Pearson's chi-square, Wilcoxon rank-sum, and logistic regression. A total of 216 women with IDP in a CNB were included. Nineteen patients (8.8%) upgraded to BC in the overall cohort, including 14 DCIS and 5 invasive carcinomas. An upgrade rate of 27% was found in atypical IDP (14 of 51 cases), while only 3% of pure IDP upgraded to BC (5 of 165 cases). Older age (>53 years) at the time of biopsy (odds ratio [OR] = 1.05, 95% confidence interval [CI] = 1.01-1.09, p = 0.027) and concomitant atypical ductal hyperplasia (ADH) (OR = 9.69, 95% CI = 3.37-27.81, p < 0.0001) were significantly associated with upgrade. Our results support surgical excision of IDP on CNB when associated with ADH or diagnosed in women aged older than 53 years. The low surgical upgrade rate of 3% for pure IDP on CNB in younger women should be part of the management discussion.

Sequencing identifies multiple early introductions of SARS-CoV-2 to the New York City region.

Effective public response to a pandemic relies upon accurate measurement of the extent and dynamics of an outbreak. Viral genome sequencing has emerged as a powerful approach to link seemingly unrelated cases, and large-scale sequencing surveillance can inform on critical epidemiological parameters. Here, we report the analysis of 864 SARS-CoV-2 sequences from cases in the New York City metropolitan area during the COVID-19 outbreak in spring 2020. The majority of cases had no recent travel history or known exposure, and genetically linked cases were spread throughout the region. Comparison to global viral sequences showed that early transmission was most linked to cases from Europe. Our data are consistent with numerous seeds from multiple sources and a prolonged period of unrecognized community spreading. This work highlights the complementary role of genomic surveillance in addition to traditional epidemiological indicators.

Genetic basis of SMARCB1 protein loss in 22 sinonasal carcinomas.

SMARCB1-deficient sinonasal carcinoma (SNC) is an aggressive malignancy characterized by INI1 loss mostly owing to homozygous SMARCB1 deletion. With the exception of a few reported cases, these tumors have not been thoroughly studied by massive parallel sequencing (MPS). A retrospective cohort of 22 SMARCB1-deficient SNCs were studied by light microscopy, immunohistochemistry, fluorescence in situ hybridization (n = 9), targeted exome MPS (n = 12), and Fraction and Allele-Specific Copy Number Estimates from Tumor Sequencing (FACETS) (n = 10), a bioinformatics pipeline for copy number/zygosity assessment. SMARCB1-deficient SNC was found in 13 (59%) men and 9 (41%) women. Most common growth patterns were the basaloid pattern (59%), occurring mostly in men (77%), and plasmacytoid/eosinophilic/rhabdoid pattern (23%), arising mostly in women (80%). The former group was significantly younger (median age = 46 years, range = 24-54, vs 79 years, range = 66-95, p < 0.0001). Clear cell, pseudoglandular, glandular, spindle cell, and sarcomatoid features were variably present. SMARCB1-deficient SNC expressed cytokeratin (100%), p63 (72%), neuroendocrine markers (52%), CDX-2 (44%), S-100 (25%), CEA (4/4 cases), Hepatocyte (2/2 cases), and aberrant nuclear ß-catenin (1/1 case). SMARCB1 showed homozygous deletion (68%), hemizygous deletion (16%), or truncating mutations associated with copy neutral loss of heterozygosity (11%). Coexisting genetic alterations were 22q loss including loss of NF2 and CHEK2 (50%), chromosome 7 gain (25%), and TP53 V157F, CDKN2A W110∗, and CTNNB1 S45F mutations. At 2 years and 5 years, the disease-specific survival and disease-free survival were 70% and 35% and 13% and 0%, respectively. SMARCB1-deficient SNCs are phenotypically and genetically diverse, and these distinctions warrant further investigation for their biological and clinical significance.

WITHDRAWN: ASSOCIATION OF INITIAL VIRAL LOAD IN SARS-CoV-2 PATIENTS WITH OUTCOME AND SYMPTOMS.

The Publisher regrets that this article is an accidental duplication of an article that has already been published, https://doi.org/10.1016/j.ajpath.2020.07.001. The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal.

Association of Initial Viral Load in Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Patients with Outcome and Symptoms.

The dynamics of viral load (VL) of the severe acute respiratory syndrome coronavirus 2 and its association with different clinical parameters remain poorly characterized in the US patient population. Herein, we investigate associations between VL and parameters, such as severity of symptoms, disposition (admission versus direct discharge), length of hospitalization, admission to the intensive care unit, length of oxygen support, and overall survival in 205 patients from a tertiary care center in New York City. VL was determined using quantitative PCR and log10 transformed for normalization. Associations were tested with univariate and multivariate regression models. Diagnostic VL was significantly lower in hospitalized patients than in patients not hospitalized (log10 VL = 3.3 versus 4.0; P = 0.018) after adjusting for age, sex, race, body mass index, and comorbidities. Higher VL was associated with shorter duration of the symptoms in all patients and hospitalized patients only and shorter hospital stay (coefficient = -2.02, -2.61, and -2.18; P < 0.001, P = 0.002, and P = 0.013, respectively). No significant association was noted between VL, admission to intensive care unit, length of oxygen support, and overall survival. Our findings suggest a higher shedding risk in less symptomatic patients, an important consideration for containment strategies. Furthermore, we identify a novel association between VL and history of cancer. Larger studies are warranted to validate our findings.

Sequencing identifies multiple early introductions of SARS-CoV-2 to the New York City Region.

Effective public response to a pandemic relies upon accurate measurement of the extent and dynamics of an outbreak. Viral genome sequencing has emerged as a powerful approach to link seemingly unrelated cases, and large-scale sequencing surveillance can inform on critical epidemiological parameters. Here, we report the analysis of 864 SARS-CoV-2 sequences from cases in the New York City metropolitan area during the COVID-19 outbreak in Spring 2020. The majority of cases had no recent travel history or known exposure, and genetically linked cases were spread throughout the region. Comparison to global viral sequences showed that early transmission was most linked to cases from Europe. Our data are consistent with numerous seeds from multiple sources and a prolonged period of unrecognized community spreading. This work highlights the complementary role of genomic surveillance in addition to traditional epidemiological indicators.