Utility of methylated DNA markers for the diagnosis of malignant biliary strictures.

BACKGROUND AND AIMS: Early identification of malignant biliary strictures (MBS) is challenging, with up to 20% classified as indeterminants after preliminary testing and tissue sampling with endoscopic retrograde cholangiopancreatography (ERCP). We aimed to evaluate the use of methylated DNA markers (MDM) from biliary brushings to enhance MBS detection in a prospective cohort. METHODS: Candidate MDMs were evaluated for their utility in MBS diagnosis through a series of discovery and validation phases. DNA was extracted from biliary brushing samples, quantified, bisulfite-converted, and then subjected to methylation-specific Droplet Digital Polymerase Chain Reaction (ddPCR).  Patients were considered to have no malignancy if the sampling was negative and there was no evidence of malignancy after 1 year or definitive negative surgical histopathology. RESULTS: Fourteen candidate MDMs were evaluated in the discovery phase, with top-performing and new markers evaluated in the technical validation phase. The top four MDMs were TWIST1, HOXA1, VSTM2B, and CLEC11A, which individually achieved AUC values of 0.82, 0.81, 0.83, and 0.78, respectively, with sensitivities of 59.4%, 53.1%, 62.5%, and 50.0%, respectively, at high specificities for malignancy of 95.2-95.3% for the final biologic validation phase. When combined as a panel, the AUC was 0.86, achieving 73.4% sensitivity and 92.9% specificity, which outperformed cytology and fluorescent in situ hybridization (FISH). CONCLUSIONS: The selected methylated DNA markers demonstrated improved performance characteristics for the detection of MBS compared to cytology and FISH. ​ Therefore, MDMs should be considered viable candidates for inclusion in diagnostic testing algorithms.​.

Recommendations for Tumor Mutational Burden Assay Validation and Reporting: A Joint Consensus Recommendation of the Association for Molecular Pathology, College of American Pathologists, and Society for Immunotherapy of Cancer.

Tumor mutational burden (TMB) has been recognized as a predictive biomarker for immunotherapy response in several tumor types. Several laboratories offer TMB testing, but there is significant variation in how TMB is calculated, reported, and interpreted among laboratories. TMB standardization efforts are underway, but no published guidance for TMB validation and reporting is currently available. Recognizing the current challenges of clinical TMB testing, the Association for Molecular Pathology convened a multidisciplinary collaborative working group with representation from the American Society of Clinical Oncology, the College of American Pathologists, and the Society for the Immunotherapy of Cancer to review the laboratory practices surrounding TMB and develop recommendations for the analytical validation and reporting of TMB testing based on survey data, literature review, and expert consensus. These recommendations encompass pre-analytical, analytical, and postanalytical factors of TMB analysis, and they emphasize the relevance of comprehensive methodological descriptions to allow comparability between assays.

Evaluating User Experience and DNA Yield from Self-Collection Devices.

BACKGROUND: The COVID-19 pandemic emphasized an urgent need for devices used in the self-collection of biospecimens in an evolving patient care system. The mailing of biospecimen self-collection kits to patients, with samples returned via mail, provides a more convenient testing regimen, but could also impart patient sampling variabilities. User compliance with device directions is central to downstream testing of collected biospecimens and clear instructions are central to this goal. METHODS: Here, we performed an evaluation of 10 oral DNA collection devices involving either swab or saliva self-collection and analyzed ease of use and comfort level with a device, as well as DNA recovery quantity/quality and sample stability. RESULTS: We show that while these DNA quality/quantity metrics are comparable between devices, users prefer direct saliva collection over swab-based devices. CONCLUSIONS: This information is useful in guiding future experiments including their use in human RNA, microbial, or viral sample collection/recovery and their use in clinical testing.

Single nucleotide polymorphism (SNP) chromosomal microarray as a diagnostic tool for mucinous tubular and spindle cell carcinoma: A validation study.

Mucinous tubular and spindle cell carcinoma (MTSCC) shows significant overlap with papillary renal cell carcinoma (PRCC), and harbor recurrent copy-number alterations (CNA). We evaluated 16 RCC with features suggestive of MTSCC using chromosomal microarrays. The cohort was comprised of 8 females and males, each, with an age range of 33-79 years (median, 59), and a tumor size range of 3.4-15.5 cm (median, 5.0). Half the tumors were high-grade (8/16, 50%) with features such as necrosis, marked cytologic atypia, and sarcomatoid differentiation, and 5/16 (31%) were high stage (≥pT3a). Three (of 16, 19%) cases had a predominant (>95%) spindle cell component, whereas 5/16 (31%) were composed of a predominant (>95%) epithelial component. Most cases (12/16, 75%) exhibited a myxoid background and/or extravasated mucin, at least focally. Twelve (of 16, 75%) cases demonstrated CNA diagnostic of MTSCC (losses of chromosomes 1, 4, 6, 8, 9, 13, 14, 15, and 22). In addition, 2 high-grade tumors showed loss of CDKN2A/B, and gain of 1q, respectively, both of which are associated with aggressive behavior. Three (of 16, 19%) cases, demonstrated nonspecific CNA, and did not meet diagnostic criteria for established RCC subtypes. One (of 16, 6%) low-grade epithelial predominant tumor (biopsy) demonstrated characteristic gains of 7, 17, and loss of Y, diagnostic of PRCC. MTSCC can be a morphologically heterogenous tumor. Our study validates the detection of characteristic chromosomal CNA for diagnostic use that may be useful in challenging cases with unusual spindle cell or epithelial predominant features, as well as in high-grade tumors.

From pandemic to endemic: Divergence of COVID-19 positive-tests and hospitalization numbers from SARS-CoV-2 RNA levels in wastewater of Rochester, Minnesota.

Traditionally, public health surveillance relied on individual-level data but recently wastewater-based epidemiology (WBE) for the detection of infectious diseases including COVID-19 became a valuable tool in the public health arsenal. Here, we use WBE to follow the course of the COVID-19 pandemic in Rochester, Minnesota (population 121,395 at the 2020 census), from February 2021 to December 2022. We monitored the impact of SARS-CoV-2 infections on public health by comparing three sets of data: quantitative measurements of viral RNA in wastewater as an unbiased reporter of virus level in the community, positive results of viral RNA or antigen tests from nasal swabs reflecting community reporting, and hospitalization data. From February 2021 to August 2022 viral RNA levels in wastewater were closely correlated with the oscillating course of COVID-19 case and hospitalization numbers. However, from September 2022 cases remained low and hospitalization numbers dropped, whereas viral RNA levels in wastewater continued to oscillate. The low reported cases may reflect virulence reduction combined with abated inclination to report, and the divergence of virus levels in wastewater from reported cases may reflect COVID-19 shifting from pandemic to endemic. WBE, which also detects asymptomatic infections, can provide an early warning of impending cases, and offers crucial insights during pandemic waves and in the transition to the endemic phase.

Genomic attributes of prostate cancer across primary and metastatic noncastrate and castrate resistant disease states: a next generation sequencing study of 183 patients.

Primary prostatic adenocarcinoma (pPC) undergoes genomic evolution secondary to therapy-related selection pressures as it transitions to metastatic noncastrate (mNC-PC) and castrate resistant (mCR-PC) disease. Next generation sequencing results were evaluated for pPC (n = 97), locally advanced disease (involving urinary bladder/rectum, n = 12), mNC-PC (n = 21), and mCR-PC (n = 54). We identified enrichment of TP53 alterations in high-grade pPC, TP53/RB1 alterations in HGNE disease, and AR alterations in metastatic and castrate resistant disease. Actionable alterations (MSI-H phenotype and HRR genes) were identified in approximately a fifth of all cases. These results help elucidate the landscape of genomic alterations across the clinical spectrum of prostate cancer.

Clinicopathologic and Molecular Characteristics of Resected Thoracic Mass Lesions in the Pediatric Population: A 25-Year Institutional Experience From a Tertiary Care Center.

CONTEXT.­: Primary thoracic neoplasms are rare in children, whereas nonneoplastic mass lesions or cysts and metastases are more common, and there is a relative paucity of comprehensive histopathologic and molecular data. OBJECTIVE.­: To define the clinicopathologic spectrum of neoplastic and nonneoplastic diseases observed in resected mass lesions in the chest of pediatric patients, and to identify somatic alterations observed in primary neoplasms. DESIGN.­: Clinicopathologic features of thoracic mass lesions (n = 385) resected from 373 patients aged ≤21 years in a 25-year period (1993-2018) were included. Primary neoplasms having sufficient material were tested by a laboratory-developed comprehensive genomic profiling assay that assesses tumor mutational burden, microsatellite instability, somatic sequence variants, gene amplifications, fusions, and specific transcript variants. RESULTS.­: The most commonly resected space-occupying lesions were nonneoplastic mass lesions and cysts or malformations, resected in 117 (31.4%) and 58 of 373 patients (15.5%) respectively. Metastatic neoplasms were observed in 169 of 373 patients (45.3%; mean age 14.4 years, range 1-21 years); the most common was osteosarcoma (68 of 169; 40.2% of metastases). Primary lung neoplasms occurred in 24 of 373 patients (6.4%; mean age 14.5 years, range 6 months-21 years), and 16 patients had primary extrapulmonary thoracic tumors. Carcinoid tumor was the most common primary lung neoplasm (7 typical, 3 atypical). Molecular testing showed a prevalence of somatic pathogenic or likely pathogenic mutations and copy-number alterations. No fusions or splice variants were identified. Tumors were microsatellite-stable with low tumor mutational burden. CONCLUSIONS.­: Resected pediatric thoracic mass lesions are more likely to be metastatic lesions, congenital cysts or malformations, or nonneoplastic lesions compared to primary thoracic neoplasms, which are encountered at a low frequency and tend to have relatively simple genetic profiles.

Sarcomas Harboring EWSR1::PATZ1 Fusions: A Clinicopathologic Study of 17 Cases.

Soft tissue sarcomas harboring EWSR1::PATZ1 are a recently recognized entity with variable morphology and a heterogeneous immunohistochemical profile. We studied 17 such tumors. The tumors occurred in 12 men and 5 women (median age, 50 years; range, 15-71 years), involved the thoracoabdominal soft tissues (14 cases; 82%), lower extremities (2 cases; 12%), and tongue (1 case; 6%), and ranged from 0.7 to 11.3 cm (median, 4.7 cm). All but 1 patient received complete surgical resection; 7 were also treated with neoadjuvant chemo/radiotherapy. All cases showed typical features of EWSR1::PATZ1 sarcoma, including uniform round to spindled cells, fibromyxoid matrix, fibrous bands, hyalinized vessels, and pseudoalveolar/microcystic spaces. Unusual features, seen in a subset of cases, included degenerative-appearing nuclear atypia, epithelioid cytomorphology, mature fat, abundant rhabdomyoblasts, high mitotic activity, and foci with increased cellularity and nuclear atypia. Positive immunohistochemical results were desmin (16/17, 94%), MyoD1 (13/14, 93%), myogenin (6/14, 43%), GFAP (10/10, 100%), S100 protein (15/17, 88%), SOX10 (7/13, 54%), keratin (10/17, 59%), CD99 (4/11, 36%), H3K27me3 (retained expression 9/9, 100%), p16 (absent expression 1/4, 25%), and p53 (wild type 3/3, 100%). Fusion events included EWSR1 exon 8::PATZ1 exon 1 (14/17, 82%), EWSR1 exon 9::PATZ1 exon 1 (2/17, 12%), and EWSR1 exon 7::PATZ1 exon 1 (1/17, 6%). No evaluated tumor had alterations of CDKN2A/B and/or TP53, or MDM2 amplification. Clinical follow-up (16 patients: median, 13.5 months; range, 1-77 months) showed distant metastases in 3 patients (1/3 at time of presentation) and no local recurrences. At the time of last follow-up, 14 patients were disease free, 1 was alive with disease, 1 was dead of disease (at 13 months), and 1 had an indeterminant pulmonary nodule. We conclude that the morphologic spectrum of EWSR1::PATZ1 is broader than has been previously appreciated. Although more long-term follow-up is needed, the prognosis of these very rare sarcomas may be more favorable than previously reported.

SARS-CoV-2 emergency use authorization published sensitivity differences do not correlate with positivity rate in a hospital/reference laboratory setting.

During the first year of the COVID-19 pandemic skyrocketing demand for testing in the United States, coupled with supply chain issues, necessitated the use of multiple SARS-CoV-2 molecular testing platforms at many health centers. At our institution these platforms consisted of 8 ordered services for sample triage, using 9 emergency use authorized (EUA) SARS-CoV-2 RNA nucleic acid amplification tests resulting in 10 possible ordered service/EAU combinations. Here we review the results of the first ∼2.9 million samples tested and note the variability in positivity rates. We conclude that differences in reported limit of detection did not translate to differences in positivity rate or show correlation to discordant results observed. This highlights the importance of balancing patient testing capacity needs with the desire to have more sensitive tests.

Pseudoendocrine sarcoma: clinicopathologic, molecular, and epigenetic features of one case.

Pseudoendocrine sarcoma (PES) is a recently described neoplasm typically arising in paravertebral soft tissues. Histologically, PES resembles well-differentiated neuroendocrine tumors but lacks expression of epithelial/neuroendocrine markers, and most show aberrant nuclear ß-catenin positivity. We describe the clinicopathological and molecular features and DNA methylation profile of one PES. A resected paraspinal soft tissue mass in a 52-year-old man showed a neuroendocrine-like neoplasm, negative for keratin, and synaptophysin and showing diffuse nuclear ß-catenin expression. Targeted NGS confirmed a CTNNB1 (p.S37C) mutation. Whole genome methylation analysis showed no match to any methylation class in the central nervous system tumor (versions 11b6 and 12b6) or sarcoma classifier (calibrated scores of ≤0.3), but clustered together with a recently reported PES in which methylation analysis was also performed. He remained disease-free for 18 months after surgery, followed by chemoradiation. As more cases are examined, our findings suggest that PES may have a unique methylation profiling signature.

Prognostic stratification of endometrial cancers with high microsatellite instability or no specific molecular profile.

Objective: To identify high-risk disease in clinicopathologic low-risk endometrial cancer (EC) with high microsatellite instability (MSI-H) or no specific molecular profile (NSMP) and therapeutic insensitivity in clinicopathologic high-risk MSI-H/NSMP EC. Methods: We searched The Cancer Genome Atlas for DNA sequencing, RNA expression, and surveillance data regarding MSI-H/NSMP EC. We used a molecular classification system of E2F1 and CCNA2 expression and sequence variations in POLE, PPP2R1A, or FBXW7 (ECPPF) to prognostically stratify MSI-H/NSMP ECs. Clinical outcomes were annotated after integrating ECPPF and sequence variations in homologous recombination (HR) genes. Results: Data were available for 239 patients with EC, which included 58 MSI-H and 89 NSMP cases. ECPPF effectively stratified MSI-H/NSMP EC into distinct molecular groups with prognostic implications: molecular low risk (MLR), with low CCNA2 and E2F1 expression, and molecular high risk (MHR), with high CCNA2 and E2F1 expression and/or PPP2R1A and/or FBXW7 variants. The 3-year disease-free survival (DFS) rate was 43.8% in the MHR group with clinicopathologic low-risk indicators and 93.9% in the MLR group (P<.001). In the MHR group, wild-type HR genes were present in 28% of cases but in 81% of documented recurrences. The 3-year DFS rate in patients with MSI-H/NSMP EC with clinicopathologic high-risk indicators was significantly higher in the MLR (94.1%) and MHR/HR variant gene (88.9%) groups than in the MHR/HR wild-type gene group (50.3%, P<.001). Conclusion: ECPPF may resolve prognostic challenges for MSI-H/NSMP EC by identifying occult high-risk disease in EC with clinicopathologic low-risk indicators and therapeutic insensitivity in EC with clinicopathologic high-risk indicators.

Evaluation of the analytical sensitivity of ACON and LumiraDx SARS-CoV-2 rapid antigen tests using samples with presumed Omicron variant.

BACKGROUND: Analytical sensitivity of 2 rapid antigen tests was evaluated for detection of presumed SARS-CoV-2 Omicron variants and earlier variants of concern. METHODS: A total of 152 SARS-CoV-2 RNA positive samples (N and ORF1ab positive but S gene negative) were tested for SARS-CoV-2 antigen by ACON lateral flow and LumiraDx fluorescence immunoassays. Sensitivity within 3 viral load ranges was compared among these 152 samples and 194 similarly characterized samples collected prior to the circulation of the Delta variant (pre-Delta). RESULTS: Antigen was detected in >95% of pre-Delta and presumed Omicron samples for both tests at viral loads >500,000 copies/mL, and 65 to 85% of samples with 50,000-500,000 copies/mL. At viral load <50,000 copies/mL, antigen tests showed better sensitivity in detecting pre-Delta compared to Omicron variants. LumiraDx was more sensitive than ACON at low viral load. CONCLUSIONS: Antigen tests had decreased sensitivity for detecting presumed Omicron compared to pre-Delta variants at low viral load.

εγ-Thalassemia, a New Hemoglobinopathy Category.

BACKGROUND: Large ß-globin gene cluster deletions (hereditary persistence of fetal hemoglobin [Hb] or ß-, δß-, γδß-, and ϵγδß-thalassemia), are associated with widely disparate phenotypes, including variable degrees of microcytic anemia and Hb F levels. When present, increased Hb A2 is used as a surrogate marker for ß-thalassemia. Notably, ϵγδß-thalassemias lack the essential regulatory locus control region (LCR) and cause severe transient perinatal anemia but normal newborn screen (NBS) results and Hb A2 levels. Herein, we report a novel deletion of the ϵ, Aγ, Gγ, and ψß loci with intact LCR, δ-, and ß-regions in 2 women and newborn twins. METHODS: Capillary electrophoresis (CE), high-performance liquid chromatography (HPLC), DNA sequencing, multiplex ligation-dependent probe amplification (MLPA), gap-polymerase chain reaction (gap-PCR), and long-read sequencing (LRS) were performed. RESULTS: NBS showed an Hb A > Hb F pattern for both twins. At 20 months, Hb A2 was increased similarly to that in the mother and an unrelated woman. Unexplained microcytosis was absent and the twins lacked severe neonatal anemia. MLPA, LRS, and gap-PCR confirmed a 32 599 base pair deletion of ϵ (HBE1) through ψß (HBBP1) loci. CONCLUSIONS: This deletion represents a hemoglobinopathy category with a distinct phenotype that has not been previously described, an ϵγ-thalassemia. Both the NBS Hb A > F pattern and the subsequent increased Hb A2 without microcytosis are unusual. A similar deletion should be considered when this pattern is encountered and appropriate test methods selected for detection. Knowledge of the clinical impact of this new category will improve genetic counselling, with distinction from the severe transient anemia associated with ϵγδß-thalassemia.

Genomic markers of recurrence risk in atypical meningioma following gross total resection.

Background: Meningiomas are the most common primary central nervous system (CNS) tumor in adults and CNS World Health Organization grade 2 (atypical) meningiomas show an intermediate risk of recurrence/progression. Molecular parameters are needed to better inform management following gross total resection (GTR). Methods: We performed comprehensive genomic analysis of tumor tissue from 63 patients who underwent radiologically confirmed GTR of a primary grade 2 meningioma, including a CLIA-certified target next-generation sequencing panel (n = 61), chromosomal microarray (n = 63), genome-wide methylation profiling (n = 62), H3K27me3 immunohistochemistry (n = 62), and RNA-sequencing (n = 19). Genomic features were correlated with long-term clinical outcomes (median follow-up: 10 years) using Cox proportional hazards regression modeling and published molecular prognostic signatures were evaluated. Results: The presence of specific copy number variants (CNVs), including -1p, -10q, -7p, and -4p, was the strongest predictor of decreased recurrence-free survival (RFS) within our cohort (P < .05). NF2 mutations were frequent (51%) but did not show a significant association with RFS. DNA methylation-based classification assigned tumors to DKFZ Heidelberg benign (52%) or intermediate (47%) meningioma subclasses and was not associated with RFS. H3K27 trimethylation (H3K27me3) was unequivocally lost in 4 tumors, insufficient for RFS analysis. Application of published integrated histologic/molecular grading systems did not improve prediction of recurrence risk over the presence of -1p or -10q alone. Conclusions: CNVs are strong predictors of RFS in grade 2 meningiomas following GTR. Our study supports incorporation of CNV profiling into clinical evaluation to better guide postoperative patient management, which can be readily implemented using existing, clinically validated technologies.

Authorized SARS-CoV-2 molecular methods show wide variability in the limit of detection.

On February 29th, 2020, the U.S. Food and Drug Administration issued the first Emergency Use Authorization (EUA) for a SARS-CoV-2 assay outside of the U.S. Centers for Disease Control and Prevention. As of May 3rd, 2021, 289 total EUAs have been granted. Like influenza, there is no standard for defining limit of detection (LoD), but rather guidance that analytical sensitivity/LoD be established as the level that gives a 95% detection rate in at least 20 replicates. Here we compare the performance characteristics of SARS-CoV-2 tests receiving EUA by standardizing sensitivity to a common unit of measure and assess the variability in LoD between tests. Additionally, we looked at factors that may impact sensitivities due to lack of standardization of the test development process and compare results for a standardized reference panel for comparative analysis within a subset of EUA tests offered by the U.S. Food and Drug Administration.

ECPPF (E2F1, CCNA2, POLE, PPP2R1A, FBXW7) stratification: Profiling high-risk subtypes of histomorphologically low-risk and treatment-insensitive endometrioid endometrial cancer.

In endometrial cancer, occult high-risk subtypes (rooted in histomorphologically low-risk disease) with insensitivity to adjuvant therapies impede improvements in therapeutic efficacy. Therefore, we aimed to assess the ability of molecular high-risk (MHR) and low-risk (MLR) ECPPF (E2F1, CCNA2, POLE, PPP2R1A, FBXW7) stratification to profile recurrence in early, low-risk endometrioid endometrial cancer (EEC) and insensitivity to platinum-based chemotherapy or radiotherapy (or both) in high-risk EEC. Using The Cancer Genome Atlas endometrial cancer database, we identified 192 EEC cases with available DNA sequencing and RNA expression data. Molecular parameters were integrated with clinicopathologic risk factors and adverse surveillance events. MHR was defined as high (-H) CCNA2 or E2F1 log2 expression (≥2.75), PPP2R1A mutations (-mu), or FBXW7mu; MLR was defined as low (-L) CCNA2 and E2F1 log2 expression (<2.75). We assessed 164 cases, plus another 28 with POLEmu for favorable-outcomes comparisons. MHR and MLR had significantly different progression-free survival (PFS) rates (P < .001), independent of traditional risk factors (eg, TP53mu), except for stage IV disease. PFS of CCNA2-L/E2F1-L paralleled that of POLEmu. ECPPF status stratified responses to adjuvant therapy in stage III-IV EEC (P < .01) and profiled stage I, grade 1-2 cases with risk of recurrence (P < .001). MHR was associated with CTNNB1mu-linked treatment failures (P < .001). Expression of homologous recombination repair (HR) and cell cycle genes was significantly elevated in CCNA2-H/E2F1-H compared with CCNA2-L/E2F1-L (P<1.0E-10), suggesting that HR deficiencies may underlie the favorable PFS in MLR. HRmu were detected in 20.7%. No treatment failures were observed in high-grade or advanced EEC with HRmu (P = .02). Favorable PFS in clinically high-risk EEC was associated with HRmu and MLR ECPPF (P < .001). In summary, MLR ECPPF and HRmu were associated with therapeutic efficacy in EEC. MHR ECPPF was associated with low-risk, early-stage recurrences and insensitivity to adjuvant therapies.