Rapid tumor DNA analysis of cerebrospinal fluid accelerates treatment of central nervous system lymphoma.

ABSTRACT: Delays and risks associated with neurosurgical biopsies preclude timely diagnosis and treatment of central nervous system (CNS) lymphoma and other CNS neoplasms. We prospectively integrated targeted rapid genotyping of cerebrospinal fluid (CSF) into the evaluation of 70 patients with CNS lesions of unknown cause. Participants underwent genotyping of CSF-derived DNA using a quantitative polymerase chain reaction-based approach for parallel detection of single-nucleotide variants in the MYD88, TERT promoter, IDH1, IDH2, BRAF, and H3F3A genes within 80 minutes of sample acquisition. Canonical mutations were detected in 42% of patients with neoplasms, including cases of primary and secondary CNS lymphoma, glioblastoma, IDH-mutant brainstem glioma, and H3K27M-mutant diffuse midline glioma. Genotyping results eliminated the need for surgical biopsies in 7 of 33 cases (21.2%) of newly diagnosed neoplasms, resulting in significantly accelerated initiation of disease-directed treatment (median, 3 vs 12 days; P = .027). This assay was then implemented in a Clinical Laboratory Improvement Amendments environment, with 2-day median turnaround for diagnosis of CNS lymphoma from 66 patients across 4 clinical sites. Our study prospectively demonstrates that targeted rapid CSF genotyping influences oncologic management for suspected CNS tumors.

Liquid biopsy-based circulating tumour (ct)DNA analysis of a spectrum of myeloid and lymphoid malignancies yields clinically actionable results.

AIMS: Liquid biopsy (LBx)-based next-generation sequencing (NGS) of circulating tumour DNA (ctDNA) can facilitate molecular profiling of haematopoietic neoplasms (HNs), particularly when tissue-based NGS is infeasible. METHODS AND RESULTS: We studied HN LBx samples tested with FoundationOne Liquid CDx, FoundationOne Liquid, or FoundationACT between July 2016 and March 2022. We identified 271 samples: 89 non-Hodgkin lymphoma (NHL), 43 plasma-cell neoplasm (PCN), 41 histiocytoses, 27 myelodysplastic syndrome (MDS), 25 diffuse large B-cell lymphoma (DLBCL), 22 myeloproliferative neoplasm (MPN), 14 Hodgkin lymphoma (HL), and 10 acute myeloid leukaemia (AML). Among 73.4% with detectable pathogenic alterations, median maximum somatic allele frequency (MSAF) was 16.6%, with AML (36.2%), MDS (19.7%), and MPN (44.5%) having higher MSAFs than DLBCL (3.9%), NHL (8.4%), HL (1.5%), PCN (2.8%), and histiocytoses (1.8%) (P = 0.001). LBx detected characteristic alterations across HNs, including in TP53, KRAS, MYD88, and BTK in NHLs; TP53, KRAS, NRAS, and BRAF in PCNs; IGH in DLBCL; TP53, ATM, and PDCD1LG2 in HL; BRAF and MAP2K1 in histiocytoses; TP53, SF3B1, DNMT3A, TET2, and ASXL1 in MDS; JAK2 in MPNs; and FLT3, IDH2, and NPM1 in AML. Among 24 samples, the positive percent agreement by LBx was 75.7% for variants present in paired buffy coat, marrow, or tissues. Also, 75.0% of pairs exhibited alterations only present on LBx. These were predominantly subclonal (clonal fraction of 3.8%), reflecting the analytical sensitivity of LBx. CONCLUSION: These data demonstrate that LBx can detect relevant genomic alterations across HNs, including at low clonal fractions, suggesting a potential clinical utility for identifying residual or emerging therapy-resistant clones that may be undetectable in site-specific tissue biopsies.

Slide-to-Slide Tissue Transfer and Array Assembly From Limited Samples for Comprehensive Molecular Profiling.

Tissue microarrays (TMA) have become an important tool in high-throughput molecular profiling of tissue samples in the translational research setting. Unfortunately, high-throughput profiling in small biopsy specimens or rare tumor samples (eg, orphan diseases or unusual tumors) is often precluded owing to limited amounts of tissue. To overcome these challenges, we devised a method that allows tissue transfer and construction of TMAs from individual 2- to 5-µm sections for subsequent molecular profiling. We named the technique slide-to-slide (STS) transfer, and it requires a series of chemical exposures (so-called xylene-methacrylate exchange) in combination with rehydrated lifting, microdissection of donor tissues into multiple small tissue fragments (methacrylate-tissue tiles), and subsequent remounting on separate recipient slides (STS array slide). We developed the STS technique by assessing the efficacy and analytical performance using the following key metrics: (a) dropout rate, (b) transfer efficacy, (c) success rates using different antigen-retrieval methods, (d) success rates of immunohistochemical stains, (e) fluorescent in situ hybridization success rates, and (f) DNA and (g) RNA extraction yields from single slides, which all functioned appropriately. The dropout rate ranged from 0.7% to 6.2%; however, we applied the same STS technique successfully to fill these dropouts ("rescue" transfer). Hematoxylin and eosin assessment of donor slides confirmed a transfer efficacy of >93%, depending on the size of the tissue (range, 76%-100%). Fluorescent in situ hybridization success rates and nucleic acid yields were comparable with those of traditional workflows. In this study, we present a quick, reliable, and cost-effective method that offers the key advantages of TMAs and other molecular techniques-even when tissue is sparse. The perspectives of this technology in biomedical sciences and clinical practice are promising, given that it allows laboratories to create more data with less tissue.

Clinical Implications and Treatment Strategies for ESR1 Fusions in Hormone Receptor-Positive Metastatic Breast Cancer: A Case Series.

In hormone receptor-positive metastatic breast cancer (HR+ MBC), endocrine resistance is commonly due to genetic alterations of ESR1, the gene encoding estrogen receptor alpha (ERα). While ESR1 point mutations (ESR1-MUT) cause acquired resistance to aromatase inhibition (AI) through constitutive activation, far less is known about the molecular functions and clinical consequences of ESR1 fusions (ESR1-FUS). This case series discusses 4 patients with HR+ MBC with ESR1-FUS in the context of the existing ESR1-FUS literature. We consider therapeutic strategies and raise the hypothesis that CDK4/6 inhibition (CDK4/6i) may be effective against ESR1-FUS with functional ligand-binding domain swaps. These cases highlight the importance of screening for ESR1-FUS in patients with HR+ MBC while continuing investigation of precision treatments for these genomic rearrangements.

Clinicopathologic Evaluation and Molecular Profiling of Recurrent Stage IA Endometrial Endometrioid Carcinoma: A Case-control Study.

Most low-grade, early-stage endometrial endometrioid carcinomas (EEC) have an excellent prognosis; however, recurrences occur in a small subset with several studies reporting an increase in CTNNB1 exon 3 mutations in this population. Herein we evaluated 10 recurrent low-grade (FIGO 1 or 2), early-stage (FIGO IA) EECs matched to 10 nonrecurrent EECs to further characterize their clinicopathologic features and molecular phenotype. Cases were matched to controls based on size, grade, and depth of invasion. All tumors were evaluated for specific clinicopathologic parameters followed by next-generation sequencing using a 1213 gene panel. Recurrent EECs demonstrated no significant clinicopathologic differences when compared with nonrecurrent EECs, in terms of age, body mass index, pattern of invasion, presence of endometrial atypical hyperplasia/endometrioid intraepithelial neoplasia, associated metaplastic changes, peritumoral lymphocytes, mitoses, and tumor-infiltrating lymphocytes. Both cohorts also showed a similar number of pathogenic mutations, including CTNNB1 exon 3 mutations, as well as tumor mutational burden and microsatellite profiles. Although in this particular study, the lack of correlation between CTNNB1 exon 3 mutation and recurrence might be secondary to a small sample size, it also suggests the presence of other contributing factors. Thus, it helps set the foundation for larger series incorporating whole genome, transcriptome, proteome, and epigenome analyses to answer this clinically important question.

Diagnostic quality model (DQM): an integrated framework for the assessment of diagnostic quality when using AI/ML.

BACKGROUND: Laboratory medicine has reached the era where promises of artificial intelligence and machine learning (AI/ML) seem palpable. Currently, the primary responsibility for risk-benefit assessment in clinical practice resides with the medical director. Unfortunately, there is no tool or concept that enables diagnostic quality assessment for the various potential AI/ML applications. Specifically, we noted that an operational definition of laboratory diagnostic quality - for the specific purpose of assessing AI/ML improvements - is currently missing. METHODS: A session at the 3rd Strategic Conference of the European Federation of Laboratory Medicine in 2022 on "AI in the Laboratory of the Future" prompted an expert roundtable discussion. Here we present a conceptual diagnostic quality framework for the specific purpose of assessing AI/ML implementations. RESULTS: The presented framework is termed diagnostic quality model (DQM) and distinguishes AI/ML improvements at the test, procedure, laboratory, or healthcare ecosystem level. The operational definition illustrates the nested relationship among these levels. The model can help to define relevant objectives for implementation and how levels come together to form coherent diagnostics. The affected levels are referred to as scope and we provide a rubric to quantify AI/ML improvements while complying with existing, mandated regulatory standards. We present 4 relevant clinical scenarios including multi-modal diagnostics and compare the model to existing quality management systems. CONCLUSIONS: A diagnostic quality model is essential to navigate the complexities of clinical AI/ML implementations. The presented diagnostic quality framework can help to specify and communicate the key implications of AI/ML solutions in laboratory diagnostics.

Implementation and Clinical Adoption of Precision Oncology Workflows Across a Healthcare Network.

BACKGROUND: Precision oncology relies on molecular diagnostics, and the value-proposition of modern healthcare networks promises a higher standard of care across partner sites. We present the results of a clinical pilot to standardize precision oncology workflows. METHODS: Workflows are defined as the development, roll-out, and updating of disease-specific molecular order sets. We tracked the timeline, composition, and effort of consensus meetings to define the combination of molecular tests. To assess clinical impact, we examined order set adoption over a two-year period (before and after roll-out) across all gastrointestinal and hepatopancreatobiliary (GI) malignancies, and by provider location within the network. RESULTS: Development of 12 disease center-specific order sets took ~9 months, and the average number of tests per indication changed from 2.9 to 2.8 (P = .74). After roll-out, we identified significant increases in requests for GI patients (17%; P < .001), compliance with testing recommendations (9%; P < .001), and the fraction of "abnormal" results (6%; P < .001). Of 1088 GI patients, only 3 received targeted agents based on findings derived from non-recommended orders (1 before and 2 after roll-out); indicating that our practice did not negatively affect patient treatments. Preliminary analysis showed 99% compliance by providers in network sites, confirming the adoption of the order sets across the network. CONCLUSION: Our study details the effort of establishing precision oncology workflows, the adoption pattern, and the absence of harm from the reduction of non-recommended orders. Establishing a modifiable communication tool for molecular testing is an essential component to optimize patient care via precision oncology.

Uterine PEComas: correlation between melanocytic marker expression and TSC alterations/TFE3 fusions.

Uterine PEComas often present a diagnostic challenge as they share morphological and immunohistochemical features with smooth muscle tumors. Herein we evaluated a series of 19 uterine PEComas to compare the degree of melanocytic marker expression with their molecular profile. Patients ranged from 32-77 (median 48) years, with six tumors classified as malignant based on the modified gynecologic-specific prognostic algorithm. All patients with malignant PEComas were alive with disease or dead  of disease at last follow-up, while all those of uncertain malignant potential were alive and well (median follow-up, 47 months).Seventeen of 19 (89%) PEComas harbored either a TSC1 or TSC2 alteration. One of the two remaining tumors showed a TFE3 rearrangement, but the other lacked alterations in all genes evaluated. All showed at least focal (usually strong) positivity for HMB-45, with 15/19 (79%) having >50% expression, while the tumor lacking TSC or TFE3 alterations was strongly positive in 10% of cells. Melan-A and MiTF were each positive in 15/19 (79%) tumors, but staining extent and intensity were much more variable than HMB-45. Five of six (83%) malignant PEComas also harbored alterations in TP53, ATRX, or RB1, findings not identified in any tumors of uncertain malignant potential. One malignant PEComa was microsatellite-unstable/mismatch repair protein-deficient.In summary, TSC alterations/TFE3 fusions and diffuse (>50%) HMB-45 expression are characteristic of uterine PEComas. In morphologically ambiguous mesenchymal neoplasms with myomelanocytic differentiation, especially those with metastatic or recurrent disease, next-generation sequencing is recommended to evaluate for TSC alterations; as such, patients can be eligible for targeted therapy.

Diagnostic Value of MAML2 Rearrangements in Mucoepidermoid Carcinoma.

Mucoepidermoid carcinoma (MEC) is often seen in salivary glands and can harbor MAML2 translocations (MAML2+). The translocation status has diagnostic utility as an objective confirmation of the MEC diagnosis, for example, when distinction from the more aggressive adenosquamous carcinoma (ASC) is not straightforward. To assess the diagnostic relevance of MAML2, we examined our 5-year experience in prospective testing of 8106 solid tumors using RNA-seq panel testing in combinations with a two-round Delphi-based scenario survey. The prevalence of MAML2+ across all tumors was 0.28% (n = 23/8106) and the majority of MAML2+ cases were found in head and neck tumors (78.3%), where the overall prevalence was 5.9% (n = 18/307). The sensitivity of MAML2 for MEC was 60% and most cases (80%) were submitted for diagnostic confirmation; in 24% of cases, the MAML2 results changed the working diagnosis. An independent survey of 15 experts showed relative importance indexes of 0.8 and 0.65 for "confirmatory MAML2 testing" in suspected MEC and ASC, respectively. Real-world evidence confirmed that the added value of MAML2 is a composite of an imperfect confirmation test for MEC and a highly specific exclusion tool for the diagnosis of ASC. Real-world evidence can help move a rare molecular-genetic biomarker from an emerging tool to the clinic.

Presymptomatic Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 Among Residents and Staff at a Skilled Nursing Facility: Results of Real-time Polymerase Chain Reaction and Serologic Testing.

High rates of asymptomatic coronavirus disease 2019 infection suggest benefits to routine testing in congregate care settings. Screening was undertaken in a single nursing facility without a known case of coronavirus disease 2019, demonstrating an 85% prevalence among residents and 37% among staff. Serology was not helpful in identifying infections.

Mosaicism for Receptor Tyrosine Kinase Activation in a Glioblastoma Involving Both PDGFRA Amplification and NTRK2 Fusion.

Rearrangements involving the neurotrophic receptor tyrosine kinase (NTRK) gene family have been reported in diverse tumor types, and NTRK-targeted therapies have recently been approved. In this article, we report a case of a 26-year-old man with an NTRK2-rearranged isocitrate dehydrogenase-wild-type glioblastoma who showed a robust but temporary response to the NTRK inhibitor larotrectinib. Rebiopsy after disease progression showed elimination of the NTRK2-rearranged tumor cell clones, with secondary emergence of a PDGFRA-amplified subclone. Retrospective examination of the initial biopsy material confirmed rare cells harboring PDGFRA amplification. Although mosaic amplification of multiple receptor tyrosine kinase genes in glioblastoma has been previously described, mosaicism involving a fusion gene driver event has not. This case highlights the potential efficacy of NTRK-targeted treatment in glioblastoma and the implications of molecular heterogeneity in the setting of targeted therapy. KEY POINTS: This case highlights the efficacy of the NTRK inhibitor larotrectinib in treating NTRK-rearranged glioblastoma. This is the first case to demonstrate mosaicism in glioblastoma involving both a fusion gene and amplification for receptor tyrosine kinases. Intratumoral heterogeneity in glioblastoma has significant implications for tumor resistance to targeted therapies.

Embryonal rhabdomyosarcoma of the uterine corpus: a clinicopathological and molecular analysis of 21 cases highlighting a frequent association with DICER1 mutations.

Herein we evaluated a series of 21 embryonal rhabdomyosarcomas of the uterine corpus (ucERMS), a rare neoplasm, to characterize their morphology, genomics, and behavior. Patients ranged from 27 to 73 (median 52) years and tumors from 4 to 15 (median 9) cm, with extrauterine disease noted in two. Follow-up (median 16 months) was available for 14/21 patients; nine were alive and well, four died of disease, and one died from other causes. Most tumors (16/21) showed predominantly classic morphology, comprised of alternating hyper- and hypocellular areas of primitive small cells and differentiating rhabdomyoblasts in a loose myxoid/edematous stroma. A cambium layer was noted in all; seven had heterologous elements (six with fetal-type cartilage) and eight displayed focal anaplasia. The remaining five neoplasms showed only a minor component (≤20%) of classic morphology, with anaplasia noted in four and tumor cell necrosis in three. The most frequent mutations detected were in DICER1 (14/21), TP53 (7/20), PI3K/AKT/mTOR pathway (7/20), and KRAS/NRAS (5/20). Copy-number alterations were present in 10/19 tumors. Overall, 8/14 DICER1-associated ucERMS showed concurrent loss of function and hotspot mutations in DICER1, which is a feature more likely to be seen in tumors associated with DICER1 syndrome. Germline data were available for two patients, both DICER1 wild type (one with concurrent loss of function and hotspot alterations). DICER1-associated ucERMS were more likely to show a classic histological appearance including heterologous elements than DICER1-independent tumors. No differences in survival were noted between the two groups, but both patients with extrauterine disease at diagnosis and two with recurrences died from disease. As no patients had a known personal or family history of DICER1 syndrome, we favor most DICER1-associated ucERMS to be sporadic.

Clinical sensitivity and interpretation of PCR and serological COVID-19 diagnostics for patients presenting to the hospital.

The diagnosis of COVID-19 requires integration of clinical and laboratory data. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostic assays play a central role in diagnosis and have fixed technical performance metrics. Interpretation becomes challenging because the clinical sensitivity changes as the virus clears and the immune response emerges. Our goal was to examine the clinical sensitivity of two most common SARS-CoV-2 diagnostic test modalities, polymerase chain reaction (PCR) and serology, over the disease course to provide insight into their clinical interpretation in patients presenting to the hospital. We conducted a single-center, retrospective study. To derive clinical sensitivity of PCR, we identified 209 PCR-positive SARS-CoV-2 patients with multiple PCR test results (624 total PCR tests) and calculated daily sensitivity from date of symptom onset or first positive test. Clinical sensitivity of PCR decreased with days post symptom onset with >90% clinical sensitivity during the first 5 days after symptom onset, 70%-71% from Days 9 to 11, and 30% at Day 21. To calculate daily clinical sensitivity by serology, we utilized 157 PCR-positive patients with a total of 197 specimens tested by enzyme-linked immunosorbent assay for IgM, IgG, and IgA anti-SARS-CoV-2 antibodies. In contrast to PCR, serological sensitivity increased with days post symptom onset with >50% of patients seropositive by at least one antibody isotype after Day 7, >80% after Day 12, and 100% by Day 21. Taken together, PCR and serology are complimentary modalities that require time-dependent interpretation. Superimposition of sensitivities over time indicate that serology can function as a reliable diagnostic aid indicating recent or prior infection.

Female adnexal tumors of probable Wolffian origin: morphological, immunohistochemical, and molecular analysis of 15 cases.

Female adnexal tumors of probable Wolffian origin are rare and present a diagnostic challenge due to their morphological and immunohistochemical overlap with more common ovarian and broad ligament entities. We evaluated the morphological, immunohistochemical, and molecular features of 15 tumors of probable Wolffian origin. Patients ranged from 32 to 69 (mean 47) years and tumors from 1.8 to 30 (mean 10) cm. All except one arose in para-adnexal soft tissues. Follow-up was available for six patients, five of whom were alive and well, while the sixth, who had extra-adnexal disease at diagnosis, died from unrelated causes. The following patterns were noted: tubular (all tumors), solid 11/15 (73%), sieve-like 7/15 (47%), and reticular 1/15 (7%). A myxoid background was present in 3/15 (20%) of tumors and eosinophilic luminal secretions in 11/15 (73%). Most tumors (12/15, 80%) had low-grade nuclear atypia, while three showed foci with scattered high-grade atypia. Mitotic index ranged from 0 to 17 (mean 4) per ten high-power fields. Tumors were positive for pankeratin and negative for TTF-1. EMA, GATA3, and PAX8 were positive in 2/10 (20%; focal), 3/15 (20%; focal), and 1/15 (7%; focal) of tumors, respectively. CD10, SF-1, calretinin, inhibin, ER, PR, cytokeratin 7, and WT1 were variably expressed. Pathogenic mutations were rare and included STK11 (n = 3), APC (n = 1), and MBD4 (n = 1). Copy number variations were detected in the three tumors with STK11 mutations and a myxoid background. These data demonstrate that female adnexal tumors of probable Wolffian origin are morphologically and immunohistochemically diverse, but infrequently harbor pathogenic mutations. However, their lack of mutations in contrast to their mimickers may be a valuable tool in diagnostically difficult cases.

Interpretation of Mismatch Repair Protein Immunohistochemistry in Endometrial Carcinoma Should Consider Both Lynch Syndrome Screening and Immunotherapy Susceptibility: An Illustrative Case Report.

We present an instructive case of FIGO grade 1 endometrioid endometrial carcinoma with a biphasic morphology, corresponding to subclonal loss of mismatch repair proteins (MMRP) MLH1 and PMS2 by immunohistochemistry and subclonal microsatellite instability. A pulmonary metastasis represented only the tumor component with retention of MMRPs. This case illustrates the need for pathologists to recognize and report heterogenous expression of MMRPs in endometrial carcinoma, to consider tumor heterogeneity when selecting foci for molecular studies, and to re-evaluate MMRP expression in tumor metastases, when clinically indicated. These considerations are particularly important as the relevance of MMRP expression in endometrial cancer expands beyond Lynch syndrome screening, into a new role as a predictive marker for immunotherapy.

Quantitative next-generation sequencing-based analysis indicates progressive accumulation of microsatellite instability between atypical hyperplasia/endometrial intraepithelial neoplasia and paired endometrioid endometrial carcinoma.

Atypical hyperplasia/endometrial intraepithelial neoplasia is an accepted precursor to endometrioid-type endometrial carcinoma. Mismatch repair-deficient endometrial carcinomas are also known to be a biologically and clinically distinct subset of tumors. However, the development of microsatellite instability in endometrial carcinogenesis has not yet been evaluated by novel next-generation sequencing-based methods. We examined 17 mismatch repair-deficient endometrioid endometrial carcinomas and their paired atypical hyperplasia/endometrial intraepithelial neoplasia precursors using a next-generation sequencing panel with quantitative microsatellite instability detection at 336 loci. Findings were compared to histological features, polymerase chain reaction-based microsatellite instability testing, immunohistochemical expression of mismatch repair proteins, and tumor mutational burden calculations. All 17 endometrial carcinomas and 8/17 atypical hyperplasia/endometrial intraepithelial neoplasia showed microsatellite instability by next-generation sequencing-based testing. Endometrial carcinoma specimens showed significantly more unstable microsatellite loci than paired atypical hyperplasia/endometrial intraepithelial neoplasia (mean: 40.0% vs 19.9 unstable loci, respectively). Out of nine microsatellite-stable atypical hyperplasia/endometrial intraepithelial neoplasia specimens, four showed mismatch repair loss by immunohistochemistry. All atypical hyperplasia/endometrial intraepithelial neoplasia and endometrial carcinoma specimens with microsatellite instability were also mismatch repair-deficient by immunohistochemistry. Tumor mutational burden was significantly greater in endometrial carcinoma than in paired atypical hyperplasia/endometrial intraepithelial neoplasia specimens, and tumor mutational burden was significantly correlated with percent unstable microsatellite loci. Paired atypical hyperplasia/endometrial intraepithelial neoplasia and endometrial carcinoma specimens show progressive accumulation of unstable microsatellite loci following loss of mismatch repair protein expression. Comprehensive next-generation sequencing-based testing of endometrial carcinomas offers new insights into endometrial carcinogenesis and opportunities for improved tumor surveillance, diagnosis, and management.

Loss of SMAD4 protein expression in gastrointestinal and extra-gastrointestinal carcinomas.

AIMS: SMAD4 (DPC4) is a tumour suppressor gene that is dysregulated in various tumour types, particularly pancreaticobiliary and gastrointestinal carcinomas. Corresponding loss of protein expression has been reported in approximately 50% of pancreatic and 25% of colonic adenocarcinomas. In the evaluation of carcinoma of unknown primary site, immunohistochemical loss of SMAD4 expression is often used to suggest pancreaticobiliary origin, but there are limited data on the spectrum of SMAD4 expression in carcinomas of other sites. This study evaluates the frequency of SMAD4 loss in a large cohort of carcinomas from diverse anatomical sites. METHODS AND RESULTS: Immunohistochemistry for SMAD4 was performed on tissue microarrays or whole tissue sections of 1210 carcinomas from various organs: gastrointestinal tract, liver, pancreas/biliary tract, lung, breast, thyroid, kidney, ovary and uterus. Expression was considered lost when there was complete absence of staining in tumour cell nuclei, in the presence of intact staining in non-neoplastic cells. SMAD4 loss was seen in 58% of pancreatic adenocarcinomas, 27% of appendiceal adenocarcinomas, 19% of colorectal adenocarcinomas, 16% of cholangiocarcinomas, 10% of lung adenocarcinomas and <5% of oesophageal, breast, gastric and mucinous ovarian adenocarcinomas. All papillary thyroid, hepatocellular, non-mucinous ovarian, endometrial and renal cell carcinomas showed intact SMAD4 nuclear expression. CONCLUSION: In addition to pancreaticobiliary, appendiceal and colonic tumours, SMAD4 loss is also seen in a small subset of other carcinomas, specifically breast, lung, oesophageal and gastric adenocarcinomas, all of which are typically CK7-positive, similar to pancreaticobiliary carcinoma. Awareness of SMAD4 loss in these other carcinoma types is helpful in the evaluation of carcinomas of unknown or uncertain primary site.

High NPM1 mutant allele burden at diagnosis correlates with minimal residual disease at first remission in de novo acute myeloid leukemia.

Acute myeloid leukemia (AML) with mutated NPM1 is a newly recognized separate entity in the revised 2016 WHO classification, and is associated with a favorable prognosis. While previous studies have evaluated NPM1 in a binary fashion, we recently demonstrated a significant independent negative prognostic effect of high NPM1 mutant allele burden (VAF) at diagnosis in a cohort of de novo AML patients. Although the importance of minimal residual disease (MRD) monitoring in NPM1-mutated AML has been well characterized, the potential relationship between diagnostic allele burden and MRD is unknown. We retrospectively evaluated for MRD at first remission (CR1). We used either next-generation sequencing (NGS) [n = 71], and/or immunohistochemistry (IHC) for mutant NPM1 (NPM1c) [n = 60], in a subset of patients from our recently examined cohort. We identified a statistically significant positive correlation between the VAF at diagnosis, and at CR1 (Spearman r = 0.4, P = .006), and enrichment for MRD in high diagnostic VAF patients (P = .05), as previously defined. IHC-positivity also correlated significantly with a higher median diagnostic NPM1 VAF (0.42 vs 0.39, P = .02), and with the VAF at CR1 (Spearman r = 0.7, P = .003). In multivariable analyses, both high diagnostic VAF (P = .003) and MRD (P = .02) were independent predictors of shorter event-free survival (EFS). Our findings suggest a relationship between the NPM1 mutant allele burden at diagnosis, and the presence of MRD at first remission. Our findings support IHC as a potentially useful adjunctive tool for disease monitoring.

GNAS mutations in primary mucinous and non-mucinous lung adenocarcinomas.

GNAS mutations have been described in mucinous and non-mucinous epithelial neoplasms of the appendix, pancreas, and colon, with hotspot GNAS mutations found in up to two-thirds of pancreatic intraductal papillary mucinous neoplasms. Additionally, many GNAS-mutated tumors have concurrent mutations in the Ras/Raf pathway. The clinicopathologic features of GNAS-mutated lung carcinomas, however, have not yet been characterized. Primary lung carcinomas from Brigham and Women's Hospital (n=1282) or Massachusetts General Hospital (n=1070) were genotyped on a targeted massively parallel sequencing panel of oncogenes and tumor suppressor genes including GNAS. Clinical and pathological features were reviewed, and TTF-1 immunohistochemistry was performed when material was available. Nineteen lung adenocarcinomas with hotspot GNAS mutations were identified (19/2352, 0.8%) including 14 at codon 201 and 5 at codon 227. GNAS-mutated lung adenocarcinomas occurred predominantly in female patients (16/19, 84%). Ten (10) were classified as invasive mucinous adenocarcinomas (IMA), and nine (9) were non-mucinous adenocarcinomas. All IMAs had GNAS codon 201 mutations and concurrent Ras/Raf pathway mutations (9 KRAS, 1 BRAF). No tumors with GNAS codon 227 mutations had mucinous histological features. 86% of GNAS-mutated non-mucinous adenocarcinomas (6/7) were positive for TTF-1 immunohistochemistry, while only 25% of GNAS-mutated IMAs (1/4) were positive for TTF-1. Patients with GNAS-mutated non-mucinous adenocarcinomas were more likely to have a history of smoking (9/9, 100%) compared to patients with GNAS-mutated IMAs (2/10, 20%) (P<0.001). Hotspot GNAS mutations can occur in primary lung adenocarcinomas. When associated with concurrent mutations in the Ras/Raf pathway, these neoplasms often present as IMAs. GNAS mutations are not specific to neoplasms of the gastrointestinal tract, and clinicopathologic correlation is necessary in GNAS-mutated adenocarcinomas in the lung to determine the primary site of origin.

Differentiated exophytic vulvar intraepithelial lesions are genetically distinct from keratinizing squamous cell carcinomas and contain mutations in PIK3CA.

Human papillomavirus-negative keratinizing vulvar cancers typically harbor TP53 mutations as do their precursors, differentiated vulvar intraepithelial neoplasia. However, atypical verruciform proliferations are also associated with these malignancies and their pathogenesis is poorly understood. This study compared 11 atypical verruciform lesions, including atypical verruciform hyperplasia, vulvar acanthosis with altered differentiation, and verruciform lichen simplex chronicus, with 14 human papillomavirus-negative keratinizing squamous cell carcinomas. Extracted tissue DNA was subjected to targeted massively parallel sequencing of the exonic regions of 300 genes. Eight (73%) and six (55%) of eleven atypical verruciform lesions contained mutations in PIK3CA and ARID2, respectively. No TP53 mutations were identified. Eleven (79%) and five (36%) of fourteen keratinizing squamous cell carcinomas tested contained TP53 and CDKN2A mutations, respectively. Keratinizing squamous cell carcinomas displayed the majority of copy number variations with some variations (7p gain and 8p loss) shared by some cases in both groups. One patient developed atypical verruciform lesions with PIK3CA mutations followed by a keratinizing carcinoma with mutations in both PIK3CA and TP53. This study, for the first time segregates atypical verruciform lesions by virtue of a unique genotype (PIK3CA mutant/TP53 wild type) illustrating an example of progression to a TP53-mutated keratinizing carcinoma. The findings indicate that although PIK3CA mutations are found in <10% of vulvar squamous cell carcinomas, they may be specific for a particular pathway involving atypical verruciform lesions, which could function as either a direct precursor or a risk factor for vulvar squamous cell carcinoma. Given the presence of a molecular signature, we propose the term 'differentiated exophytic vulvar intraepithelial lesion' for this group. Whether they function as direct precursors to a less common form of squamous cell carcinoma will require further study, but carcinomas associated with these lesions might warrant testing for PIK3CA mutations to address this question.