60 kD Ro and nRNP A Frequently Initiate Human Lupus Autoimmunity

Systemic lupus erythematosus (SLE) is a clinically heterogeneous, humoral autoimmune disorder. The unifying feature among SLE patients is the production of large quantities of autoantibodies. Serum samples from 129 patients collected before the onset of SLE and while in the United States military were evaluated for early pre-clinical serologic events. The first available positive serum sample frequently already contained multiple autoantibody specificities (65%). However, in 34 SLE patients the earliest pre-clinical serum sample positive for any detectable common autoantibody bound only a single autoantigen, most commonly 60 kD Ro (29%), nRNP A (24%), anti-phospholipids (18%) or rheumatoid factor (15%). We identified several recurrent patterns of autoantibody onset using these pre-diagnostic samples. In the serum samples available, anti-nRNP A appeared before or simultaneously with anti-nRNP 70 K in 96% of the patients who had both autoantibodies at diagnosis. Anti-60 kD Ro antibodies appeared before or simultaneously with anti-La (98%) or anti-52 kD Ro (95%). The autoantibody response in SLE patients begins simply, often binding a single specific autoantigen years before disease onset, followed by epitope spreading to additional autoantigenic specificities that are accrued in recurring patterns

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

CASE REPORT Open Access

of a case with a unique intratumoral sarcoid-like reaction Ritterhouse et al

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

Germline BRCA-Associated Endometrial Carcinoma Is a Distinct Clinicopathologic Entity

Purpose: Whether endometrial carcinoma (EC) should be considered part of the gBRCA1/2-associated hereditary breast and ovarian cancer (HBOC) syndrome is topic of debate. We sought to assess whether ECs occurring in gBRCA carriers are enriched for clinicopathologic and molecular characteristics, thereby supporting a causal relationship. Experimental Design: Thirty-eight gBRCA carriers that developed EC were selected from the nationwide cohort study on hereditary breast and ovarian cancer in the Netherlands (HEBON), and these were supplemented with four institutional cases. Tumor tissue was retrieved via PALGA (Dutch Pathology Registry). Nineteen morphologic features were scored and histotype was determined by three expert gyneco-logic pathologists, blinded for molecular analyses (UCM-OncoPlus Assay including 1213 genes). ECs with LOH of the gBRCA-wild-type allele (gBRCA/LOHpos) were defined "gBRCA-associated," those without LOH (gBRCA/LOHneg) were defined "sporadic." Results: LOH could be assessed for 40 ECs (30 gBRCA1, 10 gBRCA2), of which 60% were gBRCA/LOHpos. gBRCA/LOHpos ECs were more frequently of nonendometrioid (58%, P = 0.001) and grade 3 histology (79%, P <0.001). All but two were in the TP53-mutated TCGA-subgroup (91.7%, P <0.001). In contrast, gBRCA/LOHneg ECs were mainly grade 1 endometrioid EC (94%) and showed a more heterogeneous distribution of TCGA-molecular subgroups: POLE-mutated (6.3%), MSI-high (25%), NSMP (62.5%), and TP53-mutated (6.3%). Conclusions: We provide novel evidence in favor of EC being part of the gBRCA-associated HBOC-syndrome. gBRCA-associated ECs are enriched for EC subtypes associated with unfavorable clinical outcome. These findings have profound therapeutic consequences as these patients may benefit from treatment strategies such as PARP inhibitors. In addition, it should influence counseling and surveillance of gBRCA carriers

Primary Resistance to Larotrectinib in a Patient With Squamous NSCLC With Subclonal NTRK1 Fusion: Case Report

The NTRK genes encode the TRK proteins. NTRK fusions lead to constitutively active, ligand-independent downstream signaling. NTRK fusions are implicated in up to 1% of all solid tumors and 0.2% of NSCLC. Larotrectinib, a highly selective small molecule inhibitor of all three TRK proteins, has a response rate of 75% across a wide range of solid tumors. Mechanisms of primary resistance to larotrectinib are not well understood. We report a case of a 75-year-old male with minimal smoking history with NTRK fusion-positive metastatic squamous NSCLC with primary resistance to larotrectinib. We suggest subclonal NTRK fusion as a possible mechanism contributing to primary resistance to larotrectinib

Secondary resistance to immunotherapy associated with β-catenin pathway activation or PTEN loss in metastatic melanoma

BACKGROUND: While cancer immunotherapies including checkpoint blockade antibodies, adoptive T cell therapy, and even some vaccines have given rise to major clinical responses with durability in many cases, a subset of patients who initially respond subsequently develop secondary resistance to therapy. Tumor-intrinsic mechanisms of acquired immunotherapy resistance are incompletely understood. METHODS: Baseline and treatment-resistant tumors underwent molecular analysis via transcriptional profiling or genomic sequencing for oncogenic alterations and histologic analysis for T cell infiltration to investigate mechanisms contributing to T cell exclusion and acquired resistance to immunotherapy. RESULTS: We describe two patients with metastatic melanoma who initially showed a durable partial response to either a melanoma-peptide/interleukin-12 vaccine or combined anti-CTLA-4 + anti-PD-1 therapy, but subsequently developed new treatment-resistant metastases. In the first case, the recurrent tumor showed new robust tumor expression of β-catenin, whereas in the second case genomic sequencing revealed acquired PTEN loss. Both cases were associated with loss of T cell infiltration, and both pathways have been mechanistically linked to immune resistance preclinically. CONCLUSION: Our results suggest that secondary resistance to immunotherapies can arise upon selection for new oncogenic variants that mediate T cell exclusion. To identify the spectrum of underlying mechanisms of therapeutic resistance, similar evaluation for the emergence of tumor-intrinsic alterations in resistant lesions should be done prospectively at the time of relapse in a range of additional patients developing secondary resistance.NCI (Award R00CA204595

Measurable residual disease monitoring for patients with acute myeloid leukemia following hematopoietic cell transplantation using error corrected hybrid capture next generation sequencing.

Improved systems for detection of measurable residual disease (MRD) in acute myeloid leukemia (AML) are urgently needed, however attempts to utilize broad-scale next-generation sequencing (NGS) panels to perform multi-gene surveillance in AML post-induction have been stymied by persistent premalignant mutation-bearing clones. We hypothesized that this technology may be more suitable for evaluation of fully engrafted patients following hematopoietic cell transplantation (HCT). To address this question, we developed a hybrid-capture NGS panel utilizing unique molecular identifiers (UMIs) to detect variants at 0.1% VAF or below across 22 genes frequently mutated in myeloid disorders and applied it to a retrospective sample set of blood and bone marrow DNA samples previously evaluated as negative for disease via standard-of-care short tandem repeat (STR)-based engraftment testing and hematopathology analysis in our laboratory. Of 30 patients who demonstrated trackable mutations in the 22 genes at eventual relapse by standard NGS analysis, we were able to definitively detect relapse-associated mutations in 18/30 (60%) at previously disease-negative timepoints collected 20-100 days prior to relapse date. MRD was detected in both bone marrow (15/28, 53.6%) and peripheral blood samples (9/18, 50%), while showing excellent technical specificity in our sample set. We also confirmed the disappearance of all MRD signal with increasing time prior to relapse (>100 days), indicating true clinical specificity, even using genes commonly associated with clonal hematopoiesis of indeterminate potential (CHIP). This study highlights the efficacy of a highly sensitive, NGS panel-based approach to early detection of relapse in AML and supports the clinical validity of extending MRD analysis across many genes in the post-transplant setting

Measurable residual disease monitoring for patients with acute myeloid leukemia following hematopoietic cell transplantation using error corrected hybrid capture next generation sequencing

Improved systems for detection of measurable residual disease (MRD) in acute myeloid leukemia (AML) are urgently needed, however attempts to utilize broad-scale next-generation sequencing (NGS) panels to perform multi-gene surveillance in AML post-induction have been stymied by persistent premalignant mutation-bearing clones. We hypothesized that this technology may be more suitable for evaluation of fully engrafted patients following hematopoietic cell transplantation (HCT). To address this question, we developed a hybrid-capture NGS panel utilizing unique molecular identifiers (UMIs) to detect variants at 0.1% VAF or below across 22 genes frequently mutated in myeloid disorders and applied it to a retrospective sample set of blood and bone marrow DNA samples previously evaluated as negative for disease via standard-of-care short tandem repeat (STR)-based engraftment testing and hematopathology analysis in our laboratory. Of 30 patients who demonstrated trackable mutations in the 22 genes at eventual relapse by standard NGS analysis, we were able to definitively detect relapse-associated mutations in 18/30 (60%) at previously disease-negative timepoints collected 20–100 days prior to relapse date. MRD was detected in both bone marrow (15/28, 53.6%) and peripheral blood samples (9/18, 50%), while showing excellent technical specificity in our sample set. We also confirmed the disappearance of all MRD signal with increasing time prior to relapse (>100 days), indicating true clinical specificity, even using genes commonly associated with clonal hematopoiesis of indeterminate potential (CHIP). This study highlights the efficacy of a highly sensitive, NGS panel-based approach to early detection of relapse in AML and supports the clinical validity of extending MRD analysis across many genes in the post-transplant setting