Morphologic Characteristics and Mutational Analysis of Fumarate Hydratase Deficient Kidney and Smooth Muscle Tumors.

OBJECTIVES: Fumarate hydratase (FH)-deficient tumors can occur due to germline or somatic mutations and have distinctive morphologic features. The aims of this study are to refine morphologic criteria and identify mutations in FH-deficient smooth muscle tumors (SMTs). METHODS: The morphology of SMTs and kidney tumors submitted to a national reference laboratory for FH immunohistochemistry (IHC) was reviewed by two gynecologic and two genitourinary pathologists, respectively. Fisher exact test was used for analysis. Fourteen SMTs were sequenced using the Illumina TruSight Oncology 500 Assay. RESULTS: Twenty-two kidney tumors (5 FH deficient) and 51 SMTs (27 FH deficient) were reviewed. FH-deficient kidney tumors exclusively showed cord-like growth, rhabdoid change, and absence of coagulative tumor necrosis and psammoma bodies. FH-deficient SMTs were significantly more likely to have staghorn vessels, eosinophilic cytoplasmic inclusions, schwannoma-like areas, or hereditary leiomyomatosis and renal cell cancer-like nuclei (P < .05 for each). Seven of 14 sequenced SMTs showed mutations of the FH gene and no other driver mutations. CONCLUSIONS: FH-deficient SMTs submitted for FH immunohistochemistry (IHC) showed distinct morphology. Although FH IHC is used for screening of FH-deficient tumors, FH mutations were identified in only 50% of FH-deficient SMTs. This highlights the need for additional exploration of mechanisms of FH protein loss in tumors lacking FH mutations.

Simultaneous detection of lung fusions using a multiplex RT-PCR next generation sequencing-based approach: a multi-institutional research study.

BACKGROUND: Gene fusion events resulting from chromosomal rearrangements play an important role in initiation of lung adenocarcinoma. The recent association of four oncogenic driver genes, ALK, ROS1, RET, and NTRK1, as lung tumor predictive biomarkers has increased the need for development of up-to-date technologies for detection of these biomarkers in limited amounts of material. METHODS: We describe here a multi-institutional study using the Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel to interrogate previously characterized lung tumor samples. RESULTS: Reproducibility between laboratories using diluted fusion-positive cell lines was 100%. A cohort of lung clinical research samples from different origins (tissue biopsies, tissue resections, lymph nodes and pleural fluid samples) were used to evaluate the panel. We observed 97% concordance for ALK (28/30 positive; 71/70 negative samples), 95% for ROS1 (3/4 positive; 19/18 negative samples), and 93% for RET (2/1 positive; 13/14 negative samples) between the AmpliSeq assay and other methodologies. CONCLUSION: This methodology enables simultaneous detection of multiple ALK, ROS1, RET, and NTRK1 gene fusion transcripts in a single panel, enhanced by an integrated analysis solution. The assay performs well on limited amounts of input RNA (10 ng) and offers an integrated single assay solution for detection of actionable fusions in lung adenocarcinoma, with potential savings in both cost and turn-around-time compared to the combination of all four assays by other methods.

Prediction of lung cancer histological types by RT-qPCR gene expression in FFPE specimens.

Lung cancer histologic diagnosis is clinically relevant because there are histology-specific treatment indications and contraindications. Histologic diagnosis can be challenging owing to tumor characteristics, and it has been shown to have less-than-ideal agreement among pathologists reviewing the same specimens. Microarray profiling studies using frozen specimens have shown that histologies exhibit different gene expression trends; however, frozen specimens are not amenable to routine clinical application. Herein, we developed a gene expression-based predictor of lung cancer histology for FFPE specimens, which are routinely available in clinical settings. Genes predictive of lung cancer histologies were derived from published cohorts that had been profiled by microarrays. Expression of these genes was measured by quantitative RT-PCR (RT-qPCR) in a cohort of patients with FFPE lung cancer. A histology expression predictor (HEP) was developed using RT-qPCR expression data for adenocarcinoma, carcinoid, small cell carcinoma, and squamous cell carcinoma. In cross-validation, the HEP exhibited mean accuracy of 84% and κ = 0.77. In separate independent validation sets, the HEP was compared with pathologist diagnoses on the same tumor block specimens, and the HEP yielded similar accuracy and precision as the pathologists. The HEP also exhibited good performance in specimens with low tumor cellularity. Therefore, RT-qPCR gene expression from FFPE specimens can be effectively used to predict lung cancer histology.

PAM50 proliferation score as a predictor of weekly paclitaxel benefit in breast cancer.

To identify a group of patients who might benefit from the addition of weekly paclitaxel to conventional anthracycline-containing chemotherapy as adjuvant therapy of node-positive operable breast cancer. The predictive value of PAM50 subtypes and the 11-gene proliferation score contained within the PAM50 assay were evaluated in 820 patients from the GEICAM/9906 randomized phase III trial comparing adjuvant FEC to FEC followed by weekly paclitaxel (FEC-P). Multivariable Cox regression analyses of the secondary endpoint of overall survival (OS) were performed to determine the significance of the interaction between treatment and the (1) PAM50 subtypes, (2) PAM50 proliferation score, and (3) clinical and pathological variables. Similar OS analyses were performed in 222 patients treated with weekly paclitaxel versus paclitaxel every 3 weeks in the CALGB/9342 and 9840 metastatic clinical trials. In GEICAM/9906, with a median follow up of 8.7 years, OS of the FEC-P arm was significantly superior compared to the FEC arm (unadjusted HR = 0.693, p = 0.013). A benefit from paclitaxel was only observed in the group of patients with a low PAM50 proliferation score (unadjusted HR = 0.23, p < 0.001; and interaction test, p = 0.006). No significant interactions between treatment and the PAM50 subtypes or the various clinical-pathological variables, including Ki-67 and histologic grade, were identified. Finally, similar OS results were obtained in the CALGB data set, although the interaction test did not reach statistical significance (p = 0.109). The PAM50 proliferation score identifies a subset of patients with a low proliferation status that may derive a larger benefit from weekly paclitaxel.

Multiple MDMA (Ecstasy) overdoses at a rave event: a case series.

Twelve patients with 3,4-methylenedioxymethamphetamine (MDMA) toxicity from a single rave event presented to multiple San Francisco Bay area hospitals with various life-threatening complications including seizures and hyperthermia. Eight required emergent endotracheal intubation and six had hypotension. Hyperkalemia, acute kidney injury, and rhabdomyolysis were present in most of the patients. In all, 2 patients died, 4 survived with permanent neurologic, musculoskeletal, and/or renal sequelae, and 6 survived without any apparent lasting deficits. Hyperthermia was present in 10 patients and was severe (40.9-43° C) in 7. Using multiple cooling methods, the average time to achieve cooling was 2.7 hours. Serum drug analysis was performed on 3 patients, demonstrating toxic MDMA concentrations without the presence of other xenobiotics. Two capsules confiscated by police at the event contained 82% and 98% MDMA, respectively, without other pharmacologically active compounds. Capsule #2 contained 270 mg MDMA, which is more than twice the amount of MDMA usually contained in 1 dose. The MDMA-induced hyperthermia significantly contributed to the morbidity and mortality in this case series. Factors contributing to the severity of the hyperthermia include ingestion of large doses of MDMA, a warm ambient environment, and physical exertion.

PAM50 breast cancer subtyping by RT-qPCR and concordance with standard clinical molecular markers.

BACKGROUND: Many methodologies have been used in research to identify the "intrinsic" subtypes of breast cancer commonly known as Luminal A, Luminal B, HER2-Enriched (HER2-E) and Basal-like. The PAM50 gene set is often used for gene expression-based subtyping; however, surrogate subtyping using panels of immunohistochemical (IHC) markers are still widely used clinically. Discrepancies between these methods may lead to different treatment decisions. METHODS: We used the PAM50 RT-qPCR assay to expression profile 814 tumors from the GEICAM/9906 phase III clinical trial that enrolled women with locally advanced primary invasive breast cancer. All samples were scored at a single site by IHC for estrogen receptor (ER), progesterone receptor (PR), and Her2/neu (HER2) protein expression. Equivocal HER2 cases were confirmed by chromogenic in situ hybridization (CISH). Single gene scores by IHC/CISH were compared with RT-qPCR continuous gene expression values and "intrinsic" subtype assignment by the PAM50. High, medium, and low expression for ESR1, PGR, ERBB2, and proliferation were selected using quartile cut-points from the continuous RT-qPCR data across the PAM50 subtype assignments. RESULTS: ESR1, PGR, and ERBB2 gene expression had high agreement with established binary IHC cut-points (area under the curve (AUC) ≥ 0.9). Estrogen receptor positivity by IHC was strongly associated with Luminal (A and B) subtypes (92%), but only 75% of ER negative tumors were classified into the HER2-E and Basal-like subtypes. Luminal A tumors more frequently expressed PR than Luminal B (94% vs 74%) and Luminal A tumors were less likely to have high proliferation (11% vs 77%). Seventy-seven percent (30/39) of ER-/HER2+ tumors by IHC were classified as the HER2-E subtype. Triple negative tumors were mainly comprised of Basal-like (57%) and HER2-E (30%) subtypes. Single gene scoring for ESR1, PGR, and ERBB2 was more prognostic than the corresponding IHC markers as shown in a multivariate analysis. CONCLUSIONS: The standard immunohistochemical panel for breast cancer (ER, PR, and HER2) does not adequately identify the PAM50 gene expression subtypes. Although there is high agreement between biomarker scoring by protein immunohistochemistry and gene expression, the gene expression determinations for ESR1 and ERBB2 status was more prognostic.

Agreement in risk prediction between the 21-gene recurrence score assay (Oncotype DX®) and the PAM50 breast cancer intrinsic Classifier™ in early-stage estrogen receptor-positive breast cancer.

PURPOSE: To compare risk assignment by PAM50 Breast Cancer Intrinsic Classifier™ and Oncotype DX_Recurrence Score (RS) in the same population. METHODS: RNA was extracted from 151 estrogen receptor (ER)+ stage I-II breast cancers and gene expression profiled using PAM50 "intrinsic" subtyping test. RESULTS: One hundred eight cases had complete molecular information; 103 (95%) were classified as luminal A (n = 76) or luminal B (n = 27). Ninety-two percent (n = 98) had a low (n = 59) or intermediate (n = 39) RS. Among luminal A cancers, 70% had low (n = 53) and the remainder (n = 23) had an intermediate RS. Among luminal B cancers, nine were high (33%) and 13 were intermediate (48%) by the RS. Almost all cancers with a high RS were classified as luminal B (90%, n = 9). One high RS cancer was identified as basal-like and had low ER/ESR1 and low human epidermal growth factor receptor 2 (HER2) expression by quantitative polymerase chain reaction in both assays. The majority of low RS cases were luminal A (83%, n = 53). Importantly, half of the intermediate RS cancers were re-categorized as low risk luminal A subtype by PAM50. CONCLUSION: There is good agreement between the two assays for high (i.e., luminal B or RS > 31) and low (i.e., luminal B or RS < 18) prognostic risk assignment but PAM50 assigns more patients to the low risk category. About half of the intermediate RS group was reclassified as luminal A by PAM50.

Purification and partial characterization of vitellogenin from shorthead redhorse (Moxostoma macrolepidotum) and copper redhorse (Moxostoma hubbsi) and detection in plasma and mucus with a heterologous antibody.

Vitellogenin (VTG), the egg yolk precursor protein, was purified from plasma of estradiol-3-benzoate (E2B)-treated male shorthead redhorse (Moxostoma macrolepidotum) and immature copper redhorse (Moxostoma hubbsi) by a two-step chromatographic procedure without precipitation. Intact VTGs appeared as dimers with apparent molecular masses, determined by gel filtration, of approximately 425 kDa (copper redhorse) and approximately 450 kDa (shorthead redhorse). In native polyacrylamide gel electrophoresis (PAGE), dimeric redhorse VTGs appeared as a 520 kDa band. Both VTGs were reduced to a single monomer of approximately 150 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and nonreducing conditions, indicating that monomers are not linked by disulfide bonds in the dimer form. The purified proteins were characterized as phospholipoglycoproteins. Isoelectric focusing of both VTGs revealed components with isoelectric points ranging from 5.3 to 6.0, suggesting charge heterogeneity. The amino acid composition of both VTGs contains a high proportion of nonpolar amino acids and was similar to those of other teleosts. An antibody developed against carp (Cyprinus carpio) VTG showed cross-reactivity with VTG from both redhorse species. Using this antibody, VTG was detected in plasma and surface mucus of E2B-treated redhorse. This is the most extensive report on purification and characterization of vitellogenin from catostomidid species.

Synaptotagmin-1 utilizes membrane bending and SNARE binding to drive fusion pore expansion.

In regulated vesicle exocytosis, SNARE protein complexes drive membrane fusion to connect the vesicle lumen with the extracellular space. The triggering of fusion pore formation by Ca(2+) is mediated by specific isoforms of synaptotagmin (Syt), which employ both SNARE complex and membrane binding. Ca(2+) also promotes fusion pore expansion and Syts have been implicated in this process but the mechanisms involved are unclear. We determined the role of Ca(2+)-dependent Syt-effector interactions in fusion pore expansion by expressing Syt-1 mutants selectively altered in Ca(2+)-dependent SNARE binding or in Ca(2+)-dependent membrane insertion in PC12 cells that lack vesicle Syts. The release of different-sized fluorescent peptide-EGFP vesicle cargo or the vesicle capture of different-sized external fluorescent probes was used to assess the extent of fusion pore dilation. We found that PC12 cells expressing partial loss-of-function Syt-1 mutants impaired in Ca(2+)-dependent SNARE binding exhibited reduced fusion pore opening probabilities and reduced fusion pore expansion. Cells with gain-of-function Syt-1 mutants for Ca(2+)-dependent membrane insertion exhibited normal fusion pore opening probabilities but the fusion pores dilated extensively. The results indicate that Syt-1 uses both Ca(2+)-dependent membrane insertion and SNARE binding to drive fusion pore expansion.

Role of phosphoinositide signaling in the control of insulin exocytosis.

Phosphoinositides (PI) are important signaling molecules involved in the regulation of vesicular trafficking. We found that phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-biphosphate [PI(4,5)P(2)] increase the secretory response triggered by 10 mum Ca(2+) in streptolysin-O-permeabilized insulin-secreting INS-1E cells. In addition, nutrient-induced exocytosis was diminished in intact cells expressing constructs that sequester PI(4,5)P(2) and in cells transfected with constructs that reduce by RNA interference the level of two enzymes involved in PI(4,5)P(2) production, type III PI4-kinase beta and type I phosphatidylinositol 4-bisphosphate 5-kinase-gamma. To clarify the mechanism of action of PI, we investigated the involvement in the regulation of insulin exocytosis of three potential PI targets, phospholipase D1, the Ca(2+)-dependent activator protein for secretion 1, and Munc18-interacting protein 1. Transfection of insulin-secreting cells with plasmids that direct the synthesis of small interfering RNAs capable of reducing the endogenous levels of these proteins inhibited hormone release elicited by glucose- and cAMP-elevating agents without affecting basal release. Our data indicate that the production of PI(4,5)P(2) is necessary for proper control of beta-cell secretion and suggest that at least part of the effect of PI on insulin exocytosis could be exerted through the activation of phospholipase D1, Ca(2+)-dependent activator protein for secretion 1, and Munc18-interacting protein 1.

CAPS acts at a prefusion step in dense-core vesicle exocytosis as a PIP2 binding protein.

CAPS-1 is required for Ca2+-triggered fusion of dense-core vesicles with the plasma membrane, but its site of action and mechanism are unknown. We analyzed the kinetics of Ca2+-triggered exocytosis reconstituted in permeable PC12 cells. CAPS-1 increased the initial rate of Ca2+-triggered vesicle exocytosis by acting at a rate-limiting, Ca2+-dependent prefusion step. CAPS-1 activity depended upon prior ATP-dependent priming during which PIP2 synthesis occurs. CAPS-1 activity and binding to the plasma membrane depended upon PIP2. Ca2+ was ineffective in triggering vesicle fusion in the absence of CAPS-1 but instead promoted desensitization to CAPS-1 resulting from decreased plasma membrane PIP2. We conclude that CAPS-1 functions following ATP-dependent priming as a PIP2 binding protein to enhance Ca2+-dependent DCV exocytosis. Essential prefusion steps in dense-core vesicle exocytosis involve sequential ATP-dependent synthesis of PIP2 and the subsequent PIP2-dependent action of CAPS-1. Regulation of PIP2 levels and CAPS-1 activity would control the secretion of neuropeptides and monoaminergic transmitters.