Comparison of 2 T-Cell Receptor-γ Clonality Assays on Skin Biopsies Suspicious for Mycosis Fungoides.

ABSTRACT: PCR-based fragment analysis of the T-cell receptor (TCR) gene is used extensively in diagnostic labs to assess clonality in T-cell populations in multiple tissue sites. Of the numerous TCR assays that have been reported, studies assessing use on biopsies suspicious for mycosis fungoides specifically are lacking. We compared clonality findings from a previously run 2-tube/2-fluorochrome dye assay to a redesigned 1-tube/1-fluorochrome dye assay on formalin-fixed skin biopsies. Overall, the accuracy of the 2-tube assay was marginally better (75.7% vs. 71.4%), when using clinical history combined with histologic diagnosis as the gold standard. The 2-tube assay had better sensitivity (73.7% vs. 65.8%), while the 1-tube assay had superior specificity (93.8% vs. 87.5%). Clonality results were easier to interpret with the 1-tube assay. In nearly 19% of cases, a change of assays on the same biopsy resulted in a change of clonality interpretation. For laboratories that change TCR-γ clonality assays, follow-up biopsies for mycosis fungoides assessment may result in a change of diagnosis.

Serum D-2-hydroxyglutarate and the ratio of D-2HG/L-2HG predict IDH mutation in acute myeloid leukemia.

This study investigates whether serum D-2HG (D-2-hydroxyglutarate) produced by the mutated isocitrate dehydrogenase (IDH) can predict IDH mutations in acute myeloid leukemia (AML) at diagnosis. D-2HG and L-2HG are measured by liquid chromatography-tandem mass spectrometry. D-2HG, total 2HG and the D/L ratio (D-2HG/L-2HG) are significantly higher in IDH mutated cases than in IDH wild cases. The optimal cutoff values to predict IDH mutations at 100% sensitivity (specificity 91%-94%) are >588 ng/mL for D-2HG and >2.33 for the D/L ratio. Our study indicates that elevated serum D-2HG and the D/L ratio may serve as noninvasive biomarkers of IDH mutation in AML.

Stepped Behavioral and Biological Screening for Oral Oncogenic HPV DNA in Middle-aged and Elderly Adults: A Feasibility Study.

Novel preventive interventions are needed to address the rising incidence of human papillomavirus (HPV)-mediated oropharyngeal cancer (HPV+ OPC). This pilot study evaluated the feasibility of a stepped, behavioral and biological screening program for oral oncogenic HPV infection, an intermediate HPV+ OPC outcome.This was a cross-sectional, feasibility study. Eligible 45-74 years old adults identified from three clinical research registries were administered a behavioral risk survey (step 1). Participant tobacco use and sexual behavior history were translated into a quantifiable risk of oral oncogenic HPV DNA, according to prior National Health and Nutrition Examination Survey analyses. Females with >2% risk and males with >7% risk were offered biological screening for oral oncogenic HPV DNA (step 2) via an oral rinse and gargle specimen.A total of 292 individuals were contacted, but only 144 (49%) were reached. Among these, 56 individuals (19%) were uninterested and 18 (13%) were ineligible. Seventy individuals began the survey and 66 completed it (step 1), among whom 46 were classified as low-risk. Among the remaining 20 participants classified as high-risk for an oral oncogenic HPV infection, 5% were current smokers and the median participant had performed oral sex on 10 unique partners. During step 2 (biological screening), 45% (9/20) completed testing, all of whom tested negative for oral oncogenic HPV DNA.In this pilot of a stepped, oral oncogenic HPV screening program, enrollment and study completion were suboptimal. These barriers to screening should be characterized and addressed before reevaluating the feasibility of this program. PREVENTION RELEVANCE: Novel preventive interventions are needed to address the rising incidence of HPV+ OPC. In this feasibility study, we characterized barriers to a two-step, behavioral and biological screening program for oral oncogenic HPV infection, an intermediate outcome for HPV+ OPC.

Lenalidomide-Associated Secondary B-Lymphoblastic Leukemia/Lymphoma-A Unique Entity.

OBJECTIVES: Autologous stem cell transplant with lenalidomide maintenance therapy has greatly improved the relapse-free and overall survival rates of patients with multiple myeloma but also has been associated with an increased risk of secondary B-lymphoblastic leukemia/lymphoma (B-ALL). METHODS: We report a comprehensive review of the clinicopathologic features of 2 patients with multiple myeloma who developed secondary B-ALL during lenalidomide maintenance. RESULTS: Our observations showed that the disease may initially present with subtle clinical, morphologic, and flow-cytometric findings. The flow cytometry findings in such cases may initially mimic an expansion of hematogones with minimal immunophenotypic variation. Both patients achieved complete remission of secondary B-ALL after standard chemotherapy; however, one patient continues to have minimal residual disease, and the other experienced relapse. Next-generation sequencing of the relapse specimen showed numerous, complex abnormalities, suggesting clonal evolution. CONCLUSIONS: Our findings suggest the need for increased awareness and further study of this unique form of secondary B-ALL.

Transcriptionally Active HPV and Targetable EGFR Mutations in Sinonasal Inverted Papilloma: An Association Between Low-risk HPV, Condylomatous Morphology, and Cancer Risk?

Sinonasal inverted papillomas (IPs) commonly recur, and transform to malignancy in 5% to 10% of patients. It has long been debated whether IPs are caused by high-risk or low-risk (lr) human papillomavirus (HPV) and whether the HPV is transcriptionally active. EGFR mutations have also been recently implicated in the pathogenesis of IP with an unclear relationship to HPV status. IP cases over a 10-year period were tested for p16 by immunohistochemistry and for transcriptionally active hrHPV and lrHPV by reverse-transcriptase real-time polymerase chain reaction and RNA in situ hybridization, respectively. EGFR tyrosine kinase domain Sanger sequencing was performed on all lrHPV RNA positive and 15 randomly selected lrHPV RNA negative IPs. Seven sinonasal nonkeratinizing squamous cell carcinomas (SCCs) without associated IP were included as controls. Of the 44 IPs, 5 (11.4%) were associated with SCC, all keratinizing type. All IPs and associated SCCs were negative for p16 and hrHPV. lrHPV RNA was detected in 5/42 (12%) cases, including 3/5 (60%) with associated SCC (P=0.009). All 5 lrHPV RNA positive IPs involved the nasal cavity, had a distinct, condylomatous morphology, and were EGFR wild-type. In contrast, 11/15 (73.3%) lrHPV RNA negative IPs that were sequenced had EGFR exon 19 or 20 mutations. All control nonkeratinizing SCCs were lrHPV RNA negative, but 5/7 (71.4%) were p16 and high-risk HPV RNA positive. This study shows that a subset of IPs involving the nasal cavity have transcriptionally active lrHPV, condylomatous morphology, and possibly increased risk of malignancy. Furthermore, lrHPV positivity is mutually exclusive with EGFR mutations, which suggests alternate mechanisms of pathogenesis.

Does Tumor FDG-PET Avidity Represent Enhanced Glycolytic Metabolism in Non-Small Cell Lung Cancer?

BACKGROUND: In non-small cell lung cancer (NSCLC), 18fluoro-2-deoxyglucose-positron emission tomography (FDG-PET) assists in diagnosis, staging, and evaluating treatment response. One variable of FDG-PET, the maximum standard uptake value (SUVm), is considered an objective measure of glucose uptake. However, little is known about the fate of glucose in FDG-avid lung tumors in vivo. This study used stable glucose isotope tracing to determine whether the SUVm predicts glycolytic metabolism or other glucose fates in tumors. METHODS: In this prospective Institutional Review Board-approved clinical trial, 52 untreated potentially resectable confirmed NSCLC patients underwent FDG-PET computed tomography. During the surgical procedure, the patients were infused with 13C-labeled glucose. Blood, tumor, and normal lung samples were analyzed by mass spectrometry to determine 13C enrichment in glycolytic intermediates. These values were compared with clinical variables, including SUVm, maximum tumor diameter, stage, grade, and MIB-1/Ki67 proliferation index. RESULTS: For each patient, 13C enrichment in each metabolite was compared between tumor and adjacent lung. Although all tumors metabolized glucose, SUVm did not correlate with glycolytic intermediate labeling. Rather, SUVm correlated with markers indicating the use of other respiratory substrates, including lactate, and with the proliferation index. CONCLUSIONS: SUVm does not correlate with glycolytic metabolism in human NSCLC but does correlate with the proliferation index, suggesting that SUVm predicts glucose use by pathways other than glycolysis. These pathways may offer alternative therapeutic targets, including biosynthetic pathways required for cell proliferation. The research techniques in this study offer the opportunity to understand the relationships between SUVm, tumor metabolism, and therapeutic vulnerabilities in human NSCLCs.

Cytogenomic characterization of double minute heterogeneity in therapy related acute myeloid leukemia.

Breast cancer patients treated with adjuvant chemotherapy regimens containing alkylating agents and anthracyclines are at an increased risk for secondary myeloid malignancies, either acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Complex genomic changes (karyotypes and/or gene amplification) accompany the development of the secondary neoplasms. Here we present a unique case of a breast cancer patient who developed secondary AML within 18 months of treatment with trastuzumab, pertuzumab, docetaxel, carboplatin (TCHP) and radiation. Leukemia cells had catastrophic alterations in chromosomes 8, 11, and 17. Genetic abnormalities in the leukemia cells included amplification of MYC and KMT2A as double minutes, and deletion and mutational inactivation of TP53 Concurrent amplification of different genes at different levels and on different double minutes, we have named "double minute heterogeneity." Clinically, this case highlights the need to identify genes amplified in secondary myeloid malignancies by cytogenomic microarray (CMA) analysis since these may have therapeutic implications.

Genome-Wide Analysis of Glioblastoma Patients with Unexpectedly Long Survival.

Glioblastoma (GBM), representing WHO grade IV astrocytoma, is a relatively common primary brain tumor in adults with an exceptionally dismal prognosis. With an incidence rate of over 10 000 cases in the United States annually, the median survival rate ranges from 10-15 months in IDH1/2-wildtype tumors and 24-31 months in IDH1/2-mutant tumors, with further variation depending on factors such as age, MGMT methylation status, and treatment regimen. We present a cohort of 4 patients, aged 37-60 at initial diagnosis, with IDH1-mutant GBMs that were associated with unusually long survival intervals after the initial diagnosis, currently ranging from 90 to 154 months (all still alive). We applied genome-wide profiling with a methylation array (Illumina EPIC Array 850k) and a next-generation sequencing panel to screen for genetic and epigenetic alterations in these tumors. All 4 tumors demonstrated methylation patterns and genomic alterations consistent with GBM. Three out of four cases showed focal amplification of the CCND2 gene or gain of the region on 12p that included CCND2, suggesting that this may be a favorable prognostic factor in GBM. As this study has a limited sample size, further evaluation of patients with similar favorable outcome is warranted to validate these findings.

Spinal Pleomorphic Xanthoastrocytoma With a QKI-RAF1 Fusion.

Pleomorphic xanthoastrocytoma (PXA) is a slow-growing neoplasm that predominantly affects the pediatric and young adult population. This neoplasm has a good prognosis, with a median 10-year survival rate of 70%. The majority of tumors are supratentorial and arise in the temporal lobe, while spinal tumors are extremely rare, with only 8 reported cases. Molecular perturbations involving the MAPK/ERK signaling pathway have been described in PXAs. The most common mutation is BRAF V600E in 60%-80% of cases. Other mechanisms activating this pathway in the absence of this mutation are rare and include CRAF (RAF1) fusion genes. We report a PXA case in the cervical spinal cord of a 49-year-old man with slowly progressive coordination difficulties and extremity numbness. The tumor was negative for the V600E mutation, but 2 RNA sequencing platforms detected a QKI-RAF1 fusion (t(6; 3)(q26; p25)), which has not been previously reported in PXAs. This fusion is known to activate MAPK/ERK and PI3K/mTOR signaling. Although first- and second-generation RAF inhibitors are predicted to be ineffective, this fusion may be targetable by the novel RAF inhibitor LY3009120 and to some extent by the MEK inhibitor trametinib. Genetic analysis to screen for MAPK/ERK pathway mutations is warranted on PXAs negative for the V600E mutation.

BRAF mutation leading to central nervous system rosai-dorfman disease.

Rosai-Dorfman disease (RDD) is an uncommon histiocytic proliferative disorder that can present in nodal, extranodal, or, extremely rarely, in central nervous system (CNS)-restricted form. RDD is characterized histologically as a non-Langerhans cell histiocytosis composed of atypical CD68+ /S-100+ /CD1a- macrophages demonstrating prominent emperipolesis and effacement of the surrounding tissue. Previously thought to represent a reactive process, recent studies have raised the possibility that RDD and other histiocytic lesions, including Erdheim-Chester and Langerhans cell histiocytosis, are clonal processes linked to somatic mutations in the mitogen-activated protein (MAP) kinase pathway. Herein, we present a fatal case of RDD isolated to the CNS and used a next-generation targeted gene panel and Sanger sequencing to uncover a pathogenic deletion in the ß3-αC loop of the kinase domain in exon 12 of BRAF. This mutation, previously described in melanoma and Langerhans cell histiocytosis, represents the first BRAF mutation of this kind identified in RDD. These findings support the idea that RDD is a neoplastic condition and raise the possibility that inhibitors of the MAP kinase pathway may be effective in RDD. Ann Neurol 2018;83:147-152.

Lactate Metabolism in Human Lung Tumors.

Cancer cells consume glucose and secrete lactate in culture. It is unknown whether lactate contributes to energy metabolism in living tumors. We previously reported that human non-small-cell lung cancers (NSCLCs) oxidize glucose in the tricarboxylic acid (TCA) cycle. Here, we show that lactate is also a TCA cycle carbon source for NSCLC. In human NSCLC, evidence of lactate utilization was most apparent in tumors with high 18fluorodeoxyglucose uptake and aggressive oncological behavior. Infusing human NSCLC patients with 13C-lactate revealed extensive labeling of TCA cycle metabolites. In mice, deleting monocarboxylate transporter-1 (MCT1) from tumor cells eliminated lactate-dependent metabolite labeling, confirming tumor-cell-autonomous lactate uptake. Strikingly, directly comparing lactate and glucose metabolism in vivo indicated that lactate's contribution to the TCA cycle predominates. The data indicate that tumors, including bona fide human NSCLC, can use lactate as a fuel in vivo.

Rapid progression to glioblastoma in a subset of IDH-mutated astrocytomas: a genome-wide analysis.

According to the recently updated World Health Organization (WHO) classification (2016), grade II-III astrocytomas are divided into IDH-wildtype and IDH-mutant groups, the latter being significantly less aggressive in terms of both progression-free and total survival. We identified a small cohort of WHO grade II-III astrocytomas that harbored the IDH1 R132H mutation, as confirmed by both immunohistochemistry and molecular sequence analysis, which nonetheless had unexpectedly rapid recurrence and subsequent progression to glioblastoma. Among these four cases, the mean time to recurrence as glioblastoma was only 16 months and the mean total survival among the three patients who have died during the follow-up was only 31 months. We hypothesized that these tumors had other, unfavorable genetic or epigenetic alterations that negated the favorable effect of the IDH mutation. We applied genome-wide profiling with a methylation array (Illumina Infinium Human Methylation 450k) to screen for genetic and epigenetic alterations in these tumors. As expected, the methylation profiles of all four tumors were found to match most closely with IDH-mutant astrocytomas. Compared with a control group of four indolent, age-similar WHO grade II-III astrocytomas, the tumors showed markedly increased levels of overall copy number changes, but no consistent specific genetic alterations were seen across all of the tumors. While most IDH-mutant WHO grade II-III astrocytomas are relatively indolent, a subset may rapidly recur and progress to glioblastoma. The precise underlying cause of the increased aggressiveness in these gliomas remains unknown, although it may be associated with increased genomic instability.

Histologic transformation of EGFR mutant lung adenocarcinoma without exposure to EGFR inhibition.

Resistance to EGFR kinase inhibitors appears to be invariable in the treatment of non-small cell lung cancer. Several mechanisms have been described. Here, we report the first case of histologic transformation of EGFR mutant lung adenocarcinoma without prior exposure to EGFR inhibition.

Non-malignant respiratory epithelial cells preferentially proliferate from resected non-small cell lung cancer specimens cultured under conditionally reprogrammed conditions.

The "conditionally reprogrammed cells" (CRC) method, using a Rho kinase inhibitor and irradiated mouse fibroblast cells has been described for the efficient growth of cells from malignant and non-malignant samples from primary tumor and non-malignant sites. Using the CRC method, four institutions independently cultured tumor tissues from 48 non-small cell lung cancers (NSCLC, mostly from primary resected tumors) and 22 non-malignant lungs. We found that epithelial cells could be cultured from tumor and non-malignant lung. However, epithelial cells cultured from tumors had features of non-malignant respiratory epithelial cells which include: 1) among 22 mutations found in the original tumors only two mutations were found in the CRC cultures with reduced frequency (31% to 13% and 92% to 15% from original tumor and CRC culture respectively); 2) copy number variation was analyzed in 9 tumor and their CRC cultures and only diploid patterns were found in CRC cultures; 3) mRNA expression profiles were similar to those of normal respiratory epithelial cells; and 4) co-culture of tumor and non-malignant lung epithelial cells resulted in mostly non-malignant cells. We conclude that CRC method is a highly selective and useful method for the growth of non-malignant respiratory epithelial cells from tumor specimens and only occasionally do such CRC cultures contain a small subpopulation of cancer cells marked by oncogenic mutations. While our findings are restricted to resected primary NSCLC, they indicated the necessity to fully characterize all CRC cultures and the need to develop culture technology that facilitates the growth of primary lung cancers.

Potential pitfalls of mass spectrometry to uncover mutations in childhood soft tissue sarcoma: A report from the Children's Oncology Group.

Mass spectrometry-based methods have been widely applied - often as the sole method - to detect mutations in human cancer specimens. We applied this approach to 52 childhood soft tissue sarcoma specimens in an attempt to identify potentially actionable mutations. This analysis revealed that 25% of the specimens harbored high-confidence calls for mutated alleles, including a mutation encoding FLT3(I836M) that was called in four cases. Given the surprisingly high frequency and unusual nature of some of the mutant alleles, we carried out ultra-deep next generation sequencing to confirm them. We confirmed only three mutations, which encoded NRAS(A18T), JAK3(V722I) and MET(R970C) in three specimens. Beyond highlighting those mutations, our findings demonstrate potential pitfalls of primarily utilizing a mass spectrometry-based approach to broadly screen for DNA sequence variants in archived, clinical-grade tumor specimens. Duplicate mass spectrometric analyses and confirmatory next generation sequencing can help diminish false positive calls, but this does not ameliorate potential false negatives due in part to evaluating a limited panel of sequence variants.

Metabolic Heterogeneity in Human Lung Tumors.

Non-small cell lung cancer (NSCLC) is heterogeneous in the genetic and environmental parameters that influence cell metabolism in culture. Here, we assessed the impact of these factors on human NSCLC metabolism in vivo using intraoperative (13)C-glucose infusions in nine NSCLC patients to compare metabolism between tumors and benign lung. While enhanced glycolysis and glucose oxidation were common among these tumors, we observed evidence for oxidation of multiple nutrients in each of them, including lactate as a potential carbon source. Moreover, metabolically heterogeneous regions were identified within and between tumors, and surprisingly, our data suggested potential contributions of non-glucose nutrients in well-perfused tumor areas. Our findings not only demonstrate the heterogeneity in tumor metabolism in vivo but also highlight the strong influence of the microenvironment on this feature.

Prospective Longitudinal Analysis of 2-Hydroxyglutarate Magnetic Resonance Spectroscopy Identifies Broad Clinical Utility for the Management of Patients With IDH-Mutant Glioma.

Purpose Proton magnetic resonance spectroscopy (MRS) of the brain can detect 2-hydroxyglutarate (2HG), the oncometabolite produced in neoplasms harboring a mutation in the gene coding for isocitrate dehydrogenase ( IDH). We conducted a prospective longitudinal imaging study to determine whether quantitative assessment of 2HG by MRS could serve as a noninvasive clinical imaging biomarker for IDH-mutated gliomas. Patients and Methods 2HG MRS was performed in 136 patients using point-resolved spectroscopy at 3 T in parallel with standard clinical magnetic resonance imaging and assessment. Data were analyzed in patient cohorts representing the major phases of the glioma clinical course and were further subgrouped by histology and treatment type to evaluate 2HG. Histologic correlations were performed. Results Quantitative 2HG MRS was technically and biologically reproducible. 2HG concentration > 1 mM could be reliably detected with high confidence. During the period of indolent disease, 2HG concentration varied by less than ± 1 mM, and it increased sharply with tumor progression. 2HG concentration was positively correlated with tumor cellularity and significantly differed between high- and lower-grade gliomas. In response to cytotoxic therapy, 2HG concentration decreased rapidly in 1p/19q codeleted oligodendrogliomas and with a slower time course in astrocytomas and mixed gliomas. The magnitude and time course of the decrease in 2HG concentration and magnitude of the decrease in tumor volume did not differ between oligodendrogliomas treated with temozolomide or carmustine. Criteria for 2HG MRS were established to make a presumptive molecular diagnosis of an IDH mutation in gliomas technically unable to undergo a surgical procedure. Conclusion 2HG concentration as measured by MRS was reproducible and reliably reflected the disease state. These data provide a basis for incorporating 2HG MRS into clinical management of IDH-mutated gliomas.