The University of Pennsylvania glioblastoma (UPenn-GBM) cohort: advanced MRI, clinical, genomics, & radiomics.

Glioblastoma is the most common aggressive adult brain tumor. Numerous studies have reported results from either private institutional data or publicly available datasets. However, current public datasets are limited in terms of: a) number of subjects, b) lack of consistent acquisition protocol, c) data quality, or d) accompanying clinical, demographic, and molecular information. Toward alleviating these limitations, we contribute the "University of Pennsylvania Glioblastoma Imaging, Genomics, and Radiomics" (UPenn-GBM) dataset, which describes the currently largest publicly available comprehensive collection of 630 patients diagnosed with de novo glioblastoma. The UPenn-GBM dataset includes (a) advanced multi-parametric magnetic resonance imaging scans acquired during routine clinical practice, at the University of Pennsylvania Health System, (b) accompanying clinical, demographic, and molecular information, (d) perfusion and diffusion derivative volumes, (e) computationally-derived and manually-revised expert annotations of tumor sub-regions, as well as (f) quantitative imaging (also known as radiomic) features corresponding to each of these regions. This collection describes our contribution towards repeatable, reproducible, and comparative quantitative studies leading to new predictive, prognostic, and diagnostic assessments.

Molecular Neuropathology in Practice: Clinical Profiling and Integrative Analysis of Molecular Alterations in Glioblastoma.

Molecular profiling of glioblastoma has revealed complex cytogenetic, epigenetic, and molecular abnormalities that are necessary for diagnosis, prognosis, and treatment. Our neuro-oncology group has developed a data-driven, institutional consensus guideline for efficient and optimal workup of glioblastomas based on our routine performance of molecular testing. We describe our institution's testing algorithm, assay development, and genetic findings in glioblastoma, to illustrate current practices and challenges in neuropathology related to molecular and genetic testing. We have found that coordination of test requisition, tissue handling, and incorporation of results into the final pathologic diagnosis by the neuropathologist improve patient care. Here, we present analysis of O6-methylguanine-DNA-methyltransferase promoter methylation and next-generation sequencing results of 189 patients, obtained utilizing our internal processes led by the neuropathology team. Our institutional pathway for neuropathologist-driven molecular testing has streamlined the management of glioblastoma samples for efficient return of results for incorporation of genomic data into the pathological diagnosis and optimal patient care.

Clonal Selection with RAS Pathway Activation Mediates Secondary Clinical Resistance to Selective FLT3 Inhibition in Acute Myeloid Leukemia.

Gilteritinib is a potent and selective FLT3 kinase inhibitor with single-agent clinical efficacy in relapsed/refractory FLT3-mutated acute myeloid leukemia (AML). In this context, however, gilteritinib is not curative, and response duration is limited by the development of secondary resistance. To evaluate resistance mechanisms, we analyzed baseline and progression samples from patients treated on clinical trials of gilteritinib. Targeted next-generation sequencing at the time of AML progression on gilteritinib identified treatment-emergent mutations that activate RAS/MAPK pathway signaling, most commonly in NRAS or KRAS. Less frequently, secondary FLT3-F691L gatekeeper mutations or BCR-ABL1 fusions were identified at progression. Single-cell targeted DNA sequencing revealed diverse patterns of clonal selection and evolution in response to FLT3 inhibition, including the emergence of RAS mutations in FLT3-mutated subclones, the expansion of alternative wild-type FLT3 subclones, or both patterns simultaneously. These data illustrate dynamic and complex changes in clonal architecture underlying response and resistance to mutation-selective tyrosine kinase inhibitor therapy in AML. SIGNIFICANCE: Comprehensive serial genotyping of AML specimens from patients treated with the selective FLT3 inhibitor gilteritinib demonstrates that complex, heterogeneous patterns of clonal selection and evolution mediate clinical resistance to tyrosine kinase inhibition in FLT3-mutated AML. Our data support the development of combinatorial targeted therapeutic approaches for advanced AML.See related commentary by Wei and Roberts, p. 998.This article is highlighted in the In This Issue feature, p. 983.

Analytical Validation of a Highly Sensitive, Multiplexed Chronic Myeloid Leukemia Monitoring System Targeting BCR-ABL1 RNA.

This study describes the analytical performance of the QuantideX qPCR BCR-ABL IS Kit, the first Food and Drug Administration-cleared assay designed to monitor breakpoint cluster region-Abelson tyrosine-protein kinase 1 (BCR-ABL1) fusion transcripts isolated from peripheral blood specimens from patients with chronic myeloid leukemia. This multiplex real-time quantitative RT-PCR assay amplifies both e13a2 and e14a2 Major BCR-ABL1 transcripts and the reference target ABL1. The test results are provided in international scale (IS) values by incorporating armored RNA-based calibrators that have defined IS values tied directly to the World Health Organization BCR-ABL1 Primary Reference Materials, without the necessity of determining and maintaining conversion factors. For each batch run, the integrated interpretive software evaluates run and specimen quality control metrics (including a sufficient amount of ABL1 control transcripts to ensure a minimal limit of detection) and calculates both molecular response (MR) and %IS values for each specimen. The test has a limit of detection of MR4.7 (0.002%IS) and a linear range from MR0.3 (50%IS) to MR4.7 (0.002%IS) for both Major transcripts. Single-site and multisite precision studies demonstrated a maximum SD of 0.13 MR (30% CV within the assay range between MR0.7 and MR3.7). The performance of this BCR-ABL1 monitoring test meets all of the clinical guideline recommendations for sensitivity and IS reporting for the management of chronic myeloid leukemia patients.

Marginal zone lymphoma-derived interfollicular diffuse large B-cell lymphoma harboring 20q12 chromosomal deletion and missense mutation of BIRC3 gene: a case report.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) typically leads to effacement of the nodal architecture by an infiltrate of malignant cells. Rarely (<1%), DLBCL can present with an interfollicular pattern (DLBCL-IF) preserving the lymphoid follicles. It has been postulated that DLBCL-IF is derived from marginal zone B cells and may represent a large-cell transformation of marginal zone lymphoma (MZL), however no direct evidence has been provided to date. Here we describe a rare case of a diagnostically challenging DLBCL-IF involving a lymph node in a patient with a prior history of lymphadenopathy for several years and MZL involving skin. CASE PRESENTATION: A 53-year old man presented to our Dermatology Clinic due to a 1-year history of generalized itching, fatigue of 2-3 month's duration, nausea and mid back rash that was biopsied. PET (positron emission tomography)/CT (computed tomography) was performed and revealed inguinal, pelvic, retroperitoneal, axillary, and cervical lymphadenopathy. The patient was referred to surgery for excisional biopsy of a right inguinal lymph node. Diagnostic H&E stained slides and ancillary studies were reviewed for the lymph node and skin specimens. B-cell clonality by PCR and sequencing studies were performed on both specimens. We demonstrate that this patient's MZL and DLBCL-IF are clonally related, strongly suggesting that transformation of MZL to DLBCL had occurred. Furthermore, we identified a novel deletion of the long arm of chromosome 20 (del(20q12)) and a missense mutation in BIRC3 (Baculoviral IAP repeat-containing protein 3) in this patient's DLBCL that are absent from his MZL, suggesting that these genetic alterations contributed to the large cell transformation. CONCLUSIONS: To our knowledge, this is the first report providing molecular evidence for a previously suspected link between MZL and DLBCL-IF. In addition, we describe for the first time del(20q12) and a missense mutation in BIRC3 in DLBCL. Our findings also raise awareness of DLBCL-IF and discuss the diagnostic pitfalls of this rare entity.

Building a Robust Tumor Profiling Program: Synergy between Next-Generation Sequencing and Targeted Single-Gene Testing.

Next-generation sequencing (NGS) is a powerful platform for identifying cancer mutations. Routine clinical adoption of NGS requires optimized quality control metrics to ensure accurate results. To assess the robustness of our clinical NGS pipeline, we analyzed the results of 304 solid tumor and hematologic malignancy specimens tested simultaneously by NGS and one or more targeted single-gene tests (EGFR, KRAS, BRAF, NPM1, FLT3, and JAK2). For samples that passed our validated tumor percentage and DNA quality and quantity thresholds, there was perfect concordance between NGS and targeted single-gene tests with the exception of two FLT3 internal tandem duplications that fell below the stringent pre-established reporting threshold but were readily detected by manual inspection. In addition, NGS identified clinically significant mutations not covered by single-gene tests. These findings confirm NGS as a reliable platform for routine clinical use when appropriate quality control metrics, such as tumor percentage and DNA quality cutoffs, are in place. Based on our findings, we suggest a simple workflow that should facilitate adoption of clinical oncologic NGS services at other institutions.

Multifocal fibrosing thyroiditis and its association with papillary thyroid carcinoma using BRAF pyrosequencing.

Multifocal fibrosing thyroiditis (MFT) is characterized by numerous foci of fibrosis in a stellate configuration with fibroelastotic and fibroblastic centers entrapping epithelial structures. MFT has been proposed as a risk factor for papillary thyroid carcinoma (PTC) development. We attempted to identify whether MFT showed such molecular changes and could possibly be related to PTC. We identified seven cases of PTC with MFT in our institutional pathology database and personal consult service of one of the authors (VAL) for the years 1999 to 2012. Areas of PTC, MFT, and normal tissue were selected for BRAF analysis. Macro-dissection, DNA extraction and PCR amplification, and pyrosequencing were performed to detect BRAF mutations in codon 600. All of the MFT lesions and normal thyroid tissue were negative for BRAF mutations. Of the seven PTCs analyzed, five (71 %) were negative for BRAF mutations, while two cases were positive. In our study, none of the MFT lesions harbored BRAF mutations, whereas 29 % (two of seven) PTCs in the same gland were positive. Hence, in this small study, we found no evidence that the MFT lesion is a direct precursor to PTC. It is likely an incidental bystander in the process and a reflection of the background thyroiditis.

Determinants of survival in advanced non--small-cell lung cancer in the era of targeted therapies.

BACKGROUND: Molecular profiling of non-small-cell lung cancer (NSCLC) samples has a profound impact on choice of therapy. However, it is less clear whether EGFR and KRAS mutations are prognostic outside of a trial-based treatment paradigm. METHODS: We performed a retrospective chart review of 513 patients with NSCLC undergoing EGFR and KRAS mutational analysis at the Hospital of the University of Pennsylvania between May 2008 and November 2011. Survival analysis was based on the 376 patients who received systemic treatment, and their survival was determined from the date of initiation of systemic therapy. RESULTS: The median overall survival (OS) was 30.8 months (95% confidence interval [CI], 24.7-36.9). Neither EGFR mutational status (P = .09) nor KRAS mutational status (0.69) was associated with OS. Female sex (P < .001), never smoker status (P = .01), better performance status (PS) (P < .001), lower Charlson Comorbidity Index (P < .001), and lower age-weighted index (P < .001) were associated with prolonged survival. The presence of bone metastases (P = .001) and liver metastases (P = .004) was also associated with a shortened survival. In a multivariable regression that adjusted for stage, we demonstrated that male gender (P = .002), worse Eastern Cooperative Oncology Group PS (P = .01), metastases to bone (P = .03), and higher age-weighted comorbidity index (P = .001) were independent prognostic factors for shorter survival. EGFR mutation status was not prognostic (P = .85). CONCLUSION: In our series, EGFR and KRAS do not function as prognostic determinants for NSCLC.

Frequency of EGFR and KRAS mutations in patients with non small cell lung cancer by racial background: do disparities exist?

INTRODUCTION: Mutations in EGFR and KRAS can impact treatment decisions for patients with NSCLC. The incidence of these mutations varies, and it is unclear whether there is a decreased frequency among African Americans (AfAs). METHODS: We performed a retrospective chart review of 513 NSCLC patients undergoing EGFR and KRAS mutational analysis at the Hospital of the University of Pennsylvania between May 2008 and November 2011. Clinical and pathologic data were abstracted from the patients' electronic medical record. RESULTS: Of 497 patients with informative EGFR mutation analyses, the frequency of EGFR mutation was 13.9%. The frequency of EGFR mutations was associated with race (p < 0.001) and was lower in AfA patients compared to Caucasian (C) patients but did not reach statistical significance (4.8% vs. 13.7%, p = 0.06). Mean Charlson Comorbidity Index and number of cigarette pack years were significantly lower in patients with EGFR mutations (p = 0.01 and p < 0.001, respectively). Multivariable logistic regression analysis showed a significant association between race and EGFR mutation (p = 0.01), even after adjusting for smoking status (p < 0.001) and gender (p = 0.03). KRAS mutation (study frequency 28.1%) was not associated with race (p = 0.08; p=0.51 for Afa vs. C patients), but was more common among smokers (p < 0.001) and females (p = 0.01). CONCLUSIONS: Based on multivariable analysis, even after adjusting for smoking status and gender, we found that race was statistically significantly associated with EGFR mutation, but not KRAS mutational status. To the best of our knowledge, this is the largest single institution series to date evaluating racial differences in EGFR and KRAS mutational status among patients with NSCLC.

A comparative analysis of molecular genetic and conventional cytogenetic detection of diagnostically important translocations in more than 400 cases of acute leukemia, highlighting the frequency of false-negative conventional cytogenetics.

In this study, we correlated the results of concurrent molecular and cytogenetic detection of entity-defining translocations in adults with acute leukemia to determine the frequency of cryptic translocations missed by conventional cytogenetics (CC) and of recurrent, prognostically relevant translocations not detectable by multiplex reverse transcriptase-polymerase chain reaction (MRP). During a 5.5-year period, 442 diagnostic acute leukemia specimens were submitted for MRP-based detection of 7 common recurrent translocations: t(8;21), t(15;17), inv(16), t(9;22), t(12;21), t(4;11), and t(1;19), with a detection rate of 15.2% (67/442). CC was performed in 330 (74.7%) of 442 cases. In 7 of these 330 cases, CC missed the translocation detected by MRP. In 50 additional cases, CC revealed 1 of the MRP-detectable translocations (all were also MRP positive), yielding a false-negative rate of 12% (7/57) for the CC assay. The remaining 140 of 190 cases with clonal cytogenetic changes harbored abnormalities that were not targeted by the MRP assay, including 8 that define specific acute myeloid leukemia entities. This study revealed the frequent occurrence of false-negative, entity-defining CC analysis and highlighted the complementary nature of MRP and CC approaches in detecting genetic abnormalities in acute leukemia.

Molecular diagnosis of acute myeloid leukemia.

The diagnosis and classification of acute myeloid leukemia is multifaceted, requiring the integration of a variety of laboratory findings, with genetic approaches now firmly established as a central component. Molecular genetic technologies continue to evolve and provide additional tiers of both clarity and complexity. Many have rapidly moved into clinical laboratories; others remain as relevant discovery tools, while some are poised to take their place in diagnostic testing menus. Here, we attempt to synthesize the role of various testing modalities and exciting nascent fundamental discoveries, with a view as to how these might be integrated into the contemporary and future evaluation of this group of aggressive hematologic malignancies.

Cloning and characterization of a novel MyoD enhancer-binding factor.

Glucocorticoid-induced gene-1 (Gig1) was identified in a yeast one-hybrid screen for factors that interact with the MyoD core enhancer. The Gig1 gene encodes a novel C2H2 zinc finger protein that shares a high degree of sequence similarity with two known DNA binding proteins in humans, Glut4 enhancer factor and papillomavirus binding factor (PBF). The mouse ortholog of PBF was also isolated in the screen. The DNA binding domain of Gig1, which contains TCF-E-tail CR1 and CR2 motifs shown to mediate promoter specificity of TCF-E-tail isoforms, was mapped to a C-terminal domain that is highly conserved in Glut4 enhancer factor and PBF. In mouse embryos, in situ hybridization revealed a restricted pattern of expression of Gig1 that overlaps with MyoD expression. A nuclear-localized lacZ knockin null allele of Gig1 was produced to study Gig1 expression with greater resolution and to assess Gig1 functions. X-gal staining of Gig1(nlacZ) heterozygous embryos revealed Gig1 expression in myotomal myocytes, skeletal muscle precursors in the limb, and in nascent muscle fibers of the body wall, head and neck, and limbs through E14.5 (latest stage examined). Gig1 was also expressed in a subset of Scleraxis-positive tendon precursors/rudiments of the limbs, but not in the earliest tendon precursors of the somite (syndetome) defined by Scleraxis expression. Additional regions of Gig1 expression included the apical ectodermal ridge, neural tube roof plate and floor plate, apparent motor neurons in the ventral neural tube, otic vesicles, notochord, and several other tissues representing all three germ layers. Gig1 expression was particularly well represented in epithelial tissues and in a number of cells/tissues of neural crest origin. Expression of both the endogenous MyoD gene and a reporter gene driven by MyoD regulatory elements was similar in wild-type and homozygous null Gig1(nlacZ) embryos, and mutant mice were viable and fertile, indicating that the functions of Gig1 are redundant with other factors.

Educating future leaders of medical research: analysis of student opinions and goals from the MD-PhD SAGE (Students' Attitudes, Goals, and Education) survey.

PURPOSE: To collect national trainee-derived data about the educational process and experience of MD-PhD students. METHOD: Eight hundred sixty-eight MD-PhD students enrolled in 15 training programs nationally were surveyed in spring 2003 via a 29-item Web-based questionnaire. Closed-ended questions assessed students' opinions, attitudes, and goals concerning education and future careers, as well as demographic characteristics. Programs were categorized by size; students were categorized by stage of training. Statistical analyses included chi-square tests and ANOVAs. RESULTS: From 13 institutions, 492 questionnaires were received, for a 57.6% response rate. Generally, satisfaction with overall education was highest at the beginning of training, lowest during graduate school, and rebounded after the PhD. Students at smaller programs were less satisfied with information received regarding future residency/career choices and coordination between training stages. Later-stage students showed greater satisfaction in obtaining the philosophical goals of their education and a different array of clinical interests than early-stage students. A majority of students chose research as their future primary activity, academic centers as their primary practice setting, and disease oriented as their future primary research activity. Many students did not agree with the current working definition of a physician-scientist. CONCLUSIONS: Findings indicate that students have clinical interests that develop over time, a variety of future career goals, and ambivalence concerning the appropriate balance of clinical and research activities. This information may provide a basis for future improvements in the education of MD-PhDs in the United States and abroad.

Educational views and attitudes, and career goals of MD-PhD students at the University of Pennsylvania School of Medicine.

PURPOSE: Despite the documented contribution of graduates of MD-PhD programs to the medical profession, few data exist concerning the views, attitudes, and career goals of students before they graduate from such programs. METHOD: All 167 students enrolled in the University of Pennsylvania School of Medicine Combined Degree Program in the spring of 2002 were invited to participate in an IRB-approved online questionnaire consisting of 81 multiple-choice questions covering students' demographics, satisfaction with their educational experience, future goals and career aspirations, and attitudes and views concerning the physician-scientist model. RESULTS: The 96 MD-PhD students (57.5%) who completed the questionnaire represented a diverse group with individuals from every stage of training. The majority of students were satisfied with their overall educational experience (90.5%). Although students reported an interest in a wide range of clinical specialties, 84.4% indicated plans to pursue a career in research and 79.2% preferred a position at an academic medical center. However, a larger percentage of males (70.7%) than females (50.0%) listed research as their primary professional activity. The range of students' views and attitudes regarding the physician-scientist model suggests that additional education and discussion are warranted. CONCLUSION: The MD-PhD students surveyed at the University of Pennsylvania were satisfied with their education and most were planning research-oriented careers. Yet, the aspirations, views and concerns of individual MD-PhD students were varied. The authors believe this information in aggregate will prove useful to current and future students, combined degree programs, policymakers, and residency directors.

MD-PhD students in a major training program show strong interest in becoming surgeon-scientists.

A wide spectrum of individuals have discussed the importance of promoting research in orthopaedics and of developing clinician-scientists (physicians who also do significant research) in the field. Although orthopaedic research may benefit from recruitment of MD-PhD students as clinician-scientists, it is unclear to what extent MD-PhD students are interested in pursuing research and surgical specialties concurrently. To better understand their professional goals, all MD-PhD students enrolled in our institution's training program were invited to complete an online questionnaire concerning training satisfaction and future career goals. Twenty-four percent of respondents (57.5% response rate of 167 recruits) reported a primary clinical interest in a surgical field (3% interest in orthopaedics); interest was strongest late in training. The majority of surgical MD-PhD students, like nonsurgical students, were planning to make research a significant part of their careers. In addition, students identified the importance of factors such as family issues and faculty role models in determining their clinical interests. The study data indicate that MD-PhD students have strong interests in becoming surgical clinician-scientists. They also suggested that active recruitment (especially early in training) that is responsive to the personal and professional needs of students has the potential to increase the number of clinician-scientists in orthopaedics.