Faculty Bibliography

Ewing sarcoma is characterized by multiple deregulated pathways that mediate cell survival and proliferation. Heat shock protein 90 (HSP90) is a critical component of the multi-chaperone complexes that regulate the disposition and activity of a large number of proteins involved in cell-signaling systems. We tested the efficacy of PU-H71, a novel HSP90 inhibitor in Ewing sarcoma cell lines, primary samples, benign mesenchymal stromal cells and hematopoietic stem cells. We performed cell cycle analysis, clonogenic assay, immunoblot analysis and reverse phase protein array in Ewing cell lines and in vivo experiments in NSG and nude mice using the A673 cell line. We noted a significant therapeutic window in the activity of PU-H71 against Ewing cell lines and benign cells. PU-H71 treatment resulted in G2/M phase arrest. Exposure to PU-H71 resulted in depletion of critical proteins including AKT, pERK, RAF-1, c-MYC, c-KIT, IGF1R, hTERT and EWS-FLI1 in Ewing cell lines. Our results indicated that Ewing sarcoma tumor growth and the metastatic burden were significantly reduced in the mice injected with PU-H71 compared to the control mice. We also investigated the effects of bortezomib, a proteasome inhibitor, alone and in combination with PU-H71 in Ewing sarcoma. Combination index (CI)-Fa plots and normalized isobolograms indicated synergism between PU-H71 and bortezomib. Ewing sarcoma xenografts were significantly inhibited when mice were treated with the combination compared to vehicle or either drug alone. This provides a strong rationale for clinical evaluation of PU-H71 alone and in combination with bortezomib in Ewing sarcoma.

The 2011 International Association for the Study of Lung Cancer, American Thoracic Society, and European Respiratory Society classification of pulmonary adenocarcinoma recognizes the prognostic significance of different histologic patterns but does not address the issue of tumor grade. We previously developed an objective and prognostic grading system for pulmonary adenocarcinomas that is based on associating patterns with their metastatic potential. The best prognostic stratification was achieved by summing the grades of the 2 most predominant patterns (histologic score). Here, we extend this work by evaluating the prognostic importance of variant patterns of adenocarcinoma, which are not recognized by the new International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society classification. Pathologic specimens from 249 resected stage I adenocarcinomas were reviewed. The proportions of standard and nonstandard patterns (cribriform and fused glands) were recorded for each case. The associations between the presence of standard and nonstandard patterns, tumor histologic score, and disease-free survival were evaluated. Cribriform and fused gland patterns were observed in 15% and 29% of tumors, respectively. These nonstandard patterns each composed 10% to 100% of the entire tumors but were the predominant pattern in only 5% and 7% of tumors, respectively. The presence of complex glandular patterns was associated with solid pattern (P < .001) and high histologic score (P < .0001). Disease-free survival for tumors with predominant complex glandular patterns was similar to that for high-grade tumors (P = .932) and was significantly worse than that for low- and intermediate-grade tumors (P = .0025). Complex glandular patterns have a significant prognostic value and should be considered patterns of high-grade adenocarcinoma.

Tunneling nanotubes (TnTs) are long, non-adherent, actin-based cellular extensions that act as conduits for transport of cellular cargo between connected cells. The mechanisms of nanotube formation and the effects of the tumor microenvironment and cellular signals on TnT formation are unknown. In the present study, we explored exosomes as potential mediators of TnT formation in mesothelioma and the potential relationship of lipid rafts to TnT formation. Mesothelioma cells co-cultured with exogenous mesothelioma-derived exosomes formed more TnTs than cells cultured without exosomes within 24-48 h; and this effect was most prominent in media conditions (low-serum, hyperglycemic medium) that support TnT formation (1.3-1.9-fold difference). Fluorescence and electron microscopy confirmed the purity of isolated exosomes and revealed that they localized predominantly at the base of and within TnTs, in addition to the extracellular environment. Time-lapse microscopic imaging demonstrated uptake of tumor exosomes by TnTs, which facilitated intercellular transfer of these exosomes between connected cells. Mesothelioma cells connected via TnTs were also significantly enriched for lipid rafts at nearly a 2-fold higher number compared with cells not connected by TnTs. Our findings provide supportive evidence of exosomes as potential chemotactic stimuli for TnT formation, and also lipid raft formation as a potential biomarker for TnT-forming cells.

INTRODUCTION: The optimal radiation therapy (RT) field design for thymomas remains unclear. Here we report the failure patterns in stage II-IV thymoma after RT at two tertiary referral centers, classified according to the new International Thymic Malignancy Interest Group definitions. METHODS: We reviewed 156 stage II-IV patients with thymoma treated with definitive (n=24) or adjuvant (n=132) RT. All RT was delivered without elective nodal irradiation (median dose 5040 cGy). Intrathoracic failures were classified as (1) in-field failures (within 100% isodose line [IDL]), (2) marginal recurrences (<100% and >/=50% IDL), and (3) out-of-field failures (outside the 50% IDL). RESULTS: The median follow-up was 61 months. Surgical margins were positive in 39%. The median tumor size was 9 cm. The 5-year cumulative incidence of all intrathoracic failures was 24% (n=34). Failures occurred within the RT field (n=5), marginally (n=1), out-of-field (n=22), and synchronously in- and out-of-field (n=6). The 5-year cumulative incidence of in-field failures was 7%. These were associated with Masaoka stage and tumor size. Macroscopically positive margins were associated with more local failures. Intrathoracic failures occurred most commonly in the pleural space (n=29) and lymph nodes (n=9). Patients with more advanced stage, and those treated with intensity-modulated radiation therapy had more intrathoracic failures. RT dose and chemotherapy did not impact failure patterns. CONCLUSIONS: Although there were few in-field failures in patients who received RT for stage II-IV thymomas, a high rate of out-of-field intrathoracic failures still occurred. Further study is necessary to identify treatment approaches that prevent pleural recurrences.

Multiple reports indicate that epidermal growth factor receptor (EGFR) mutations are associated with lepidic-pattern lung adenocarcinoma and that KRAS mutations are associated with invasive mucinous adenocarcinoma. We sought to investigate the association between EGFR and KRAS mutations and specific morphologic characteristics, such as predominant histologic subtype and mucinous features. Clinical data for 864 patients with resected lung adenocarcinoma that underwent molecular testing for EGFR and KRAS mutations were collected. Histologic subtyping was performed according to the IASLC/ATS/ERS lung adenocarcinoma classification, with attention given to signet-ring cell feature and extracellular mucin. EGFR mutations were detected using a polymerase chain reaction-based sizing assay, KRAS mutations were detected using Sanger sequencing, and ALK expression was detected using immunohistochemistry. Invasive mucinous adenocarcinoma was associated with KRAS mutation (P<0.001). Among invasive mucinous adenocarcinomas with KRAS mutation, a pure mucinous pattern was more common than a mixed mucinous/nonmucinous pattern (P=0.002). Invasive mucinous adenocarcinoma was associated with KRAS transition mutations (G-->A) but not transversion mutations (G-->T or G-->C) compared with nonmucinous tumors (P=0.009). The lepidic-predominant group was associated with EGFR mutation compared with nonlepidic-predominant tumors (P=0.011). Extracellular mucin was associated with KRAS mutation (P<0.001), whereas signet-ring cell feature was not associated with EGFR or KRAS mutation (P=0.517). ALK expression was associated with signet-ring cell feature (P=0.001) but not with extracellular mucin (P=0.089). Our study shows that histologic patterns of mucin in lung adenocarcinoma-including invasive mucinous adenocarcinoma and extracellular mucin-are associated with KRAS mutation.

INTRODUCTION: The 2004 World Health Organization classification of lung cancer contained three major forms of non-small-cell lung cancer: squamous cell carcinoma (SqCC), adenocarcinoma (AdC), and large cell carcinoma. The goal of this study was first, to assess the reproducibility of a set of histopathological features for SqCC in relation to other poorly differentiated non-small-cell lung cancers and second, to assess the value of immunohistochemistry in improving the diagnosis. METHODS: Resection specimens (n = 37) with SqCC, large cell carcinoma, basaloid carcinoma, sarcomatoid carcinoma, lymphoepithelial-like carcinoma, and solid AdC, were contributed by the participating pathologists. Hematoxylin and eosin (H&E) stained slides were digitized. The diagnoses were evaluated in two ways. First, the histological criteria were evaluated and the (differential) diagnosis on H&E alone was scored. Second, the added value of additional stains to make an integrated diagnosis was examined. RESULTS: The histologic criteria defining SqCC were consistently used, but in poorly differentiated cases they were infrequently present, rendering the diagnosis more difficult. Kappa scores on H&E alone were for SqCC 0.46, large cell carcinoma 0.25, basaloid carcinoma 0.27, sarcomatoid carcinoma 0.52, lymphoepithelial-like carcinoma 0.56, and solid AdC 0.21. The kappa score improved with the use of additional stains for SqCC (combined with basaloid carcinoma) to 0.57, for solid AdC to 0.63. CONCLUSION: The histologic criteria that may be used in the differential diagnosis of poorly differentiated lung cancer were more precisely refined. Furthermore, additional stains improved the reproducibility of histological diagnosis of SqCC and AdC, uncovering information that was not present in routine H&E stained slides.

BACKGROUND: Thymic carcinomas are rare cancers with limited data regarding outcomes, particularly for those patients with advanced disease. METHODS: We identified patients with thymic carcinomas diagnosed between 1993 and 2012. Patient characteristics, recurrence-free survival (RFS), and overall survival (OS) were analyzed. RESULTS: One hundred twenty-one patients with thymic carcinomas were identified. Higher Masaoka stage was associated with worse OS and RFS (5-year OS of 100%, 81%, 51%, 24%, and 17% for stage I, II, III, IVa, and IVb respectively, p < 0.001 and 5-year RFS of 80%, 28%, and 7% for stage I/II, III, and IV respectively, p < 0.001). Patients with stage IVb lymph node (LN) only disease had a better 5-year OS as compared with patients with distant metastasis (24% versus 7%, p = 0.025). Of the 61 patients with stage IVb disease, 22 of 29 patients (76%) with LN-only disease underwent curative intent resection versus 3 of 32 patients (9%) with distant metastasis. Twenty-two patients with LN involvement were treated with multimodality therapy. Three (14%) remain free of disease with long-term follow-up (range, 3.4+ years- to 6.8+ years). CONCLUSIONS: We describe the clinical features of a large series of patients with thymic carcinoma in North America. The Masaoka staging system effectively prognosticated OS and RFS. Patients with stage IVb LN-only disease had significantly better OS as compared with patients with distant metastasis with a subset of patients sustaining long-term RFS with multimodality therapy. If validated, these data would support a revised staging system with subclassification of stage IVb disease into two groups.

Malignant pleural mesothelioma is a particularly aggressive and locally invasive malignancy with a poor prognosis despite advances in understanding of cancer cell biology and development of new therapies. At the cellular level, cultured mesothelioma cells present a mesenchymal appearance and a strong capacity for local cellular invasion. One important but underexplored area of mesothelioma cell biology is intercellular communication. Our group has previously characterized in multiple histological subtypes of mesothelioma a unique cellular protrusion known as tunneling nanotubes (TnTs). TnTs are long, actin filament-based, narrow cytoplasmic extensions that are non-adherent when cultured in vitro and are capable of shuttling cellular cargo between connected cells. Our prior work confirmed the presence of nanotube structures in tumors resected from patients with human mesothelioma. In our current study, we quantified the number of TnTs/cell among various mesothelioma subtypes and normal mesothelial cells using confocal microscopic techniques. We also examined changes in TnT length over time in comparison to cell proliferation. We further examined potential approaches to the in vivo study of TnTs in animal models of cancer. We have developed novel approaches to study TnTs in aggressive solid tumor malignancies and define fundamental characteristics of TnTs in malignant mesothelioma. There is mounting evidence that TnTs play an important role in intercellular communication in mesothelioma and thus merit further investigation of their role in vivo.

The past decade has seen an enormous advancement in the therapy for lung cancer, predominantly seen in adenocarcinoma, ranging from the introduction of histology-based drugs to the discovery of targetable mutations. These events have led to a personalized therapeutic approach with the delivery of drugs that target specific oncogenic pathways active in a given tumor with the intent of acquiring the best response rate. The discovery of sensitizing mutation in the epidermal growth factor receptor gene as the basis for clinical response to tyrosine kinase inhibitors led to a systematic search for other molecular targets in lung cancer. Currently, there are several molecular alterations that can be targeted by experimental drugs. These new discoveries would not be possible without a parallel technological evolution in diagnostic molecular pathology. Next-generation sequencing (NGS) is a technology that allows for the evaluation of multiple molecular alterations in the same sample using a small amount of tissue. Selective evaluation of targeted cancer genes, instead of whole-genome evaluation, is the approach that is best suited to enter clinical practice. This technology allows for the detection of most molecular alteration with a single test, thus saving tissue for future discoveries. The use of NGS is expected to increase and gain importance in clinical and experimental approaches, since it can be used as a diagnostic tool as well as for new discoveries. The technique may also help us elucidate the interplay of several genes and their alteration in the mechanism of drug response and resistance.

Context.-Thymic epithelial tumors are rare, constituting interpretive challenges for pathologists. Digital imaging can be useful as an educational tool for these rare tumors. Objectives.-To evaluate the diagnostic reproducibility of thymic tumors among thoracic pathologists. Design.-Twenty cases of thymoma or thymic carcinoma were scanned into the Aperio system. The images were sent to pathologists with expertise in thoracic pathology at 6 different centers, who were asked to classify the tumors according to the 2004 World Heath Organization classification and to diagnose invasion. Interobserver agreement was evaluated. After discussion of the first 20 cases, a second set of 10 cases was evaluated. Results.-There was agreement for the diagnosis of thymoma and thymic carcinoma in 70% of cases (n = 14); in the remaining 6 cases, there was disagreement for cases of B3 thymoma (n = 5) and type A thymoma (n = 1) and thymic carcinoma. The overall kappa was 0.39. When invasion was evaluated, the overall kappa was 0.45. In the second round of the study, after discussion of diagnostic criteria, the interobserver agreement for the diagnosis of thymoma versus thymic carcinoma was 0.67 and that for the determination of invasion was 0.57-suggesting interpretative improvement. Conclusion.-The reproducibility of diagnosis of thymic epithelial tumors, using digital imaging, is comparable to that in previous studies using glass slides. Digital imaging is a good tool for remote consultation and for educational purposes. This technology could be used to train pathologists with low-level experience in thymic epithelial tumors and to foster collaborative work in the field.