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.

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.

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.

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.

KRAS mutations define a clinically distinct subgroup of lung adenocarcinoma patients, characterized by smoking history, resistance to EGFR-targeted therapies, and adverse prognosis. Whether KRAS-mutated lung adenocarcinomas also have distinct histopathological features is not well established. We tested 180 resected lung adenocarcinomas for KRAS and EGFR mutations by high-sensitivity mass spectrometry-based genotyping (Sequenom) and PCR-based sizing assays. All tumors were assessed for the proportion of standard histological patterns (lepidic, acinar, papillary, micropapillary, solid, and mucinous), several other histological and clinical parameters, and TTF-1 expression by immunohistochemistry. Among 180 carcinomas, 63 (35%) had KRAS mutations (KRAS+), 35 (19%) had EGFR mutations (EGFR+), and 82 (46%) had neither mutation (KRAS-/EGFR-). Solid growth pattern was significantly over-represented in KRAS+ carcinomas: the mean+/-s.d. for the amount of solid pattern in KRAS+ carcinomas was 27+/-34% compared with 3+/-10% in EGFR+ (P<0.001) and 15+/-27% in KRAS-/EGFR- (P=0.033) tumors. Furthermore, at least focal (>/=20%) solid component was more common in KRAS+ (28/63; 44%) compared with EGFR+ (2/35; 6%; P<0.001) and KRAS-/EGFR- (21/82; 26%; P=0.022) carcinomas. KRAS mutations were also over-represented in mucinous carcinomas and were significantly associated with the presence of tumor-infiltrating leukocytes and heavier smoking history. EGFR mutations were associated with non-mucinous non-solid patterns, particularly lepidic and papillary, lack of necrosis, lack of cytological atypia, hobnail cytology, TTF-1 expression, and never/light smoking history. In conclusion, extended molecular and clinicopathological analysis of lung adenocarcinomas reveals a novel association of KRAS mutations with solid histology and tumor-infiltrating inflammatory cells and expands on several previously recognized morphological and clinical associations of KRAS and EGFR mutations. Solid growth pattern was recently shown to be a strong predictor of aggressive behavior in lung adenocarcinomas, which may underlie the unfavorable prognosis associated with KRAS mutations in these tumors.

We previously demonstrated a high specificity of immunohistochemistry using epidermal growth factor receptor (EGFR) mutation-specific antibodies in lung adenocarcinoma and correlation with EGFR mutation analysis. In this study, we assessed EGFR mutation status by immunohistochemistry in a variety of extrapulmonary malignancies, especially those that frequently show EGFR overexpression. Tissue microarrays containing triplicate cores of breast carcinomas (n=300), colorectal carcinomas (n=65), pancreatic adenocarcinoma (n=145), and uterine carcinosarcoma or malignant mixed mullerian tumors (n=25) were included in the study. Tissue microarray of lung adenocarcinoma with known EGFR mutation status was used as reference. Immunohistochemistry was performed using antibodies specific for the E746-A750del and L858R mutations. In pulmonary adenocarcinoma, a staining intensity of 2+ or 3+ correlates with mutation status and is therefore considered as positive. Out of 300 breast carcinomas, 293 (98%) scored 0, 5 (2%) had 1+ staining, 2 (1%) were 2+ for the L858R antibody. All breast carcinomas scored 0 with the E746-A750 antibody. All the colorectal, pancreatic carcinomas and malignant mixed mullerian tumors were negative (0) for both antibodies. Molecular analysis of the breast carcinomas that scored 2+ for L858R showed no mutation. Our results show that EGFR mutation-specific antibodies could be an additional tool distinguishing primary versus metastatic carcinomas in the lung. False-positivity can be seen in breast carcinoma but is extremely rare (1%).

Pulmonary large-cell carcinoma-a diagnostically and clinically controversial entity-is defined as a non-small-cell carcinoma lacking morphologic differentiation of either adenocarcinoma or squamous cell carcinoma, but suspected to represent an end stage of poor differentiation of these tumor types. Given the recent advances in immunohistochemistry to distinguish adenocarcinoma and squamous cell carcinoma, and the recent insights that several therapeutically relevant genetic alterations are distributed differentially in these tumors, we hypothesized that immunophenotyping may stratify large-cell carcinomas into subsets with distinct profiles of targetable driver mutations. We therefore analyzed 102 large-cell carcinomas by immunohistochemistry for TTF-1 and DeltaNp63/p40 as classifiers for adenocarcinoma and squamous cell carcinoma, respectively, and correlated the resulting subtypes with nine therapeutically relevant genetic alterations characteristic of adenocarcinoma (EGFR, KRAS, BRAF, MAP2K1/MEK1, NRAS, ERBB2/HER2 mutations and ALK rearrangements) or more common in squamous cell carcinoma (PIK3CA and AKT1 mutations). The immunomarkers classified large-cell carcinomas as variants of adenocarcinoma (n=62; 60%), squamous cell carcinoma (n=20; 20%) or marker-null (n=20; 20%). Genetic alterations were found in 38 cases (37%), including EGFR (n=1), KRAS (n=30), BRAF (n=2), MAP2K1 (n=1), ALK (n=3) and PIK3CA (n=1). All molecular alterations characteristic of adenocarcinoma occurred in tumors with immunoprofiles of adenocarcinoma or marker-null, but not in tumors with squamous immunoprofiles (combined mutation rate 50% vs 30% vs 0%, respectively; P<0.001), whereas the sole PIK3CA mutation occurred in a tumor with squamous profile (5%). Furthermore, marker-null large-cell carcinomas were associated with significantly inferior disease-free (P<0.001) and overall (P=0.001) survival. In conclusion, the majority (80%) of large-cell carcinomas can be classified by immunomarkers as variants of adenocarcinoma or squamous cell carcinoma, which stratifies these tumors into subsets with a distinct distribution of driver mutations and distinct prognoses. These findings have practical implications for diagnosis, predictive molecular testing and therapy selection.

Histological subtyping of pulmonary adenocarcinoma has recently been updated based on predominant pattern, but data on reproducibility are required for validation. This study first assesses reproducibility in subtyping adenocarcinomas and then assesses further the distinction between invasive and non-invasive (wholly lepidic) pattern of adenocarcinoma, among an international group of pulmonary pathologists. Two ring studies were performed using a micro-photographic image-based method, evaluating selected images of lung adenocarcinoma histologic patterns. In the first study, 26 pathologists reviewed representative images of typical and 'difficult' histologic patterns. A total number of scores for the typical patterns combined (n=94) and the difficult cases (n=21) were 2444 and 546, respectively. The mean kappa score (+/-s.d.) for the five typical patterns combined and for difficult cases were 0.77+/-0.07 and 0.38+/-0.14, respectively. Although 70% of the observers identified 12-65% of typical images as single pattern, highest for solid and least for micropapillary, recognizing the predominant pattern was achieved in 92-100%, of the images except for micropapillary pattern (62%). For the second study on invasion, identified as a key problem area from the first study, 28 pathologists submitted and reviewed 64 images representing typical as well as 'difficult' examples. The kappa for typical and difficult cases was 0.55+/-0.06 and 0.08+/-0.02, respectively, with consistent subdivision by the same pathologists into invasive and non-invasive categories, due to differing interpretation of terminology defining invasion. In pulmonary adenocarcinomas with classic morphology, which comprise the majority of cases, there is good reproducibility in identifying a predominant pattern and fair reproducibility distinguishing invasive from in-situ (wholly lepidic) patterns. However, more precise definitions and better education on interpretation of existing terminology are required to improve recognition of purely in-situ disease, this being an area of increasing importance.

We previously reported that although EGFR mutations are not a feature of pure squamous cell carcinomas (SCC) of the lung, these mutations do occur in adenosquamous carcinomas (AD-SCC) and in rare solid adenocarcinomas, both of which can mimic SCC in small samples. Here we present an expanded series of these cases with a focus on sensitivity to erlotinib. The study included 13 patients with EGFR mutant lung carcinomas, which after detailed pathologic review were classified as AD-SCC (n = 11) or solid adenocarcinoma (n = 2). The majority received a diagnosis of SCC in at least 1 sample. All patients were treated with erlotinib. Eight of 11 patients with AD-SCC were evaluable for response. Their overall response rate was 88% (7/8; 95% CI, 47% to 99%). One of 2 solid adenocarcinoma patients responded to erlotinib. As a group, median progression-free survival was 12 months (95% CI, 8 to not reached); median overall survival was 29 months (95% CI, 27 to not reached). In conclusion, EGFR mutant AD-SCC and solid adenocarcinoma show a response to erlotinib that is comparable to that seen in patients with conventional adenocarcinoma. These tumors can mimic SCC in small samples. We propose an approach to increase the capture of these rare histology patients for EGFR mutation testing.