Faculty Bibliography

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%).

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.

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.

The discovery of targetable driver mutations in pulmonary adenocarcinoma has revolutionized the field of thoracic oncology by the introduction of oral small molecule tyrosine kinase inhibitors that target specific EGFR mutations and EML-4/ALK rearrangement. Therefore, testing for EGFR gene mutations and ALK rearrangements has become part of everyday clinical practice. The majority of lung cancer patients present at an advanced stage, where small biopsy and cytology specimens are often the only available material for diagnostic workup and molecular characterization. Mutation testing has become the standard of care. Nonetheless, the technical complexity and relative high cost of the test have challenged the widespread use of molecular techniques in everyday clinical settings. Recently, antibodies to specific molecular alterations have become available and have the potential to become instruments for the molecular characterization of tumors. In this review article we will discuss practical issues in molecular characterization of lung adenocarcinoma on cytology material and the use of immunocytochemical stains for the detection of mutant protein as an alternative or adjunct to molecular techniques.

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.

PURPOSE: Dynamic contrast-enhanced MRI (DCE-MRI) can provide information regarding tumor perfusion and permeability and has shown prognostic value in certain tumors types. The goal of this study was to assess the prognostic value of pretreatment DCE-MRI in head and neck squamous cell carcinoma (HNSCC) patients with nodal disease undergoing chemoradiation therapy or surgery. METHODS AND MATERIALS: Seventy-four patients with histologically proven squamous cell carcinoma and neck nodal metastases were eligible for the study. Pretreatment DCE-MRI was performed on a 1.5T MRI. Clinical follow-up was a minimum of 12 months. DCE-MRI data were analyzed using the Tofts model. DCE-MRI parameters were related to treatment outcome (progression-free survival [PFS] and overall survival [OS]). Patients were grouped as no evidence of disease (NED), alive with disease (AWD), dead with disease (DOD), or dead of other causes (DOC). Prognostic significance was assessed using the log-rank test for single variables and Cox proportional hazards regression for combinations of variables. RESULTS: At last clinical follow-up, for Stage III, all 12 patients were NED. For Stage IV, 43 patients were NED, 4 were AWD, 11 were DOD, and 4 were DOC. K(trans) is volume transfer constant. In a stepwise Cox regression, skewness of K(trans) (volume transfer constant) was the strongest predictor for Stage IV patients (PFS and OS: p <0.001). CONCLUSION: Our study shows that skewness of K(trans) was the strongest predictor of PFS and OS in Stage IV HNSCC patients with nodal disease. This study suggests an important role for pretreatment DCE-MRI parameter K(trans) as a predictor of outcome in these patients.

BACKGROUND: Pulmonary adenocarcinoma (AD) has a variety of architectural patterns. Recently, a 3-tiered histological pattern-based grading system was developed for stage I lung AD, stratifying patients into low, intermediate, and high risk for recurrence. However, cytology may serve as the primary method for diagnosis in patients with inoperable disease. Attempts to correlate architecture between parallel cytological and histological preparations have not been successful. Therefore, we evaluated cytomorphologic features of previously histologically graded AD to identify features of potential prognostic significance. METHODS: One hundred and thirteen fine-needle aspirations with excised adenocarcinomas were reviewed. In the liquid-based preparation, we evaluated cell arrangements(flat sheets vs 3-D clusters vs single cells), nuclear features (size variability, shape, and contour),nucleoli (prominent or inconspicuous), presence of nuclear inclusions, chromatin (fine, coarse,or clumped), and quality of background. The features were tested by multivariate analysis to identify associations with histological grade and disease-free survival (DFS), and a cytological score was generated. RESULTS: Nuclear size, chromatin pattern, and nuclear contours showed a significant association with histological grade and DFS. These features were included in the composite cytological score (range,0-5). By grouping the cytological scores, we stratified the tumors into low (median DFS, 100%), intermediate(median DFS, 78%), and high (median DFS, 55%) rate of recurrence (P (1/4) .008). There was a good correlation with the histological grading system. CONCLUSIONS: In liquid-based preparations, distinctive cytological features of pulmonary adenocarcinoma correlate with levels of histological differentiation and can be combined into a score with prognostic significance.

The aim of the present study is to correlate non-invasive, pretreatment biological imaging (dynamic contrast enhanced-MRI [DCE-MRI] and proton magnetic resonance spectroscopy [(1)H-MRS]) findings with specific molecular marker data in neck nodal metastases of head and neck squamous cell carcinoma (HNSCC) patients. Pretreatment DCE-MRI and (1)H-MRS were performed on neck nodal metastases of 12 patients who underwent surgery. Surgical specimens were analyzed with immunohistochemistry (IHC) assays for: Ki-67 (reflecting cellular proliferation), vascular endothelial growth factor (VEGF) (the "endogenous marker" of tumor vessel growth), carbonic anhydrase (CAIX), hypoxia inducible transcription factor (HIF-1alpha), and human papillomavirus (HPV). Additionally, necrosis was estimated based on H&E staining. The Spearman correlation was used to compare DCE-MRI, (1)H-MRS, and molecular marker data. A significant correlation was observed between DCE-MRI parameter std(k(ep)) and VEGF IHC expression level (rho=0.81, p=0.0001). Furthermore, IHC expression levels of Ki-67 inversely correlated with std(K(trans)) and std(v(e)) (rho=-0.71; p=0.004, and rho=-0.73; p=0.003, respectively). Other DCE-MRI, (1)H-MRS and IHC values did not show significant correlation. The results of this preliminary study indicate that the level of heterogeneity of perfusion in metastatic HNSCC seems positively correlated with angiogenesis, and inversely correlated with proliferation. These results are preliminary in nature and are indicative, and not definitive, trends portrayed in HNSCC patients with nodal disease. Future studies with larger patient populations need to be carried out to validate and clarify our preliminary findings.

Tunneling nanotubes are long, non-adherent F-actin-based cytoplasmic extensions which connect proximal or distant cells and facilitate intercellular transfer. The identification of nanotubes has been limited to cell lines, and their role in cancer remains unclear. We detected tunneling nanotubes in mesothelioma cell lines and primary human mesothelioma cells. Using a low serum, hyperglycemic, acidic growth medium, we stimulated nanotube formation and bidirectional transfer of vesicles, proteins, and mitochondria between cells. Notably, nanotubes developed between malignant cells or between normal mesothelial cells, but not between malignant and normal cells. Immunofluorescent staining revealed their actin-based assembly and structure. Metformin and an mTor inhibitor, Everolimus, effectively suppressed nanotube formation. Confocal microscopy with 3-dimensional reconstructions of sectioned surgical specimens demonstrated for the first time the presence of nanotubes in human mesothelioma and lung adenocarcinoma tumor specimens. We provide the first evidence of tunneling nanotubes in human primary tumors and cancer cells and propose that these structures play an important role in cancer cell pathogenesis and invasion.