Faculty Bibliography

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.

The International Association for the Study of Lung Cancer (IASLC)/American Thoracic Society (ATS)/European Respiratory Society (ERS) has recently proposed a new lung adenocarcinoma classification. We investigated whether nuclear features can stratify prognostic subsets. Slides of 485 stage I lung adenocarcinoma patients were reviewed. We evaluated nuclear diameter, nuclear atypia, nuclear/cytoplasmic ratio, chromatin pattern, prominence of nucleoli, intranuclear inclusions, mitotic count/10 high-power fields (HPFs) or 2.4 mm(2), and atypical mitoses. Tumors were classified into histologic subtypes according to the IASLC/ATS/ERS classification and grouped by architectural grade into low (adenocarcinoma in situ, minimally invasive adenocarcinoma, or lepidic predominant), intermediate (papillary or acinar), and high (micropapillary or solid). Log-rank tests and Cox regression models evaluated the ability of clinicopathologic factors to predict recurrence-free probability. In univariate analyses, nuclear diameter (P=0.007), nuclear atypia (P=0.006), mitotic count (P<0.001), and atypical mitoses (P<0.001) were significant predictors of recurrence. The recurrence-free probability of patients with high mitotic count (>/=5/10 HPF: n=175) was the lowest (5-year recurrence-free probability=73%), followed by intermediate (2-4/10 HPF: n=106, 80%), and low (0-1/10 HPF: n=204, 91%, P<0.001). Combined architectural/mitotic grading system stratified patient outcomes (P<0.001): low grade (low architectural grade with any mitotic count and intermediate architectural grade with low mitotic count: n=201, 5-year recurrence-free probability=92%), intermediate grade (intermediate architectural grade with intermediate-high mitotic counts: n=206, 78%), and high grade (high architectural grade with any mitotic count: n=78, 68%). The advantage of adding mitotic count to architectural grade is in stratifying patients with intermediate architectural grade into two prognostically distinct categories (P=0.001). After adjusting for clinicopathologic factors including sex, stage, pleural/lymphovascular invasion, and necrosis, mitotic count was not an independent predictor of recurrence (P=0.178). However, patients with the high architectural/mitotic grade remained at significantly increased risk of recurrence (high vs low: P=0.005) after adjusting for clinical factors. We proposed this combined architectural/mitotic grade for lung adenocarcinoma as a practical method that can be applied in routine practice.

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.

PURPOSE: There is persistent controversy as to whether EGFR and KRAS mutations occur in pulmonary squamous cell carcinoma (SQCC). We hypothesized that the reported variability may reflect difficulties in the pathologic distinction of true SQCC from adenosquamous carcinoma (AD-SQC) and poorly differentiated adenocarcinoma due to incomplete sampling or morphologic overlap. The recent development of a robust immunohistochemical approach for distinguishing squamous versus glandular differentiation provides an opportunity to reassess EGFR/KRAS and other targetable kinase mutation frequencies in a pathologically homogeneous series of SQCC. EXPERIMENTAL DESIGN: Ninety-five resected SQCCs, verified by immunohistochemistry as DeltaNp63(+)/TTF-1(-), were tested for activating mutations in EGFR, KRAS, BRAF, PIK3CA, NRAS, AKT1, ERBB2/HER2, and MAP2K1/MEK1. In addition, all tissue samples from rare patients with the diagnosis of EGFR/KRAS-mutant "SQCC" encountered during 5 years of routine clinical genotyping were reassessed pathologically. RESULTS: The screen of 95 biomarker-verified SQCCs revealed no EGFR/KRAS [0%; 95% confidence interval (CI), 0%-3.8%], four PIK3CA (4%; 95% CI, 1%-10%), and one AKT1 (1%; 95% CI, 0%-5.7%) mutations. Detailed morphologic and immunohistochemical reevaluation of EGFR/KRAS-mutant "SQCC" identified during clinical genotyping (n = 16) resulted in reclassification of 10 (63%) cases as AD-SQC and five (31%) cases as poorly differentiated adenocarcinoma morphologically mimicking SQCC (i.e., adenocarcinoma with "squamoid" morphology). One (6%) case had no follow-up. CONCLUSIONS: Our findings suggest that EGFR/KRAS mutations do not occur in pure pulmonary SQCC, and occasional detection of these mutations in samples diagnosed as "SQCC" is due to challenges with the diagnosis of AD-SQC and adenocarcinoma, which can be largely resolved by comprehensive pathologic assessment incorporating immunohistochemical biomarkers.

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.

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.

BACKGROUND: There is growing evidence that lung adenocarcinoma and squamous cell carcinoma (SQCC) have distinct oncogenic mutations and divergent therapeutic responses, which is driving the heightened emphasis on accurate pathologic subtyping of non-small cell lung carcinoma (NSCLC). The relative feasibility and accuracy of NSCLC subtyping by small biopsy versus cytology is not well established, particularly in current practice where immunohistochemistry (IHC) is becoming routinely used to aid in this distinction. METHODS: Concurrent biopsy and cytology specimens obtained during a single procedure and diagnosed as NSCLC during a 2-year period (n = 101) were reviewed. Concordance of diagnoses in the two methods was assessed. Accuracy was determined based on subsequent resection or autopsy diagnosis (n = 21) or IHC for thyroid transcription factor 1 and p63 on a subset of cases (n = 43). RESULTS: The distribution of definitive versus favored versus unclassified categories (reflecting the degree of diagnostic certainty) was similar for biopsy (71% versus 23% versus 6%, respectively) and cytology (69% versus 19% versus 12%, respectively), p = 0.29. When results from paired specimens were combined, the rate of definitive diagnoses by at least one method was increased to 84% and the unclassified rate was decreased to 4%. NSCLC subtype concordance between biopsy and cytology was 93%. Kappa coefficient (95% confidence interval) for agreement between methods was 0.88 (0.60-0.89) for adenocarcinoma and 0.76 (0.63-0.89) for SQCC. In pairs with discordant diagnoses (n = 7) the correct tumor type was identified with a similar frequency by biopsy (n = 4) and cytology (n = 3). Factors contributing to mistyping were poor differentiation, necrosis, low cellularity, and lack of supporting IHC. All concordant diagnoses for which verification was available (n = 57) were correct. IHC was used more frequently to subtype NSCLC in biopsy than cytology (32% versus 6%; p = 0.0001). CONCLUSIONS: Small biopsy and cytology achieve comparable rates of definitive and accurate NSCLC subtyping, and the optimal results are attained when the two modalities are considered jointly. The lower requirement for IHC in cytology highlights the strength of cytomorphology in NSCLC subtyping. Whenever clinically feasible, obtaining parallel biopsy and cytology specimens is encouraged.

Immunohistochemistry is increasingly utilized to differentiate lung adenocarcinoma and squamous cell carcinoma. However, detailed analysis of coexpression profiles of commonly used markers in large series of whole-tissue sections is lacking. Furthermore, the optimal diagnostic algorithm, particularly the minimal-marker combination, is not firmly established. We therefore studied whole-tissue sections of resected adenocarcinoma and squamous cell carcinoma (n=315) with markers commonly used to identify adenocarcinoma (TTF-1) and squamous cell carcinoma (p63, CK5/6, 34betaE12), and prospectively validated the devised algorithm in morphologically unclassifiable small biopsy/cytology specimens (n=38). Analysis of whole-tissue sections showed that squamous cell carcinoma had a highly consistent immunoprofile (TTF-1-negative and p63/CK5/6/34betaE12-diffuse) with only rare variation. In contrast, adenocarcinoma showed significant immunoheterogenetity for all 'squamous markers' (p63 (32%), CK5/6 (18%), 34betaE12 (82%)) and TTF-1 (89%). As a single marker, only diffuse TTF-1 was specific for adenocarcinoma whereas none of the 'squamous markers,' even if diffuse, were entirely specific for squamous cell carcinoma. In contrast, coexpression profiles of TTF-1/p63 had only minimal overlap between adenocarcinoma and squamous cell carcinoma, and there was no overlap if CK5/6 was added as a third marker. An algorithm was devised in which TTF-1/p63 were used as the first-line panel, and CK5/6 was added for rare indeterminate cases. Prospective validation of this algorithm in small specimens showed 100% accuracy of adenocarcinoma vs squamous cell carcinoma prediction as determined by subsequent resection. In conclusion, although reactivity for 'squamous markers' is common in lung adenocarcinoma, a two-marker panel of TTF-1/p63 is sufficient for subtyping of the majority of tumors as adenocarcinomas vs squamous cell carcinoma, and addition of CK5/6 is needed in only a small subset of cases. This simple algorithm achieves excellent accuracy in small specimens while conserving the tissue for potential predictive marker testing, which is now an essential consideration in advanced lung cancer specimens.

OBJECTIVE: : To describe pathologic findings in symptomatic World Trade Center-exposed local workers, residents, and cleanup workers enrolled in a treatment program. METHODS: : Twelve patients underwent surgical lung biopsy for suspected interstitial lung disease (group 1, n = 6) or abnormal pulmonary function tests (group 2, n = 6). High-resolution computed axial tomography and pathologic findings were coded. Scanning electron microscopy with energy-dispersive x-ray spectroscopy was performed. RESULTS: : High-resolution computed axial tomography showed reticular findings (group 1) or normal or airway-related findings (group 2). Pulmonary function tests were predominantly restrictive. Interstitial fibrosis, emphysematous change, and small airway abnormalities were seen. All cases had opaque and birefringent particles within macrophages, and examined particles contained silica, aluminum silicates, titanium dioxide, talc, and metals. CONCLUSIONS: : In symptomatic World Trade Center-exposed individuals, pathologic findings suggest a common exposure resulting in alveolar loss and a diverse response to injury