Whole-mitochondrial genome sequencing in primary open-angle glaucoma using massively parallel sequencing identifies novel and known pathogenic variants.

PURPOSE: The aim of this study was to determine whether mutations in mitochondrial DNA play a role in high-pressure primary open-angle glaucoma (OMIM 137760) by analyzing new data from massively parallel sequencing of mitochondrial DNA. METHODS: Glaucoma patients with high-tension primary open-angle glaucoma and ethnically matched and age-matched control subjects without glaucoma were recruited. The entire human mitochondrial genome was amplified in two overlapping fragments by long-range polymerase chain reaction and used as a template for massively parallel sequencing on an Ion Torrent Personal Genome Machine. All variants were confirmed by conventional Sanger sequencing. RESULTS: Whole-mitochondrial genome sequencing was performed in 32 patients with primary open-angle glaucoma from India (n = 16) and Ireland (n = 16). In 16 of the 32 patients with primary open-angle glaucoma (50% of cases), there were 22 mitochondrial DNA mutations consisting of 7 novel mutations and 8 previously reported disease-associated sequence variants. Eight of 22 (36.4%) of the mitochondrial DNA mutations were in complex I mitochondrial genes. CONCLUSION: Massively parallel sequencing using the Ion Torrent Personal Genome Machine with confirmation by Sanger sequencing detected a pathogenic mitochondrial DNA mutation in 50% of the primary open-angle glaucoma cohort. Our findings support the emerging concept that mitochondrial dysfunction results in the development of glaucoma and, more specifically, that complex I defects play a significant role in primary open-angle glaucoma pathogenesis.

Chromosome 9p deletions identify an aggressive phenotype of clear cell renal cell carcinoma.

BACKGROUND: The authors investigated whether deletion of chromosome 9p in clear cell renal cell carcinoma (ccRCC) predicted worse disease-specific survival (DSS) and recurrence-free survival (RFS) and whether it was associated with more aggressive behavior in small renal masses. METHODS: In total, 703 ccRCC tumors were analyzed using fluorescence in situ hybridization (316 tumors) and cytogenetics (388 tumors). Tumor grade, classification, and size; 9p status; Eastern Cooperative Oncology Group performance status (ECOG PS); lymph node involvement; and the presence of metastasis were recorded. Outcomes were stratified by 9p status, and a Cox proportional hazards models was constructed using TNM staging, ECOG PS, tumor size, tumor grade, and 9p status. RESULTS: Deletions of 9p were detected in 97 tumors (13.8%). At presentation, 9p-deleted tumors were larger and were more likely to be high grade (grade 3 or 4), to have a high tumor (T) classification (T3-T4), and to have lymph node or distant metastases (P < .01). The median DSS for patients with and without 9p deletions was 37 months and 82 months, respectively (P < .01). In patients with localized disease, the median RFS in those who had 9p deletions was 53 months and was not reached in those without 9p deletions (P < .01). In patients who had localized lesions that measured ≤4 cm in greatest dimension, 9p-deleted tumors were more likely to recur (19% vs 2%; P = .01). CONCLUSIONS: Deletion of chromosome 9p in ccRCC occurred in 14% of patients and was associated with higher grade and T classification, and the presence of lymph node and distant metastases. In addition, 9p deletion independently conferred a worse prognosis for patients with localized ccRCC, and most noteworthy, in patients with localized, small renal masses. Preoperatively identifying patients with 9p deletions will improve risk stratification and will help to select appropriate patients for surveillance protocols or aggressive treatment.

Comparative analysis of size estimation by mapping and counting number of blocks with ductal carcinoma in situ in breast excision specimens.

CONTEXT: The size of ductal carcinoma in situ (DCIS) is a significant predictor of local tumor recurrence and is used for selection of patients for conservative versus aggressive therapy. A standardized method for size assessment is lacking. OBJECTIVE: To evaluate 2 commonly used methods for measurement of DCIS extent: one based on the distribution of the lesion in sequential series of sections (mapping method) and the other on the number of sections with DCIS (block method). DESIGN: Ninety-eight consecutive cases of DCIS, measuring at least 1.0 cm, were retrieved from our files. All specimens were serially sectioned along the long axis. The size of DCIS was calculated for each case by 2 different methods: (1) mapping method (average thickness of each slice x number of consecutive slices with DCIS) and (2) block method (number of blocks with DCIS x 0.3 cm). Additional calculations were performed by using 0.35, 0.4, and 0.5 cm as multiplication factors for the block method in order to improve concordance. RESULTS: The block method underestimated the size in 71 cases (72%) by 4.5% to 81.3% (mean, 33%). Using 0.4 cm as the multiplication factor improved concordance, while multiplying by 0.5 cm led to an overestimation of size. CONCLUSIONS: Assessment of DCIS size by the block method is inaccurate and underestimates size in most cases (72%), with an average reduction of 33%. Using 0.4 cm as the multiplication factor improves concordance. A standardized method for size estimation is necessary for effective patient management.

What can we learn from the errors in the frozen section diagnosis of pulmonary carcinoid tumors? An evidence-based approach.

The intraoperative diagnosis of pulmonary neuroendocrine tumors can be difficult. Frozen section diagnosis was requested on 87 neuroendocrine tumors including 58 typical carcinoids, 8 atypical carcinoids, 18 large cell neuroendocrine carcinomas, and 3 small cell carcinomas from 2405 patients that underwent frozen section diagnosis at Cedars-Sinai Medical Center from 2002 to 2007. The deferral and error rate for carcinoid tumors was 4.13% and 7.5%, respectively, and resulted in 4 unnecessary lobectomies and 2 second thoracotomies. The most common errors included misdiagnoses as lymphoma, squamous carcinoma or metastasis from breast carcinoma. Thirty one pathologic features were evaluated in the 66 carcinoid tumors and 10 frozen sections each of lymphoma, squamous cell carcinoma, and metastatic breast carcinoma. Seven pathologic features were significant by chi square test at P > .05. Positive likelihood ratios identified 11 pathologic features that were useful for the diagnosis of carcinoid tumor from other neoplasms. The applicability of the 11 pathologic features was tested with a group of pathologists, resulting in significant improvement in diagnostic accuracy as measured by pre and posttests. The value of evidence-based pathology and Bayesian statistics to evaluate complex differential diagnoses in pathology is discussed.

The predictive value of epidermal growth factor receptor tests in patients with pulmonary adenocarcinoma: review of current "best evidence" with meta-analysis.

Epidermal growth factor receptor signaling pathway plays an important role in pulmonary adenocarcinoma biology. Targeted therapy with tyrosine kinase inhibitors like gefitinib and erlotinib are being used in selected patients with variable response rates. Several RCT and other studies have evaluated the value of various tests such as immunohistochemistry, polymerase chain reaction, and fluorescent in situ hybridization for epidermal growth factor receptor detection. The clinical validity and applicability of these tests remain controversial. Evidence-based pathology promotes the use of systematic review of the literature and meta-analysis rather than subjective appraisal of the literature. We performed a systematic review of the literature to identify the "best evidence" regarding the use of these tests. The data were analyzed using Comprehensive meta-analysis software (Biostat, Inc, Englewood, NJ). Most of the information regarding epidermal growth factor receptor tests has been published in retrospective case series with few double-blind and prospective RCT. Estimated positive predictive values of immunohistochemistry, polymerase chain reaction, and fluorescent in situ hybridization range from 6.5% to 82%%, 7% to 100%, and 11% to 89%, respectively. Meta-analysis of nearly 5000 cases in the literature estimates that all 3 tests significantly predict response to gefitinib in patients with lung cancer. It shows lack of heterogeneity within the study results, although the current best evidence is limited by variations in study methodologies, patient ethnicity, test interpretation criteria, and variable definitions of treatment response. There is only one study evaluating the value of epidermal growth factor receptor tests in predicting response to erlotinib. Further studies are needed to clarify the predictive value of epidermal growth factor receptor tests in patients with pulmonary adenocarcinoma.

Evaluation of EGFR abnormalities in patients with pulmonary adenocarcinoma: the need to test neoplasms with more than one method.

Patients with advanced pulmonary adenocarcinoma exhibiting overexpression or mutation of epidermal growth factor receptor tend to respond better to targeted therapy with tyrosine kinase inhibitors such as gefitinib and erlotinib. There is no consensus regarding how these neoplasms should be routinely tested for epidermal growth factor receptor (EGFR) and whether the results of immunohistochemistry (IHC), mutation analysis and fluorescent in situ hybridization correlate with each other or are independent predictive variables. We tested 100 pulmonary adenocarcinomas from patients with stage III or IV disease for EGFR abnormalities using IHC, PCR and fluorescent in situ hybridization (FISH) and compared the results using kappa and other statistical methods. The sensitivity of each test to detect an EGFR abnormality and its negative predictive value to estimate the presence of an abnormal test result by the other two methods were calculated. Abnormal EGFR test results were found in 62, 40 and 24% by IHC, FISH and PCR, respectively. kappa statistics yielded poor concordance between the results of the EGFR tests (kappa=0.3, and 0.2 for IHC and PCR and for PCR and FISH, respectively). Strong membranous immunoreactivity in more than 90% of the tumor cells was found to correlate with amplification or polysomy. PCR when used as a single test is likely to underestimate the presence of EGFR abnormalities that may significantly predict response to tyrosine kinase inhibitors. The need to standardize the approach to EGFR testing in patients with advanced pulmonary adenocarcinoma is discussed.