Histologic and Molecular Characterization of Non-Small Cell Lung Carcinoma With Discordant ROS1 Immunohistochemistry and Fluorescence In Situ Hybridization.

INTRODUCTION: ROS1 immunohistochemical (IHC) positivity requires follow-up with confirmatory testing such as fluorescence in situ hybridization (FISH). Identifying predictive characteristics of false positive ROS1 IHC cases could aid in optimizing testing algorithms, decrease testing costs and preserve tissue. MATERIALS AND METHODS: Retrospective results were retrieved for 2054 patients with non-small cell lung carcinoma submitted to our laboratory for molecular testing. Reflex ROS1 FISH was done on all ROS1 immunoreactive cases using ROS1 D4D6 antibody. Staining intensity and histo-score was recorded for all ROS1 immunoreactive cases. Results of any additional molecular testing (KRAS, BRAF, EGFR, ALK FISH, RET FISH, MET FISH) were also tabulated. RESULTS: ROS1 immunoreactivity was seen in 305/2054 (14.8%) cases. Immunoreactivity was weak in majority of the cases with only 4.6% cases having an histo-score >100 and 5.9% of cases had moderate staining intensity. FISH was negative in 99% (302/305) cases with any degree of IHC expression (discordant cases) while 3 cases were positive by FISH. Diffuse strong IHC staining in greater than 90% of the tumor was noted in 6 cases, 3 (0.98%) of which were confirmed to have ROS1 rearrangement by FISH. The discordant cases had significantly higher rates of EGFR mutations (P<0.0005) in comparison to ROS1 IHC negative cases, were seen more often in adenocarcinoma and adenosquamous cell carcinoma (P<0.0005) with lepidic and acinar patterns, and more likely to occur in primary lung carcinomas (P<0.0005). CONCLUSIONS: False positive ROS1 immunoreactivity was very frequent, occurred more commonly in primary NSCLC cases with acinar and/or lepidic histologies and was more likely in EGFR mutated cases. Using higher positivity thresholds for ROS1 IHC and incorporating the histologic and molecular correlates into algorithmic strategies could result in increased specificity and clinical utility of ROS1 IHC assay.

Spatially-resolved quantification of proteins in triple negative breast cancers reveals differences in the immune microenvironment associated with prognosis.

Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype. Recent studies have shown that MHC class II (MHCII) expression and tumor infiltrating lymphocytes are important prognostic factors in patients with TNBC, although the relative importance of lymphocyte subsets and associated protein expression is incompletely understood. NanoString Digital Spatial Profiling (DSP) allows for spatially resolved, highly multiplexed quantification of proteins in clinical samples. In this study, we sought to determine if DSP could be used to characterize expression of MHCII and other immune related proteins in tumor epithelial versus stromal compartments of patient-derived TNBCs (N = 10) using a panel of 39 markers. We confirmed that a subset of TNBCs have elevated expression of HLA-DR in tumor epithelial cells; HLA-DR expression was also significantly higher in the tumors of patients with long-term disease-free survival when compared to patients that relapsed. HLA-DR expression in the epithelial compartment was correlated with high expression of CD4 and ICOS in the stromal compartment of the same tumors. We also identified candidate protein biomarkers with significant differential expression between patients that relapsed versus those that did not. In conclusion, DSP is a powerful method that allows for quantification of proteins in the immune microenvironment of TNBCs.

Successful lung cancer EGFR sequencing from DNA extracted from TTF-1 immunohistochemistry slides: a new means to extend insufficient tissue.

Lung cancer biopsy material is limited and is used for morphologic diagnosis and immunohistochemical and molecular testing. This can lead to tissue exhaustion, resulting in repeat biopsies (when clinically possible), delayed testing, and increased risks. Consequently, there is a need to optimize preanalytical specimen use for molecular testing. Although hematoxylin/eosin can be used for as a DNA source for molecular testing, little is known regarding the potential use of immunohistochemistry (IHC) slides, as these are subject to harsh conditions that can lead to DNA degradation. Our aim was to evaluate whether DNA extracted from TTF-1 IHC slides, a common stain for lung adenocarcinoma, can be tested for EGFR mutations. Twenty-two lung adenocarcinoma samples (11 EGFR wild type and 11 mutated) were selected. Slides were stained for TTF-1 IHC. Following TTF-1 staining, tissue underwent DNA extraction. Pyrosequencing for mutations in exons 18, 19, 20, and 21 of EGFR was performed, and results were compared to clinical EGFR testing data. All 22 TTF-1 samples produced successful results, and 21 were concordant. Of the 11 originally EGFR-mutated cases, 10 TTF-1 samples showed identical mutations in all exons of interest. One case with an L858R mutation on original testing was negative on sequencing of the TTF-1 sample, possibly due to lower tumor burden on the TTF-1 stained slide. All 11 originally EGFR wild-type cases showed identical results on the TTF-1 samples. TTF-1 IHC slides can be a viable DNA source for molecular testing, especially important in lung biopsies with insufficient material following diagnostic evaluation.

Peutz-Jeghers Type Polyp of the Appendix with Review of Literature.

Hamartomatous polyps of Peutz-Jeghers type are strongly associated with Peutz-Jeghers polyposis syndrome and are predominantly encountered in the small intestine. Sporadic cases are uncommonly reported. We report a case of a polyp identified incidentally in the appendix of a patient undergoing diagnostic imaging due to a history of hepatitis C infection. Histopathologic evaluation after appendectomy showed a polyp with bands of muscularis mucosae bundles with arborizing architecture and variable amounts of inspissated mucin, morphologically indistinguishable from Peutz-Jeghers type hamartomatous polyp. A family or personal history of abdominal cancers was not reported by the patient, suggesting a sporadic occurrence. Next generation sequencing revealed only two pathogenic low-level STK11 mutations, presumed to be somatic. In conclusion, this is an unusual case of a sporadic Peutz-Jeghers type polyp occurring in the appendix.

Molecular Fingerprinting of Anatomically and Temporally Distinct B-Cell Lymphoma Samples by Next-Generation Sequencing to Establish Clonal Relatedness.

CONTEXT.­: B-cell lymphomas exhibit balanced translocations that involve immunoglobulin loci and result from aberrant V(D)J recombination, class switch recombination, or somatic hypermutation. Although most of the breakpoints in the immunoglobulin loci occur in defined regions, those in the partner genes vary; therefore, it is unlikely that 2 independent clones would share identical breakpoints in both partners. Establishing whether a new lesion in a patient with history of lymphoma represents recurrence or a new process can be relevant. Polymerase chain reaction (PCR)-based clonality assays used in this setting rely only on evaluating the length of a given rearrangement. In contrast, next-generation sequencing (NGS) provides the exact translocation breakpoint at single-base resolution. OBJECTIVE.­: To determine if translocation breakpoint coordinates can serve as a molecular fingerprint unique to a distinct clonal population. DESIGN.­: Thirty-eight follicular lymphoma/diffuse large B-cell lymphoma samples collected from different anatomic sites and/or at different time points from 18 patients were analyzed by NGS. For comparison, PCR-based B-cell clonality and fluorescence in situ hybridization studies were performed on a subset of cases. RESULTS.­: IGH-BCL2 rearrangements were detected in all samples. The breakpoint coordinates on derivative chromosome(s) were identical in all samples from a given patient, but distinct between samples derived from different patients. Additionally, 5 patients carried a second rearrangement also with conserved breakpoint coordinates in the follow-up sample(s). CONCLUSIONS.­: Breakpoint coordinates in the immunoglobulin and partner genes can be used to establish clonal relatedness of anatomically/temporally distinct lesions. Additionally, an NGS-based approach has the potential to detect secondary translocations that may have prognostic and therapeutic significance.

A novel approach to quantitating leukemia fusion transcripts by qRT-PCR without the need for standard curves.

Acute myeloid leukemia patients with recurrent cytogenetic abnormalities including inv(16);CBFB-MYH11 and t(15;17);PML-RARA may be assessed by monitoring the levels of the corresponding abnormal fusion transcripts by quantitative reverse transcription-PCR (qRT-PCR). Such testing is important for evaluating the response to therapy and for the detection of early relapse. Existing qRT-PCR methods are well established and in widespread use in clinical laboratories but they are laborious and require the generation of standard curves. Here, we describe a new method to quantitate fusion transcripts in acute myeloid leukemia by qRT-PCR without the need for standard curves. Our approach uses a plasmid calibrator containing both a fusion transcript sequence and a reference gene sequence, representing a perfect normalized copy number (fusion transcript copy number/reference gene transcript copy number; NCN) of 1.0. The NCN of patient specimens can be calculated relative to that of the single plasmid calibrator using experimentally derived PCR efficiency values. We compared the data obtained using the plasmid calibrator method to commercially available assays using standard curves and found that the results obtained by both methods are comparable over a broad range of values with similar sensitivities. Our method has the advantage of simplicity and is therefore lower in cost and may be less subject to errors that may be introduced during the generation of standard curves.

Molecular genetic biomarkers in myeloid malignancies.

CONTEXT: Recent studies using massively parallel sequencing technologies, so-called next-generation sequencing, have uncovered numerous recurrent, single-gene variants or mutations across the spectrum of myeloid malignancies. OBJECTIVES: To review the recent advances in the understanding of the molecular basis of myeloid neoplasms, including their significance for diagnostic and prognostic purposes and the possible implications for the development of novel therapeutic strategies. DATA SOURCES: Literature review. CONCLUSIONS: The recurrent mutations found in myeloid malignancies fall into distinct functional categories. These include (1) cell signaling factors, (2) transcription factors, (3) regulators of the cell cycle, (4) regulators of DNA methylation, (5) regulators of histone modification, (6) RNA-splicing factors, and (7) components of the cohesin complex. As the clinical significance of these mutations and mutation combinations is established, testing for their presence is likely to become a routine part of the diagnostic workup. This review will attempt to establish a framework for understanding these mutations in the context of myeloproliferative neoplasms, myelodysplastic syndromes, and acute myeloid leukemia.

Impact of rapid on-site evaluation on the adequacy of endoscopic-ultrasound guided fine-needle aspiration of solid pancreatic lesions: a systematic review and meta-analysis.

BACKGROUND: Rapid on-site evaluation (ROSE) has the potential to improve adequacy rates for endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) of solid pancreatic lesions, but its impact is context-dependent. No studies exist that summarize the relationship between ROSE, number of needle passes, and resulting adequacy rates. AIMS: To analyze data from previous studies to establish if ROSE is associated with improved adequacy rates; to evaluate the relationship between ROSE, number of needle passes, and the resulting adequacy rates of EUS-FNA for solid pancreatic lesions. METHODS: Systematic review and meta-analysis of studies reporting the adequacy rates for EUS-FNA of solid pancreatic lesions. RESULTS: The search produced 3822 original studies, of which 70 studies met our inclusion criteria. The overall average adequacy rate was 96.2% (95% confidence interval: 95.5, 96.9). ROSE was associated with a statistically significant improvement of up to 3.5% in adequacy rates. There was heterogeneity in adequacy rates across all subgroups. No association between the assessor type and adequacy rates was found. Studies with ROSE have high per-case adequacy and a relatively high number of needle passes in contrast to non-ROSE studies. ROSE is an effect modifier of the relationship between number of needle passes and adequacy. CONCLUSIONS: ROSE is associated with up to 3.5% improvement in adequacy rates for EUS-FNA of solid pancreatic lesions. ROSE assessor type has no impact on adequacy rates. ROSE is an effect modifier on the relationship between needle passes and per-case adequacy for EUS-FNA of solid pancreatic lesions.

Rapid on-site evaluation increases endoscopic ultrasound-guided fine-needle aspiration adequacy for pancreatic lesions.

BACKGROUND: Rapid on-site evaluation (ROSE) has the potential to improve adequacy rates and affect other outcomes; however, there have been few comparative studies to assess the impact of ROSE in the setting of ultrasound-guided endoscopic fine-needle aspiration cytology for pancreatic lesions. AIMS: To determine whether ROSE improves adequacy rates of endoscopic fine-needle aspiration cytology for pancreatic lesions. METHODS: Systematic review and meta-analysis of studies reporting a head-to-head comparison of adequacy or diagnostic accuracy (with ROSE vs. without ROSE) at a single site. RESULTS: ROSE was associated with a statistically significant (p < 0.001) improvement in the adequacy rate (average 10 %, 95 % CI: 5-24 %). The impact of ROSE depends on the per-pass adequacy rate without ROSE. ROSE had no impact on diagnostic yield (p < 0.76). CONCLUSIONS: ROSE is associated with an improvement in adequacy rates when implemented at sites where the per-case adequacy rate without ROSE is low (<90 %). It is unclear whether the type of assessor (pathologist vs. non-pathologist) has a significant impact on the success rate of ROSE. ROSE has no impact on diagnostic yield. Studies should employ head-to-head comparisons of cohorts with and without ROSE at a single location.

Needle size has only a limited effect on outcomes in EUS-guided fine needle aspiration: a systematic review and meta-analysis.

BACKGROUND: Several recent studies have investigated the utility of 19-, 22-, and 25-gauge needles in endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) of pancreatic and peri-pancreatic tumors. AIM: The objective of this study was to summarize data from these studies and estimate the effect of needle size on reported outcomes such as accuracy, adequacy, and complications. METHODS: Systematic review and meta-analysis comparing the effect of needle size (19, 22, and 25G) on diagnostic accuracy, adequacy, number of needle passes, and complications. RESULTS: 25G appear to confer an advantage in adequacy rates relative to 22G needles (risk difference = 0.12 %, 95 % CI 0.01, 0.25). There was no significant difference in accuracy with an overall sensitivity and specificity for 22G being 0.78 (95 % CI 0.74-0.81) and 1.00 (95 % CI 0.98-1.00) and an overall sensitivity and specificity for 25G being 0.91 (95 % CI 0.87-0.94) and 1.00 (95 % CI 0.97-1.00). There was no difference in number of passes or complications between 25 and 22G. The limited data available regarding 19G needles do not show evidence of improved outcomes with these devices. CONCLUSIONS: In the evaluation of pancreatic and peri-pancreatic lesions by EUS-FNA, 25G needles may confer an advantage in adequacy relative to 22G needles but confer no advantages with respect to accuracy, number of passes, or complications.