David H. Hwang

KRAS and NKX2-1 Mutations in Invasive Mucinous Adenocarcinoma of the Lung

Introduction: Mucinous differentiation is observed in a subset of lung adenocarcinomas with unique clinical and pathological features, but the biology of these neoplasms is poorly understood. Methods: We apply targeted next‐generation sequencing to characterize the mutational profiles of 21 invasive mucinous adenocarcinomas, mixed mucinous/nonmucinous adenocarcinomas, and adenocarcinomas with mucinous features of the lung and validate key findings on 954 additional lung adenocarcinomas from our institution and 514 lung adenocarcinomas from The Cancer Genome Atlas. Results: Sequencing identifies pathogenic mutations in the oncogenes Kirsten rat sarcoma viral oncogene homolog (KRAS), phosphatidylinositol‐4,5‐bisphosphate 3‐kinase catalytic subunit alpha (PIK3CA), erb‐b2 receptor tyrosine kinase 2 (ERBB2), and anaplastic lymphoma receptor tyrosine kinase (ALK) and recurrent mutations in tumor protein p53 (TP53), serine/threonine kinase 11 (STK11), NK2 homeobox 1 (NKX2‐1), and SET domain containing 2 (SETD2). In the combined discovery and validation cohorts, we identify nine neoplasms with distinct molecular and pathological features. All are invasive mucinous adenocarcinomas or mixed mucinous/nonmucinous adenocarcinomas with mutations of KRAS and frameshift or nonsense mutations of NKX2‐1. Immunohistochemical analysis shows that these neoplasms are associated with altered differentiation states, including loss of expression of the pulmonary marker thyroid transcription factor 1 (also called Nkx2.1) and expression of gastrointestinal markers. Conclusions: These findings describe recurrent NKX2‐1 mutations in invasive mucinous adenocarcinomas of the lung and support NKX2‐1 as a lineage‐specific tumor suppressor gene in lung carcinogenesis.

18F-Florbetapir Binds Specifically to Myocardial Light Chain and Transthyretin Amyloid Deposits: Autoradiography Study.

Background—18F-florbetapir is a promising imaging biomarker for cardiac light chain amyloidosis (AL) and transthyretin amyloidosis (ATTR). Our aim, using human autopsy myocardial specimens, was to test the hypothesis that 18F-florbetapir binds specifically to myocardial AL and ATTR amyloid deposits. Methods and Results—We studied myocardial sections from 30 subjects with autopsy-documented AL (n=10), ATTR (n=10), and nonamyloid controls (n=10) using 18F-florbetapir and cold florbetapir compound and digital autoradiography. Total and nonspecific binding of 18F-florbetapir was determined using the maximum signal intensity values. Specific binding of 18F-florbetapir was calculated by subtracting nonspecific from total binding measurements (in decays per minute/mm2, DPM mm2) and was compared with cardiac structure and function on echocardiography and the histological extent of amyloid deposits. Diffuse or focally increased 18F-florbetapir uptake was noted in all AL and ATTR samples and in none of the control samples. Compared with control samples, mean 18F-florbetapir–specific uptake was significantly higher in the amyloid samples (0.94±0.43 versus 2.00±0.58 DPM/mm2; P<0.001), and in the AL compared with the ATTR samples (2.48±0.40 versus 1.52±0.22 DPM/mm2; P<0.001). The samples from subjects with atypical echocardiographic features of amyloidosis showed quantitatively more intense 18F-florbetapir–specific uptake compared with control samples (1.50±0.17 versus 0.94±0.43 DPM/mm2; P=0.004), despite smaller amyloid extent than in subjects with typical echocardiograms. Conclusions—18F-florbetapir specifically binds to myocardial AL and ATTR deposits in humans and offers the potential to screen for the 2 most common types of myocardial amyloid.

Consistency and reproducibility of next-generation sequencing and other multigene mutational assays: A worldwide ring trial study on quantitative cytological molecular reference specimens

Molecular testing of cytological lung cancer specimens includes, beyond epidermal growth factor receptor (EGFR), emerging predictive/prognostic genomic biomarkers such as Kirsten rat sarcoma viral oncogene homolog (KRAS), neuroblastoma RAS viral [v‐ras] oncogene homolog (NRAS), B‐Raf proto‐oncogene, serine/threonine kinase (BRAF), and phosphatidylinositol‐4,5‐bisphosphate 3‐kinase catalytic subunit α (PIK3CA). Next‐generation sequencing (NGS) and other multigene mutational assays are suitable for cytological specimens, including smears. However, the current literature reflects single‐institution studies rather than multicenter experiences.

Next-generation sequencing of cytologic preparations: An analysis of quality metrics

Next‐generation sequencing (NGS) fails for many small biopsies (BXs) because of a low overall DNA concentration or tumor percentage. Cytology smears and liquid‐based preparations (LBPs), or smears/LBPs, often contain abundant tumor cells and may provide adequate material for molecular testing when other materials are insufficient. This study examined the performance of smears/LBPs on a clinical NGS assay.

Osseous and chondromatous metaplasia in calcific aortic valve stenosis

BACKGROUND Aortic valve replacement for calcific aortic valve stenosis is one of the more common cardiac surgical procedures. However, the underlying pathophysiology of calcific aortic valve stenosis is poorly understood. We therefore investigated the histologic findings of aortic valves excised for calcific aortic valve stenosis and correlated these findings with their associated clinical features. RESULTS AND METHODS We performed a retrospective analysis on 6685 native aortic valves excised for calcific stenosis and 312 prosthetic tissue aortic valves with calcific degeneration at a single institution between 1987 and 2013. Patient demographics were correlated with valvular histologic features diagnosed on formalin-fixed, decalcified, and paraffin embedded hematoxylin and eosin stained sections. Of the analyzed aortic valves, 5200 (77.8%) were tricuspid, 1473 (22%) were bicuspid, 11 (0.2%) were unicuspid, and 1 was quadricuspid. The overall prevalence of osseous and/or chondromatous metaplasia was 15.6%. Compared to tricuspid valves, bicuspid valves had a higher prevalence of metaplasia (30.1% vs. 11.5%) and had an earlier mean age of excision (60.2 vs. 75.1 years old). In addition, the frequency of osseous metaplasia and/or chondromatous metaplasia increased with age at time of excision of bicuspid aortic valves, while tricuspid aortic valves showed the same incidence regardless of patient age. Males had a higher prevalence of metaplasia in both bicuspid (33.5% vs. 22.3%) and tricuspid (13.8% vs. 8.6%) aortic valves compared to females. Osseous metaplasia and/or chondromatous metaplasia was also more common in patients with bicuspid aortic valves and concurrent chronic kidney disease or atherosclerosis than in those without (33.6% vs. 28.3%). No osseous or chondromatous metaplasia was observed within the cusps of any of the prosthetic tissue valves. CONCLUSIONS Osseous and chondromatous metaplasia are common findings in native aortic valves but do not occur in prosthetic tissue aortic valves. Bicuspid valves appear to have an inherent proclivity for metaplasia, as demonstrated by their higher rates of osseous metaplasia and/or chondromatous metaplasia both overall and at earlier age compared to tricuspid and prosthetic tissue aortic valves. This predilection could be due to aberrant hemodynamic forces on bicuspid valves, as well as intrinsic genetic changes associated with bicuspid valve formation. Aortic valve interstitial cells may play a central role in this process. Calcification of prosthetic tissue valves is most likely a primarily dystrophic phenomenon.

Myc protein expression correlates with MYC amplification in small-cell lung carcinoma.

Myc family members are important contributors to oncogenesis in a variety of tumours. Identification of therapeutic targets is needed in small‐cell lung carcinoma (SCLC), an aggressive disease with limited treatment options. Sequencing studies have identified MYC amplification in 2–7% of SCLCs. This study aims to determine the rate of MYC gene amplification and its correlation with Myc protein overexpression in SCLC.

Automated Nucleated RBC Measurement Using the Sysmex XE-5000 Hematology Analyzer: Frequency and Clinical Significance of the Nucleated RBCs.

OBJECTIVES We validated the automatic nucleated RBC (nRBC) count on a Sysmex XE-5000 hematology analyzer (Sysmex Corporation, Kobe, Japan) and then evaluated the frequency of nRBCs in our patient population. METHODS We correlated automated nRBC enumeration by the Sysmex XE-5000 hematology analyzer on 463 peripheral blood (PB) samples with the manual nRBC count. Results from 360,504 consecutive blood samples were reviewed to determine the frequency of nRBCs in various patient populations in our hospital. RESULTS There was a strong correlation between the automated and manual nRBC count (Pearsons r = 0.97). Frequency of nRBCs varied in different patient populations and was significantly higher in the presence of other morphology flags or abnormal CBC parameters. Low-level nRBCs (0.2%-1.3%) were detected in 0.5% of samples with otherwise normal parameters. CONCLUSIONS The automated method offers many advantages for high-throughput laboratories, including faster turnaround time, labor savings, and high reliability. Automated nRBC measurement allowed us to recognize a group of individuals who have low-level circulating nRBCs with otherwise normal CBC parameters. Nucleated RBC levels below 1.5% as detected by the automated count may be present in patients without increased erythropoiesis or a pathologic bone marrow process.

Pulmonary Clinicopathological Correlation after Allogeneic Hematopoietic Stem Cell Transplantation: An Autopsy Series

Pulmonary complications are a significant cause of morbidity, mortality, and resource utilization after hematopoietic stem cell transplantation (HSCT). The objective of this study was to compare antemortem clinical suspicion of pulmonary complications and postmortem findings in a modern HSCT cohort. All patients who underwent allogeneic HSCT at our institution (n = 1854) between January 1, 2000 and June 30, 2010 were reviewed and patients who died of any cause greater than 1 year after HSCT and had an unrestricted autopsy available for analysis were included. Presence of pulmonary graft-versus-host disease (GVHD) was assessed by a pathologist blinded to the autopsy report, as previously described by Yousem (1995). A total of 35 (1.9%) patients had autopsies available for review. Airway disease, vascular disease, and interstitial disease were all clinically under-recognized compared with the pathological findings on autopsy. Varying degrees of pathological changes were detected, including 10 (28.6%) patients having bronchiolitis obliterans (BO) and 12 (34.3%) patients having pulmonary veno-occlusive disease (PVOD). Pulmonary manifestations of chronic GVHD, particularly BO and PVOD, were clinically under-recognized in our cohort. Our results suggest that PVOD, which has traditionally been considered a rare complication, may be clinically and histologically under-recognized.

Liquid biopsy of fine-needle aspiration supernatant for lung cancer genotyping

BACKGROUND Tumor genotyping is transforming lung cancer care but requires adequate tumor tissue. Advances in minimally invasive biopsy techniques have increased access to difficult-to-access lesions, but often result in smaller samples. With the advent of highly sensitive DNA genotyping methods used for plasma analysis, we hypothesized that these same methods might allow genotyping of free DNA derived from fine needle aspiration supernatant (FNA-S). METHODS We studied patients with known or suspected lung cancer undergoing fine needle aspirate (FNA). After spinning the sample for cellblock, the FNA-S (usually discarded) was saved for genotyping. Supernatant cell-free DNA (SN-cfDNA) was extracted and tested by both droplet digital PCR (EGFR, BRAF, KRAS mutations) and highly sensitive amplicon-based next-generation sequencing (NGS). RESULTS 17 samples were studied, including 11 FNAs from patients with suspected lung cancer and 6 FNAs from patients with lung cancer and acquired drug resistance. Of 6 newly diagnosed adenocarcinomas, 4 had a driver mutations (1 EGFR, 2 KRAS, 1 HER2) found on tissue; all of these could be detected in SN-cfDNA. The EGFR driver mutation was detected in all 5 adenocarcinomas with acquired EGFR resistance and the EGFR T790 M in three cases, in agreement with cellblock. CONCLUSIONS FNA-S is a rich source of fresh tumor DNA, potentially increasing the diagnostic yield from small FNAs. Through use of emerging techniques for highly sensitive genotyping, this widely available biospecimen has potential for facilitating rapid cancer genotyping at diagnosis and after drug resistance.

18F-Florbetapir Binds Specifically to Myocardial Light Chain and Transthyretin Amyloid DepositsCLINICAL PERSPECTIVE

Background—18F-florbetapir is a promising imaging biomarker for cardiac light chain amyloidosis (AL) and transthyretin amyloidosis (ATTR). Our aim, using human autopsy myocardial specimens, was to test the hypothesis that 18F-florbetapir binds specifically to myocardial AL and ATTR amyloid deposits. Methods and Results—We studied myocardial sections from 30 subjects with autopsy-documented AL (n=10), ATTR (n=10), and nonamyloid controls (n=10) using 18F-florbetapir and cold florbetapir compound and digital autoradiography. Total and nonspecific binding of 18F-florbetapir was determined using the maximum signal intensity values. Specific binding of 18F-florbetapir was calculated by subtracting nonspecific from total binding measurements (in decays per minute/mm2, DPM mm2) and was compared with cardiac structure and function on echocardiography and the histological extent of amyloid deposits. Diffuse or focally increased 18F-florbetapir uptake was noted in all AL and ATTR samples and in none of the control samples. Compared with control samples, mean 18F-florbetapir–specific uptake was significantly higher in the amyloid samples (0.94±0.43 versus 2.00±0.58 DPM/mm2; P<0.001), and in the AL compared with the ATTR samples (2.48±0.40 versus 1.52±0.22 DPM/mm2; P<0.001). The samples from subjects with atypical echocardiographic features of amyloidosis showed quantitatively more intense 18F-florbetapir–specific uptake compared with control samples (1.50±0.17 versus 0.94±0.43 DPM/mm2; P=0.004), despite smaller amyloid extent than in subjects with typical echocardiograms. Conclusions—18F-florbetapir specifically binds to myocardial AL and ATTR deposits in humans and offers the potential to screen for the 2 most common types of myocardial amyloid.