Measuring Digital Pathology Throughput and Tissue Dropouts.

BACKGROUND: Digital pathology operations that precede viewing by a pathologist have a substantial impact on costs and fidelity of the digital image. Scan time and file size determine throughput and storage costs, whereas tissue omission during digital capture ("dropouts") compromises downstream interpretation. We compared how these variables differ across scanners. METHODS: A 212 slide set randomly selected from a gynecologic-gestational pathology practice was used to benchmark scan time, file size, and image completeness. Workflows included the Hamamatsu S210 scanner (operated under default and optimized profiles) and the Leica GT450. Digital tissue dropouts were detected by the aligned overlay of macroscopic glass slide camera images (reference) with images created by the slide scanners whole slide images. RESULTS: File size and scan time were highly correlated within each platform. Differences in GT450, default S210, and optimized S210 performance were seen in average file size (1.4 vs. 2.5 vs. 3.4 GB) and scan time (93 vs. 376 vs. 721 s). Dropouts were seen in 29.5% (186/631) of successful scans overall: from a low of 13.7% (29/212) for the optimized S210 profile, followed by 34.6% (73/211) for the GT450 and 40.4% (84/208) for the default profile S210 profile. Small dislodged fragments, "shards," were dropped in 22.2% (140/631) of slides, followed by tissue marginalized at the glass slide edges, 6.2% (39/631). "Unique dropouts," those for which no equivalent appeared elsewhere in the scan, occurred in only three slides. Of these, 67% (2/3) were "floaters" or contaminants from other cases. CONCLUSIONS: Scanning speed and resultant file size vary greatly by scanner type, scanner operation settings, and clinical specimen mix (tissue type, tissue area). Digital image fidelity as measured by tissue dropout frequency and dropout type also varies according to the tissue type and scanner. Dropped tissues very rarely (1/631) represent actual specimen tissues that are not represented elsewhere in the scan, so in most cases cannot alter the diagnosis. Digital pathology platforms vary in their output efficiency and image fidelity to the glass original and should be matched to the intended application.

Twists and turns from "tumor in tumor" profiling: surveillance of chronic lymphocytic leukemia (CLL) leads to detection of a lung adenocarcinoma, whose genomic characterization alters the original hematologic diagnosis.

Comprehensive characterization of somatic genomic alterations has led to fundamental shifts in our understanding of tumor biology. In clinical practice, these studies can lead to modifications of diagnosis and/or specific treatment implications, fulfilling the promise of personalized medicine. Herein, we describe a 78-yr-old woman under surveillance for long-standing untreated chronic lymphocytic leukemia (CLL). Molecular studies from a peripheral blood specimen revealed a TP53 p.V157F mutation, whereas karyotype and fluorescence in situ hybridization (FISH) identified a 17p deletion, trisomy 12, and no evidence of IGH-CCND1 rearrangement. Positron emission tomography-computed tomography scan identified multistation intra-abdominal lymphadenopathy and a pulmonary nodule, and subsequent pulmonary wedge resection confirmed the presence of a concurrent lung adenocarcinoma. Targeted next-generation sequencing of the lung tumor identified an EGFR in-frame exon 19 deletion, two TP53 mutations (p.P152Q, p.V157F), and, unexpectedly, a IGH-CCND1 rearrangement. Follow-up immunohistochemistry (IHC) studies demonstrated a cyclin D1-positive lymphoid aggregate within the lung adenocarcinoma. The presence of the TP53 p.V157F mutation in the lung resection, detection of an IGH-CCND1 rearrangement, and cyclin D1 positivity by IHC led to revision of the patient's hematologic diagnosis and confirmed the extranodal presence of mantle cell lymphoma within the lung mass, thus representing a "tumor in tumor." Manual review of the sequencing data suggested the IGH-CCND1 rearrangement occurred via an insertional event, whose size precluded detection by original FISH studies. Thus, routine imaging for this patient's known hematologic malignancy led to detection of an unexpected solid tumor, whose subsequent precision medicine studies in the solid tumor redefined the original hematological diagnosis.

Consistency and reproducibility of next-generation sequencing in cytopathology: A second worldwide ring trial study on improved cytological molecular reference specimens.

BACKGROUND: Artificial genomic reference standards in a cytocentrifuge/cytospin format with well-annotated genomic data are useful for validating next-generation sequencing (NGS) on routine cytopreparations. Here, reference standards were optimized to be stained by different laboratories before DNA extraction and to contain a lower number of cells (2 × 105 ). This was done to better reflect the clinical challenge of working with insufficient cytological material. METHODS: A total of 17 worldwide laboratories analyzed customized reference standard slides (slides A-D). Each laboratory applied its standard workflow. The sample slides were engineered to harbor epidermal growth factor receptor (EGFR) c.2235_2249del15 p.E746_A750delELREA, EGFR c.2369C>T p.T790M, Kirsten rat sarcoma viral oncogene homolog (KRAS) c.38G>A p.G13D, and B-Raf proto-oncogene, serine/threonine kinase (BRAF) c.1798_1799GT>AA p.V600K mutations at various allele frequencies (AFs). RESULTS: EGFR and KRAS mutation detection showed excellent interlaboratory reproducibility, especially on slides A and B (10% and 5% AFs). On slide C (1% AF), either the EGFR mutation or the KRAS mutation was undetected by 10 of the 17 laboratories (58.82%). A reassessment of the raw data in a second-look analysis highlighted the mutations (n = 10) that had been missed in the first-look analysis. BRAF c.1798_1799GT>AA p.V600K showed a lower concordance rate for mutation detection and AF quantification. CONCLUSIONS: The data show that the detection of low-abundance mutations is still clinically challenging and may require a visual inspection of sequencing reads to detect. Genomic reference standards in a cytocentrifuge/cytospin format are a valid tool for regular quality assessment of laboratories performing molecular studies on cytology with low-AF mutations.

Implementing the DICOM Standard for Digital Pathology.

BACKGROUND: Digital Imaging and Communications in Medicine (DICOM®) is the standard for the representation, storage, and communication of medical images and related information. A DICOM file format and communication protocol for pathology have been defined; however, adoption by vendors and in the field is pending. Here, we implemented the essential aspects of the standard and assessed its capabilities and limitations in a multisite, multivendor healthcare network. METHODS: We selected relevant DICOM attributes, developed a program that extracts pixel data and pixel-related metadata, integrated patient and specimen-related metadata, populated and encoded DICOM attributes, and stored DICOM files. We generated the files using image data from four vendor-specific image file formats and clinical metadata from two departments with different laboratory information systems. We validated the generated DICOM files using recognized DICOM validation tools and measured encoding, storage, and access efficiency for three image compression methods. Finally, we evaluated storing, querying, and retrieving data over the web using existing DICOM archive software. RESULTS: Whole slide image data can be encoded together with relevant patient and specimen-related metadata as DICOM objects. These objects can be accessed efficiently from files or through RESTful web services using existing software implementations. Performance measurements show that the choice of image compression method has a major impact on data access efficiency. For lossy compression, JPEG achieves the fastest compression/decompression rates. For lossless compression, JPEG-LS significantly outperforms JPEG 2000 with respect to data encoding and decoding speed. CONCLUSION: Implementation of DICOM allows efficient access to image data as well as associated metadata. By leveraging a wealth of existing infrastructure solutions, the use of DICOM facilitates enterprise integration and data exchange for digital pathology.

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.

Preoperative cytologic interpretation of noninvasive follicular thyroid neoplasm with papillary-like nuclear features: a 1-year multi-institutional experience.

INTRODUCTION: Encapsulated follicular variant of papillary thyroid carcinoma (PTC) has an indolent behavior; hence, a change in terminology to "noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP)" has been proposed. Data are scant on the fine-needle aspiration (FNA) diagnosis of nodules proven to be NIFTP upon resection. The aim was to evaluate the FNA diagnosis of nodules diagnosed as NIFTP upon resection. MATERIALS AND METHODS: The archives of 8 participating institutions were searched for thyroid resection specimens obtained in a 1-year period, and pertinent demographic and pathology data were recorded. RESULTS: 2226 thyroid surgeries were performed over the indicated time period. NIFTP was diagnosed in 6.3% of cases; 118 patients (119 nodules) with NIFTP and available preoperative thyroid FNA were included. Preoperative cytologic diagnosis were: non-diagnostic: 0.8%; benign: 5.9%; atypia of undetermined significance/follicular lesion of undetermined significance: 42.9%; follicular neoplasm/suspicious for a follicular neoplasm: 31.0%; suspicious for malignancy: 15.9%; malignant: 3.4%. Molecular data was available for 49 cases, either by Afirma or ThyGenX/ThyroSeq. Of the Afirma cases, 11% were classified as "benign", 2% as "indeterminate", and 87% as "suspicious"; of the ThyGenX/ThyroSeq cases, 50% had NRAS mutations, 20% demonstrated KRAS mutations, 20% showed HRAS mutations, and 10% showed a BRAF mutation (K601E). CONCLUSIONS: NIFTP are tumors demonstrating nuclear features similar to those seen in PTC. Our series shows that a preoperative diagnosis of "suspicious for malignancy" or "malignant" is uncommon in NIFTP, suggesting that there are sufficient cytomorphologic differences between PTC and NIFTP to allow for the suspicion of NIFTP on FNA specimens.

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

BACKGROUND: 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. METHODS: This study retrospectively reviewed quality metrics from consecutive smear/LBP, core BX, and cell block (CB) cases run on a hybrid-capture NGS assay interrogating 309 cancer-related genes. The following quality metrics were compared: adequacy rate, initial DNA concentration, postshearing fragment size, post-library preparation fragment size, fragment size difference, insert size, total reads, passing-filter reads aligned, percent passing-filter unique reads aligned, mean target coverage, percentage of loci with >100× coverage, percent duplication rate, percent selected bases, and percent usable bases on bait. RESULTS: Twenty-three of 26 smears/LBPs (88%) were successfully sequenced, whereas 77 of 87 core BXs (89%) and 29 of 30 CBs (97%) were. The mean target coverage, median insert size, and percent usable bases were significantly higher in the smear/LBP category. The postshearing fragment size and the percent duplication were significantly lower for smears/LBPs. CONCLUSIONS: The adequacy rate of cytology smears/LBPs for NGS is comparable to that of core BXs or CBs. Increased values for the mean insert size, mean target coverage, and percent usable bases, along with a lower duplication rate, suggest that smears/LBPs provide higher quality DNA than formalin-fixed material. Cytology smears/LBPs can serve as a valuable source of material for molecular testing. Cancer Cytopathol 2017;125:786-94. © 2017 American Cancer Society.

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

BACKGROUND: 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. METHODS: Quantitative cytological molecular reference slides were produced with cell lines designed to harbor concurrent mutations in the EGFR, KRAS, NRAS, BRAF, and PIK3CA genes at various allelic ratios, including low allele frequencies (AFs; 1%). This interlaboratory ring trial study included 14 institutions across the world that performed multigene mutational assays, from tissue extraction to data analysis, on these reference slides, with each laboratory using its own mutation analysis platform and methodology. RESULTS: All laboratories using NGS (n = 11) successfully detected the study's set of mutations with minimal variations in the means and standard errors of variant fractions at dilution points of 10% (P = .171) and 5% (P = .063) despite the use of different sequencing platforms (Illumina, Ion Torrent/Proton, and Roche). However, when mutations at a low AF of 1% were analyzed, the concordance of the NGS results was low, and this reflected the use of different thresholds for variant calling among the institutions. In contrast, laboratories using matrix-assisted laser desorption/ionization-time of flight (n = 2) showed lower concordance in terms of mutation detection and mutant AF quantification. CONCLUSIONS: Quantitative molecular reference slides are a useful tool for monitoring the performance of different multigene mutational assays, and this could lead to better standardization of molecular cytopathology procedures. Cancer Cytopathol 2017;125:615-26. © 2017 American Cancer Society.

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.

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 (Pearson's 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.

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.

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

BACKGROUND: (18)F-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 (18)F-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 (18)F-florbetapir and cold florbetapir compound and digital autoradiography. Total and nonspecific binding of (18)F-florbetapir was determined using the maximum signal intensity values. Specific binding of (18)F-florbetapir was calculated by subtracting nonspecific from total binding measurements (in decays per minute/mm(2), DPM mm(2)) and was compared with cardiac structure and function on echocardiography and the histological extent of amyloid deposits. Diffuse or focally increased (18)F-florbetapir uptake was noted in all AL and ATTR samples and in none of the control samples. Compared with control samples, mean (18)F-florbetapir-specific uptake was significantly higher in the amyloid samples (0.94±0.43 versus 2.00±0.58 DPM/mm(2); P<0.001), and in the AL compared with the ATTR samples (2.48±0.40 versus 1.52±0.22 DPM/mm(2); P<0.001). The samples from subjects with atypical echocardiographic features of amyloidosis showed quantitatively more intense (18)F-florbetapir-specific uptake compared with control samples (1.50±0.17 versus 0.94±0.43 DPM/mm(2); P=0.004), despite smaller amyloid extent than in subjects with typical echocardiograms. CONCLUSIONS: (18)F-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.

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

AIMS: 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. METHODS AND RESULTS: One hundred and three cases of formalin-fixed, paraffin-embedded SCLC were examined. Myc protein expression was scored according to the extent of immunohistochemical staining. MYC copy number (CN) was evaluated with dual-colour chromogenic in-situ hybridization (CISH) for the MYC locus and a chromosome 8 (Chr8) centromeric control. Amplification was defined as a MYC/Chr8 ratio of ≥2. Thirty-eight per cent of SCLCs had some degree of Myc protein expression, and 9% of cases were MYC-amplified. MYC CN was significantly correlated with the extent of Myc protein expression (Spearman's ρ = 0.57, P < 0.01). There was no significant association between Myc expression or CN and clinicopathological features. CONCLUSIONS: MYC amplification by CISH was identified in 9% of SCLCs, and correlated with protein expression. As novel Myc-targeted therapies are developed, CISH and IHC should be considered as biomarkers of Myc pathway dysregulation in SCLC.

Pulmonary large cell carcinoma lacking squamous differentiation is clinicopathologically indistinguishable from solid-subtype adenocarcinoma.

CONTEXT: Pulmonary large cell carcinoma (LCC) includes tumors not readily diagnosed as adenocarcinoma (ADC) or squamous cell carcinoma on morphologic grounds, without regard to immunophenotype, according to the World Health Organization (WHO). This ambiguous designation may cause confusion over selection of mutation testing and directed therapies. Several groups have proposed the use of immunohistochemistry (IHC) to recategorize LCC as ADC or squamous cell carcinoma; however, it remains unclear if strictly defined LCCs are a clinicopathologically distinct lung tumor subset. OBJECTIVE: To compare the pathologic, molecular, and clinical features of 2 morphologically similar tumors: solid-subtype ADC and LCC. DESIGN: Tumors were included on the basis of solid growth pattern; tumors with squamous or neuroendocrine differentiation were excluded. Solid ADC (n = 42) and LCC (n = 57) were diagnosed by using WHO criteria (5 intracellular mucin droplets in ≥2 high-power fields for solid ADC) and tested for KRAS, EGFR, and ALK alterations. RESULTS: Both solid ADC and LCC groups were dominated by tumors with "undifferentiated"-type morphology and both had a high frequency of thyroid transcription factor 1 expression. KRAS was mutated in 38% of solid ADCs versus 43% of LCCs (P = .62). One ALK-rearranged and 1 EGFR-mutated tumor were detected in the solid ADC and LCC groups, respectively. There were no significant differences in clinical features or outcomes; the prevalence of smoking in both groups was greater than 95%. CONCLUSIONS: Other than a paucity of intracellular mucin, LCC lacking squamous or neuroendocrine differentiation is indistinguishable from solid-subtype ADC. We propose the reclassification of these tumors as mucin-poor solid adenocarcinomas.

The outcome of abstracts presented at the United States and Canadian Academy of Pathology annual meetings.

Many abstracts presented at scientific meetings are never published as articles in peer-reviewed journals. Using PubMed search and custom computer programs, we retrospectively reviewed all 4824 abstracts presented at the United States and Canadian Academy of Pathology annual meetings from 2005 to 2007, and found an overall publication rate of 36% for a 3-year maximal follow-up. This rate is comparable with that of other medical societies with published data. The publication rate varied from 10 to 62% among different subspecialties. The format of presentation, either platform or poster, was also a significant predictor of outcome, with 42-50% publication rate for platform abstracts and 32-36% for poster abstracts. Country of origin and the use of statistical methods did not seem to affect outcome significantly. The average time from abstract submission to article publication was 18 months. Seven journals accounted for over half of all publications, and the top three journals were American Journal of Surgical Pathology (16.2%), Modern Pathology (9.1%), and American Journal of Clinical Pathology (8.3%).

Antitumor effects of an imidazoquinoline in renal cell carcinoma.

OBJECTIVES: To evaluate the effects of imidazoquinolines in renal cell carcinoma (RCC). METHODS: In vitro experiments were carried out using mouse (RENCA) and human (CAKI-1, CAKI-2, and A-498) RCC cell lines. Toll-like receptor-7 (TLR7) expression was assessed by Western blot. We determined the ability of imidazoquinolines to induce apoptosis and inhibit cell viability in vitro. For in vivo experiments, RENCA cells were injected into the tail vein of syngeneic mice. One week after injection, mice were given oral imidazoquinoline or placebo for 14 days. Mice were then sacrificed, and lungs were inspected for tumor nodules. Immunohistochemical staining was used to assess apoptosis in vivo. RESULTS: Toll-like receptor-7 was expressed in all cell lines tested, with RENCA cells showing the highest level of expression. Imidazoquinolines inhibited in vitro cell viability of RENCA, CAKI-2, and A-498 cell lines in a time-dependent manner. Viability of CAKI-1 was not inhibited significantly. Apoptosis induction was pronounced in RENCA cells treated with imidazoquinoline. Compared with placebo, oral imidazoquinoline significantly reduced the number of pulmonary metastasis and increased cell death in vivo. CONCLUSIONS: Imidazoquinolines inhibit cell viability and cause deoxyribonucleic acid fragmentation leading to apoptosis in RCC cell lines, potentially working through the TLR7 expressed by RCC cell lines. Preliminary data from a mouse model of metastatic RCC also suggest antitumor effects and induction of apoptosis in vivo.

Negative influence of changing biopsy practice patterns on the predictive value of prostate-specific antigen for cancer detection on prostate biopsy.

BACKGROUND: A correlation between prostate specific antigen (PSA) level and positive prostate biopsy rate was established in an era when biopsy practice patterns were different from what they are today. We evaluated if changes in biopsy practice patterns have affected the ability of PSA to predict cancer detection on prostate biopsy in the current era. METHODS: Of 3634 prostate biopsies performed from 1993-2005, 1607 met criteria for analysis. Biopsy data were divided into 3 time-cohorts (1993-1997, 1998-2001, and 2002-2005) to assess for practice patterns shifts and correlation between PSA and biopsy results. RESULTS: Significant changes in biopsy practice patterns included an increase in biopsy cores and more frequent use of PSA 2.5-3.99 ng/mL as a biopsy indication. In men with normal DRE, a moderate correlation between PSA and positive biopsy rate did exist from 1993-1997, but was subsequently lost. On multivariate analysis, PSA was not a significant predictor of biopsy result in men with normal DRE. CONCLUSIONS: Early in the PSA era, the predictive power of PSA depended on multiple factors: high prevalence of disease, higher prevalence of high-grade disease, and low likelihood of prostate cancer diagnosis in men with low PSA. Now, beyond the culling effect of increased biopsy incidence and with shifted biopsy practice patterns, the correlation between PSA and biopsy result is lost in men with normal DRE. Diagnosing a higher proportion of tumors in men with a PSA between 2.0-4.0 ng/mL has negatively influenced the predictive value of PSA for cancer detection.

Tumour growth inhibition by an imidazoquinoline is associated with c-Myc down-regulation in urothelial cell carcinoma.

OBJECTIVE: To detect and characterize the potential role of c-Myc in the inhibition of proliferation and induction of cell death of urothelial cell carcinoma by imidazoquinolines, Toll-like receptor-7 (TLR7) agonists, that are thought to exert their immunogenic effects through the MyD88/NF-kappaB pathway. MATERIALS AND METHODS: Human (T24) and murine (MBT-2) bladder cancer cell lines were cultured in normal culture medium or medium supplemented with imidazoquinoline. The effects of imidazoquinoline on gene expression, transcription and tumorigenesis were then evaluated. Effects of imidazoquinoline on in vivo bladder tumour growth and gene expression were investigated using a mouse model of orthotopic bladder cancer. RESULTS: There was a dose-dependent decrease in c-Myc expression in bladder cancer cells treated with imidazoquinoline; the transcriptional activity of c-Myc was also significantly reduced. Furthermore, the in vitro proliferation and tumorigenesis of MBT-2 cells were suppressed in a dose-dependent manner. For in vivo experiments, a third of mice with bladder cancer treated with intravesical imidazoquinoline showed evidence of residual bladder tumour, vs all the placebo-treated mice. In vivo expression of c-Myc, cyclin D2 and proliferating cell nuclear antigen in the bladder tumour tissue were also down-regulated. CONCLUSIONS: Imidazoquinolines can inhibit c-Myc expression and directly affect cell growth and tumorigenesis of bladder cancer cells, independent of an immune response. These direct effects might be synergistic with previously described immunogenic actions of imidazoquinolines. Our findings could broaden the potential application of imidazoquinoline therapy beyond dermatological malignancies, and further clinical studies are warranted.