Search

There have been few clinically useful targetable biomarkers in uterine cervical carcinomas. Estrogen receptor (ER), HER2, and fibroblast activation protein (FAP) are potential therapeutic or theranostic targets in other gynecologic and genitourinary carcinoma types. We determined the immunohistochemical expression patterns of these markers in treatment-naive cervical carcinoma, and whether expression correlated with clinical outcomes after definitive chemoradiation therapy. Tissue microarrays were created from 71 patient samples taken before therapy (57 squamous cell carcinomas and 14 nonsquamous cell carcinomas) and stained for ER, HER2, and FAP. ER was positive in 25/70 cases (36%). Of 66 tumors with evaluable HER2 staining, only 1 had positive (3+) staining (3%, positive for HER2 amplification by fluorescence in situhybridization), and 1 had equivocal (2+) staining (negative for amplification by fluorescence in situhybridization). The remainder were negative for HER2 overexpression. FAP expression was widely variably in the tumor stroma. ER positivity and FAP expression did not correlate with cervical recurrence, pelvic recurrence, distant recurrence, or cancer death. In conclusion, HER2 amplification is very rare in nonmetastatic treatment-naive cervical carcinomas, but if present, could represent a target for antibody therapy. ER and FAP were expressed in a subset of tumors, but expression did not correlate with clinical outcomes. These immunohistochemical markers do not demonstrate prognostic significance in treatment-naive cervical cancer, but they may have utility in targeted therapy or imaging.

The detection of recurrent gene fusions can help confirm diagnoses in solid tumors, particularly when the morphology and staining are unusual or nonspecific, and can guide therapeutic decisions. Although fluorescence in situhybridization and PCR are often used to identify fusions, the rearrangement must be suspected, with only a few prioritized probes run. It was hypothesized that the Illumina TruSight RNA Fusion Panel, which detects fusions of 507 genes and their partners, would uncover fusions with greater sensitivity than other approaches, leading to changes in diagnosis, prognosis, or therapy. Targeted RNA sequencing was performed on formalin-fixed, paraffin-embedded sarcoma and carcinoma cases in which fluorescence in situhybridization, RT-PCR, or DNA-based sequencing was conducted during the diagnostic workup. Of the 153 cases, 138 (90%) were sequenced with adequate quality control metrics. A total of 101 of 138 (73%) cases were concordant by RNA sequencing and the prior test method. RNA sequencing identified an additional 30 cases (22%) with fusions that were not detected by conventional methods. In seven cases (5%), the additional fusion information provided by RNA sequencing would have altered diagnosis and management. A total of 19 novel fusion pairs (not previously described in the literature) were discovered (14%). Overall, the findings show that a targeted RNA-sequencing method can detect gene fusions in formalin-fixed, paraffin-embedded specimens with high sensitivity.

Severe congenital neutropenia (SCN) is a collection of diverse disorders characterized by chronically low absolute neutrophil count in the peripheral blood, increased susceptibility to infection, and a significant predisposition to the development of myeloid malignancies. SCN can be acquired or inherited. Inherited forms have been linked to variants in a group of diverse genes involved in the neutrophil-differentiation process. Variants that promote resistance to treatment have also been identified. Thus, genetic testing is important for the diagnosis, prognosis, and management of SCN. Herein we describe clinically validated assay developed for assessing patients with suspected SCN. The assay is performed from a whole-exome backbone. Variants are called across all coding exons, and results are filtered to focus on 48 genes that are clinically relevant to SCN. Validation results indicated 100% analytical sensitivity and specificity for the detection of constitutional variants among the 48 reportable genes. To date, 34 individuals have been referred for testing (age range: birth to 67 years). Several pathogenic and likely pathogenic variants have been identified, including one in a patient with late-onset disease. The pattern of cases referred for testing suggests that this assay has clinical utility in a broader spectrum of patients beyond those in the pediatric population who have classic early-onset symptoms characteristic of SCN.

NUT midline carcinoma (NMC) is a rare, aggressive poorly differentiated carcinoma genetically defined by NUTM1gene rearrangement. The purpose of this study was to determine the tumor mutational burden (TMB) and the expression of immunohistochemical (IHC) markers in NMCs that are generally used to identify patients that might benefit from checkpoint immunotherapy. Three cases in a 39-year-old male (case 1) and two 13-year-old females (cases 2, 3) were identified from departmental files, with confirmation by NUT IHC and 15q14 rearrangement by fluorescent in situ hybridization. Normal-tumor paired whole exome sequencing (WES) was applied to determine TMB. IHC for DNA mismatch repair proteins, Programmed cell death ligand 1, programmed cell death 1 (PD1), and CD8 was also performed. WES yielded a TMB of 7.61 and 1.52 per Mbp in the primary and pulmonary metastasis in case 1, respectively, and a TMB of 1.04 per Mbp in the primary tumor of case 2. Programmed cell death ligand 1 tumor proportion score was 20%, 1%, and 0% and combined positive score was 25, 5, and 0 in cases 1, 2, and 3, respectively; PD1 stain counts were 25, 52, and 35 per high-power field and the PD1/CD8 ratio was 95%, 95%, and 99% in cases 1, 2, and 3, respectively. The CD8 count per high-power field was 15, 33, and 30 per high-power field in cases 1, 2, and 3, respectively. Mismatch repair IHCs showed retained staining. Although the number of cases is limited, this study is the first to investigate checkpoint immunotherapy markers in NMCs and the results demonstrate no clear biomarker association. However, the results suggest that, if checkpoint therapy is under consideration, a comprehensive workup utilizing WES and IHC is warranted.

PCR amplification, a key step in next-generation sequencing (NGS) library construction, can generate an unlimited amount of product from limited input; however, it cannot create more information than was present in the original template. Thus, NGS libraries can be made from very little DNA, but reducing the input may compromise assay sensitivity in ways that are difficult to ascertain unless library complexity (ie, the number of unique DNA molecules represented in the library) and depth of coverage with unique sequence reads (those derived from input DNA molecules) versus duplicate sequence reads (those resulting from overamplification of particular molecules) are discretely measured. A series of experiments was performed to explore the impact of low DNA input on an amplicon-based NGS assay using unique molecular identifiers to track unique versus duplicate reads. At high sequencing depths, unique and total (unique plus duplicate) read coverage are not well correlated, so increasing the number of sequenced reads does not necessarily improve sensitivity. Unique coverage depth tends to improve with more input, but improvements are not consistent. Fluctuations in library complexity complicated variant detection using both standardized and clinical specimens, often resulting in technical replicates with vastly different estimates of variant allelic fraction. In conclusion, depth of coverage with unique reads must be tracked in clinical NGS to ensure that sensitivity and accuracy are maintained.

Clinical next-generation sequencing assays are often run on tumor specimens without a matched normal specimen, which complicates the differentiation of germline from somatic variants. In tumor-only testing, population data are often used to infer germline status, though no consensus exists on the exact population frequency (PF) cutoff above which a variant should be considered likely germline. In this study, five population databases plus the Catalog of Somatic Mutations in Cancer were used to demonstrate the impact of changing the PF cutoff on assignment of variants as germline versus somatic. The 1% to 2% PF cutoffs widely used in bioinformatic pipelines resulted in high sensitivity for classification of somatic variants, but unnecessarily reduced sensitivity for germline variants. Using optimized PF cutoffs, the source of variants in The Cancer Genome Atlas (TCGA) data could be predicted with >95% accuracy. Further exploration of four TCGA cancer data sets indicated that the optimal cutoff is influenced by both cancer type and the assay region of interest. Comparing TCGA data to data generated from a clinical, hybridization capture test (approximately 615 kb capture space) showed that PF cutoffs may not be transferable between assays, even when the gene set is held constant. Thus, filtering approaches need to be carefully designed and optimized, and should be assay-specific to support tumor-only testing until tumor-normal testing becomes routine in the clinical setting.

Molecular diagnostic techniques are part of the ancillary arsenal of anatomic pathologists. Advances in technology and knowledge regarding disease pathogenesis, tumorigenesis, and immune function contribute to the development of these assays. However, each technique, if applied incorrectly or in ignorance, can lead to difficulties in execution or errors in interpretation. In this review of commonly used molecular diagnostic tests, including immunohistochemistry, microsatellite instability testing, chromosomal microarray testing, and conventional and next-generation sequencing, the emphasis will be on potential pitfalls and considerations for each platform. Emerging technologies that may be used in clinical applications in the near future will also be discussed. An understanding of the methodologies, advantages, and drawbacks of molecular assays will help pathologists aid in diagnostic and therapeutic decisions.

From a technical perspective, specimen identity determination in surgical pathology over the last several decades has primarily focused on analysis of repetitive DNA sequences, specifically microsatellite repeats. However, a number of techniques have recently been developed that have similar, if not greater, utility in surgical pathology, most notably analysis of single nucleotide polymorphism (SNPs) and gene panels by next generation sequencing (NGS). For cases with an extremely limited sample or a degraded sample, sequence analysis of mitochondrial DNA continues to be the method of choice. From a diagnostic perspective, interest in identity determination in surgical pathology is usually centered on resolving issues of specimen provenance due to specimen labeling/accessioning deficiencies and possible contamination, but is also frequently performed in cases for which the patient's clinical course following definitive therapy is remarkably atypical, in cases of an unexpected diagnosis, and by patient request for “peace of mind”. However, the methods used for identity determination have a much broader range of applications in surgical pathology beyond tissue provenance analysis. The methods can be used to provide ancillary information for cases in which the histomorphology is not definitively diagnostic, as for example for tumors that have a virtually identical microscopic appearance but for which the differential diagnosis includes synchronous/metachronous tumors versus a metastasis, and for the diagnosis of hydropic early gestations versus hydatidiform molar pregnancies. The methods also have utility in several other clinical settings, for example to rule out a donor-transmitted malignancy in a transplant recipient, to monitor bone marrow transplant engraftment, and to evaluate natural chimerism.

The majority of papillary thyroid carcinoma (PTC) cases comprise classic papillary (C-PTC) and follicular variant (FV-PTC) histologic sub-types. Historically, clinical equivalency was assumed, but recent data suggest C-PTC may have poorer outcomes. However, large single-institution series with long-term outcomes of C-PTC and FV-PTC, using modern pathologic criteria for FV-PTC, are needed. Our objective was to compare prevalence and impact of clinicopathologic factors, including BRAFmutation status, on long-term outcomes of C-PTC and FV-PTC. We hypothesized that patients with C-PTC would have higher risk disease features and worse survival outcomes. This retrospective study included 1293 patients treated at a single, US academic institution between 1943 and 2009 with mean follow-up of 8.6 years. All patients underwent either partial or total thyroidectomy and had invasive C-PTC or FV-PTC per modern pathology criteria. Primary study measurements included differences in recurrence-free survival (RFS), disease-specific survival (DSS) and associations with clinicopathologic factors including the BRAFmutation. Compared to FV-PTC, C-PTC was associated with multiple features of high-risk disease (P< 0.05) and significantly reduced RFS and DSS. Survival differences were consistent across univariate, multivariate and Kaplan–Meier analyses. BRAFmutations were more common in C-PTC (P= 0.002). However, on Kaplan–Meier analysis, mutational status did not significantly impact RFS or DSS for patients with either histologic sub-type. C-PTC therefore indicates higher-risk disease and predicts for significantly poorer long-term outcomes when compared to FV-PTC. The nature of this difference in outcome is not explained by traditional histopathologic findings or by the BRAFmutation.

In lung adenocarcinoma, canonical EML4-ALKinversion results in a fusion protein with a constitutively active ALK kinase domain. Evidence of ALKrearrangement occurs in a minority (2–7%) of lung adenocarcinoma, and only ~60% of these patients will respond to targeted ALK inhibition by drugs such as crizotinib and ceritinib. Clinically, targeted anti-ALK therapy is often initiated based on evidence of an ALKgenomic rearrangement detected by fluorescence in situhybridization (FISH) of interphase cells in formalin-fixed, paraffin-embedded tissue sections. At the genomic level, however, ALKrearrangements are heterogeneous, with multiple potential breakpoints in EML4, and alternate fusion partners. Using next-generation sequencing of DNA and RNA together with ALK immunohistochemistry, we comprehensively characterized genomic breakpoints in 33 FISH-positive lung adenocarcinomas. Of these 33 cases, 29 (88%) had detectable DNA level ALK rearrangements involving EML4, KIF5B, or non-canonical partners including ASXL2, ATP6V1B1, PRKAR1A, and SPDYA. A subset of 12 cases had material available for RNA-Seq. Of these, eight of eight (100%) cases with DNA rearrangements showed ALK fusion transcripts from RNA-Seq; three of four cases (75%) without detectable DNA rearrangements were similarly negative by RNA-Seq, and one case was positive by RNA-Seq but negative by DNA next-generation sequencing. By immunohistochemistry, 17 of 19 (89%) tested cases were clearly positive for ALK protein expression; the remaining cases had no detectable DNA level rearrangement or had a non-canonical rearrangement not predicted to form a fusion protein. Survival analysis of patients treated with targeted ALK inhibitors demonstrates a significant difference in mean survival between patients with next-generation sequencing confirmed EML4-ALKrearrangements, and those without (20.6 months vs5.4 months, P<0.01). Together, these data demonstrate abundant genomic heterogeneity among ALK-rearranged lung adenocarcinoma, which may account for differences in treatment response with targeted ALK inhibitors.

In lung adenocarcinoma, canonical EML4-ALKinversion results in a fusion protein with a constitutively active ALK kinase domain. Evidence of ALKrearrangement occurs in a minority (2–7%) of lung adenocarcinoma, and only ~60% of these patients will respond to targeted ALK inhibition by drugs such as crizotinib and ceritinib. Clinically, targeted anti-ALK therapy is often initiated based on evidence of an ALKgenomic rearrangement detected by fluorescence in situhybridization (FISH) of interphase cells in formalin-fixed, paraffin-embedded tissue sections. At the genomic level, however, ALKrearrangements are heterogeneous, with multiple potential breakpoints in EML4, and alternate fusion partners. Using next-generation sequencing of DNA and RNA together with ALK immunohistochemistry, we comprehensively characterized genomic breakpoints in 33 FISH-positive lung adenocarcinomas. Of these 33 cases, 29 (88%) had detectable DNA level ALK rearrangements involving EML4, KIF5B, or non-canonical partners including ASXL2, ATP6V1B1, PRKAR1A, and SPDYA. A subset of 12 cases had material available for RNA-Seq. Of these, eight of eight (100%) cases with DNA rearrangements showed ALK fusion transcripts from RNA-Seq; three of four cases (75%) without detectable DNA rearrangements were similarly negative by RNA-Seq, and one case was positive by RNA-Seq but negative by DNA next-generation sequencing. By immunohistochemistry, 17 of 19 (89%) tested cases were clearly positive for ALK protein expression; the remaining cases had no detectable DNA level rearrangement or had a non-canonical rearrangement not predicted to form a fusion protein. Survival analysis of patients treated with targeted ALK inhibitors demonstrates a significant difference in mean survival between patients with next-generation sequencing confirmed EML4-ALKrearrangements, and those without (20.6 months vs5.4 months, P<0.01). Together, these data demonstrate abundant genomic heterogeneity among ALK-rearranged lung adenocarcinoma, which may account for differences in treatment response with targeted ALK inhibitors.

Next-generation sequencing is increasingly used for clinical evaluation of patients presenting with thrombotic microangiopathies because it allows for simultaneous interrogation of multiple complement and coagulation pathway genes known to be associated with disease. However, the diagnostic yield is undefined in routine clinical practice. Historic studies relied on case–control cohorts, did not apply current guidelines for variant pathogenicity assessment, and used targeted gene enrichment combined with next-generation sequencing. A clinically enhanced exome, targeting ~54 Mb, was sequenced for 73 patients. Variant analysis and interpretation were performed on genes with biological relevance in thrombotic microangiopathy (C3,CD46, CFB, CFH, CFI, DGKE, and THBD). CFHR3-CFHR1 deletion status was also assessed using multiplex ligation-dependent probe amplification. Variants were classified using American College of Medical Genetics and Genomics guidelines. We identified 5 unique novel and 14 unique rare variants in 25% (18/73) of patients, including a total of 5 pathogenic, 4 likely pathogenic, and 15 variants of uncertain clinical significance. Nine patients had homozygous deletions in CFHR3-CFHR1. The diagnostic yield, defined as the presence of a pathogenic variant, likely pathogenic variant or homozygous deletion of CFHR3-CFHR1, was 25% for all patients tested. Variants of uncertain clinical significance were identified in 21% (15/73) of patients.These results illustrate the expected diagnositic yield in the setting of thrombotic microangiopathies through the application of standardized variant interpretation, and highlight the utility of such an approach. Sequencing a clinically enhanced exome to enable targeted, disease-specific variant analysis is a viable approach. The moderate rate of variants of uncertain clinical significance highlights the paucity of data surrounding the variants in our cohort and illustrates the need for expanded variant curation resources to aid in thrombotic microangiopathy-related disease variant classification.

Next-generation sequencing is increasingly used for clinical evaluation of patients presenting with thrombotic microangiopathies because it allows for simultaneous interrogation of multiple complement and coagulation pathway genes known to be associated with disease. However, the diagnostic yield is undefined in routine clinical practice. Historic studies relied on case–control cohorts, did not apply current guidelines for variant pathogenicity assessment, and used targeted gene enrichment combined with next-generation sequencing. A clinically enhanced exome, targeting ~54 Mb, was sequenced for 73 patients. Variant analysis and interpretation were performed on genes with biological relevance in thrombotic microangiopathy (C3,CD46, CFB, CFH, CFI, DGKE, and THBD). CFHR3-CFHR1deletion status was also assessed using multiplex ligation-dependent probe amplification. Variants were classified using American College of Medical Genetics and Genomics guidelines. We identified 5 unique novel and 14 unique rare variants in 25% (18/73) of patients, including a total of 5 pathogenic, 4 likely pathogenic, and 15 variants of uncertain clinical significance. Nine patients had homozygous deletions in CFHR3-CFHR1. The diagnostic yield, defined as the presence of a pathogenic variant, likely pathogenic variant or homozygous deletion of CFHR3-CFHR1, was 25% for all patients tested. Variants of uncertain clinical significance were identified in 21% (15/73) of patients.These results illustrate the expected diagnositic yield in the setting of thrombotic microangiopathies through the application of standardized variant interpretation, and highlight the utility of such an approach. Sequencing a clinically enhanced exome to enable targeted, disease-specific variant analysis is a viable approach. The moderate rate of variants of uncertain clinical significance highlights the paucity of data surrounding the variants in our cohort and illustrates the need for expanded variant curation resources to aid in thrombotic microangiopathy-related disease variant classification.

Supplemental Digital Content is available in the text.Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT), is a rare, highly lethal malignancy predominantly affecting young adult females. We report a patient with widely metastatic SCCOHT and concurrent uterine cervical pleomorphic liposarcoma. Clinical targeted next-generation sequencing was performed on both neoplasms and demonstrated hemizygous stop-gain TP53mutations (p.R196*), and wild-type SMARCA4in both tumors. Microarray analyses of both tumors revealed similar but not identical widespread loss of heterozygosity over most chromosomes associated with loss of chromosomal copy number in the SCCOHT and pleomorphic liposarcoma tumors, amplification of FGFR1in both tumors, and amplification of MYCin the SCCOHT. Immunohistochemistry demonstrated that SMARCA4 and SMARCB1 were retained in both tumors, and that SMARCA2 expression was retained but TP53 expression was lost in the SCCOHT. Germline testing using Sanger sequencing showed heterozygous TP53mutation, confirming the diagnosis of Li-Fraumeni syndrome. These findings are novel and for the first time associate SCCOHT with Li-Fraumeni syndrome.

A national workgroup convened by the Centers for Disease Control and Prevention identified principles and made recommendations for standardizing the description of sequence data contained within the variant file generated during the course of clinical next-generation sequence analysis for diagnosing human heritable conditions. The specifications for variant files were initially developed to be flexible with regard to content representation to support a variety of research applications. This flexibility permits variation with regard to how sequence findings are described and this depends, in part, on the conventions used. For clinical laboratory testing, this poses a problem because these differences can compromise the capability to compare sequence findings among laboratories to confirm results and to query databases to identify clinically relevant variants. To provide for a more consistent representation of sequence findings described within variant files, the workgroup made several recommendations that considered alignment to a common reference sequence, variant caller settings, use of genomic coordinates, and gene and variant naming conventions. These recommendations were considered with regard to the existing variant file specifications presently used in the clinical setting. Adoption of these recommendations is anticipated to reduce the potential for ambiguity in describing sequence findings and facilitate the sharing of genomic data among clinical laboratories and other entities.

Quality assurance for clinical next-generation sequencing (NGS)–based assays is difficult given the complex methods and the range of sequence variants such assays can detect. As the number and range of mutations detected by clinical NGS assays has increased, it is difficult to apply standard analyte-specific proficiency testing (PT). Most current proficiency testing challenges for NGS are methods-based PT surveys that use DNA from reference samples engineered to harbor specific mutations that test both sequence generation and bioinformatics analysis. These methods-based PTs are limited by the number and types of mutations that can be physically introduced into a single DNA sample. In silicoproficiency testing, which evaluates only the bioinformatics component of NGS assays, is a recently introduced PT method that allows for evaluation of numerous mutations spanning a range of variant classes. In silicoPT data sets can be generated from simulated or actual sequencing data and are used to test alignment through variant detection and annotation steps. In silicoPT has several advantages over the use of physical samples, including greater flexibility in tested variants, the ability to design laboratory-specific challenges, and lower costs. Herein, we review the use of in silicoPT as an alternative to traditional methods-based PT as it is evolving in oncology applications and discuss how the approach is applicable more broadly.

Merkel cell carcinoma is a highly aggressive cutaneous neuroendocrine tumor that has been associated with Merkel cell polyomavirus in up to 80% of cases. Merkel cell polyomavirus is believed to influence pathogenesis, at least in part, through expression of the large T antigen, which includes a retinoblastoma protein-binding domain. However, there appears to be significant clinical and morphological overlap between polyomavirus-positive and polyomavirus-negative Merkel cell carcinoma cases. Although much of the recent focus of Merkel cell carcinoma pathogenesis has been on polyomavirus, the pathogenesis of polyomavirus-negative cases is still poorly understood. We hypothesized that there are underlying human somatic mutations that unify Merkel cell carcinoma pathogenesis across polyomavirus status, and to investigate we performed whole exome sequencing on five polyomavirus-positive cases and three polyomavirus-negative cases. We found that there were no significant differences in the overall number of single-nucleotide variations, copy number variations, insertion/deletions, and chromosomal rearrangements when comparing polyomavirus-positive to polyomavirus-negative cases. However, we did find that the retinoblastoma pathway genes harbored a high number of mutations in Merkel cell carcinoma. Furthermore, the retinoblastoma gene (RB1) was found to have nonsense truncating protein mutations in all three polyomavirus-negative cases; no such mutations were found in the polyomavirus-positive cases. In all eight cases, the retinoblastoma pathway dysregulation was confirmed by immunohistochemistry. Although polyomavirus-positive Merkel cell carcinoma is believed to undergo retinoblastoma dysregulation through viral large T antigen expression, our findings demonstrate that somatic mutations in polyomavirus-negative Merkel cell carcinoma lead to retinoblastoma dysregulation through an alternative pathway. This novel finding suggests that the retinoblastoma pathway dysregulation leads to an overlapping Merkel cell carcinoma phenotype and that oncogenesis occurs through either a polyomavirus-dependent (viral large T antigen expression) or polyomavirus-independent (host somatic mutation) mechanism.

Merkel cell carcinoma is a highly aggressive cutaneous neuroendocrine tumor that has been associated with Merkel cell polyomavirus in up to 80% of cases. Merkel cell polyomavirus is believed to influence pathogenesis, at least in part, through expression of the large T antigen, which includes a retinoblastoma protein-binding domain. However, there appears to be significant clinical and morphological overlap between polyomavirus-positive and polyomavirus-negative Merkel cell carcinoma cases. Although much of the recent focus of Merkel cell carcinoma pathogenesis has been on polyomavirus, the pathogenesis of polyomavirus-negative cases is still poorly understood. We hypothesized that there are underlying human somatic mutations that unify Merkel cell carcinoma pathogenesis across polyomavirus status, and to investigate we performed whole exome sequencing on five polyomavirus-positive cases and three polyomavirus-negative cases. We found that there were no significant differences in the overall number of single-nucleotide variations, copy number variations, insertion/deletions, and chromosomal rearrangements when comparing polyomavirus-positive to polyomavirus-negative cases. However, we did find that the retinoblastoma pathway genes harbored a high number of mutations in Merkel cell carcinoma. Furthermore, the retinoblastoma gene (RB1) was found to have nonsense truncating protein mutations in all three polyomavirus-negative cases; no such mutations were found in the polyomavirus-positive cases. In all eight cases, the retinoblastoma pathway dysregulation was confirmed by immunohistochemistry. Although polyomavirus-positive Merkel cell carcinoma is believed to undergo retinoblastoma dysregulation through viral large T antigen expression, our findings demonstrate that somatic mutations in polyomavirus-negative Merkel cell carcinoma lead to retinoblastoma dysregulation through an alternative pathway. This novel finding suggests that the retinoblastoma pathway dysregulation leads to an overlapping Merkel cell carcinoma phenotype and that oncogenesis occurs through either a polyomavirus-dependent (viral large T antigen expression) or polyomavirus-independent (host somatic mutation) mechanism.

The identification of recurrent gene rearrangements in the clinical laboratory is the cornerstone for risk stratification and treatment decisions in many malignant tumors. Studies have reported that targeted next-generation sequencing assays have the potential to identify such rearrangements; however, their utility in the clinical laboratory is unknown. We examine the sensitivity and specificity of ALKand KMT2A (MLL)rearrangement detection by next-generation sequencing in the clinical laboratory. We analyzed a series of seven ALKrearranged cancers, six KMT2Arearranged leukemias, and 77 ALK/KMT2Arearrangement–negative cancers, previously tested by fluorescence in situ hybridization (FISH). Rearrangement detection was tested using publicly available software tools, including Breakdancer, ClusterFAST, CREST, and Hydra. Using Breakdancer and ClusterFAST, we detected ALKrearrangements in seven of seven FISH-positive cases and KMT2Arearrangements in six of six FISH-positive cases. Among the 77 ALK/KMT2AFISH-negative cases, no false-positive identifications were made by Breakdancer or ClusterFAST. Further, we identified one ALKrearranged case with a noncanonical intron 16 breakpoint, which is likely to affect its response to targeted inhibitors. We report that clinically relevant chromosomal rearrangements can be detected from targeted gene panel–based next-generation sequencing with sensitivity and specificity equivalent to that of FISH while providing finer-scale information and increased efficiency for molecular oncology testing.

Targeted next-generation sequencing (NGS) provides predictive and prognostic information in the routine care of patients with cancer. However, with increasing knowledge of the biological basis of cancer, NGS of the same gene sets can also provide diagnostic information in challenging cases, on the basis of identification of both known and novel variants, including single-nucleotide variants, insertions and deletions, copy number alterations, and translocations. Here, we present 3 clinical cases in which targeted NGS of hybrid-capture–enriched DNA from formalin-fixed, paraffin-embedded tumor samples provided unique and clinically important diagnostic andor staging information in 3 different challenging clinical scenarios. In the first patient, NGS played a key role in both diagnosis and staging in a patient with multiple tumors of the same histologic type. The second case demonstrates the ability of NGS to clarify the tumor tissue type in a single mass involving multiple organs, and thereby guide appropriate chemotherapy. The third case illustrates that information regarding susceptibility to targeted therapeutics can also clarify the original histologic diagnosis.

The microcystic elongated and fragmented (MELF) pattern of myoinvasion is a feature of some well-differentiated endometrial endometrioid adenocarcinomas that has been associated with poor prognosis. The myoinvasion in MELF-pattern tumors can be subtle and lead to underestimation of the depth of myometrial invasion resulting in tumor understaging; the presence of lymphvascular space invasion (LVSI) and lymph node metastasis in MELF-pattern tumors can also be subtle and lead to tumor understaging. To investigate the association of MELF-pattern invasion and lymph node metastasis, we reviewed a series of well-differentiated endometrioid adenocarcinomas and correlated the presence of MELF-pattern myoinvasion and LVSI with lymph node metastasis. Cases of T1 stage well-differentiated endometrioid adenocarcinomas with LVSI and a concurrent lymph node dissection were identified from departmental files. Hematoxylin and eosin-stained slides from the hysterectomy specimen and lymph nodes were reviewed for the presence of MELF-pattern myoinvasion, LVSI, and nodal metastasis. MELF-pattern myoinvasion was identified at least focally in 36 of cases. The pattern of LVSI differed between cases with MELF-pattern invasion and conventional-type invasion, as did the pattern of nodal metastasis. A statistically significantly higher rate of lymph node metastasis was present in cases with MELF-pattern invasion than in cases with conventional invasion, and the rate stratified with the proportion of MELF-pattern adenocarcinomas. MELF-pattern cases carry an increased rate of lymph node metastasis even within the subset of endometrioid tumors with LVSI, which has implications in routine clinical practice as it signals the importance of recognizing MELF-pattern myoinvasion.

Currently, oncology testing includes molecular studies and cytogenetic analysis to detect genetic aberrations of clinical significance. Next-generation sequencing (NGS) allows rapid analysis of multiple genes for clinically actionable somatic variants. The WUCaMP assay uses targeted capture for NGS analysis of 25 cancer-associated genes to detect mutations at actionable loci. We present clinical validation of the assay and a detailed framework for design and validation of similar clinical assays. Deep sequencing of 78 tumor specimens (≥1000× average unique coverage across the capture region) achieved high sensitivity for detecting somatic variants at low allele fraction (AF). Validation revealed sensitivities and specificities of 100% for detection of single-nucleotide variants (SNVs) within coding regions, compared with SNP array sequence data (95% CI = 83.4–100.0 for sensitivity and 94.2–100.0 for specificity) or whole-genome sequencing (95% CI = 89.1–100.0 for sensitivity and 99.9–100.0 for specificity) of HapMap samples. Sensitivity for detecting variants at an observed 10% AF was 100% (95% CI = 93.2–100.0) in HapMap mixes. Analysis of 15 masked specimens harboring clinically reported variants yielded concordant calls for 13/13 variants at AF of ≥15%. The WUCaMP assay is a robust and sensitive method to detect somatic variants of clinical significance in molecular oncology laboratories, with reduced time and cost of genetic analysis allowing for strategic patient management.

Next-generation sequencing (NGS) is becoming a common approach for clinical testing of oncology specimens for mutations in cancer genes. Unlike inherited variants, cancer mutations may occur at low frequencies because of contamination from normal cells or tumor heterogeneity and can therefore be challenging to detect using common NGS analysis tools, which are often designed for constitutional genomic studies. We generated high-coverage (1000×) NGS data from synthetic DNA mixtures with variant allele fractions (VAFs) of 25% to 2.5% to assess the performance of four variant callers, SAMtools, Genome Analysis Toolkit, VarScan2, and SPLINTER, in detecting low-frequency variants. SAMtools had the lowest sensitivity and detected only 49% of variants with VAFs of approximately 25%; whereas the Genome Analysis Toolkit, VarScan2, and SPLINTER detected at least 94% of variants with VAFs of approximately 10%. VarScan2 and SPLINTER achieved sensitivities of 97% and 89%, respectively, for variants with observed VAFs of 1% to 8%, with 98% sensitivity and 99% positive predictive value in coding regions. Coverage analysis demonstrated that 500× coverage was required for optimal performance. The specificity of SPLINTER improved with higher coverage, whereas VarScan2 yielded more false positive results at high coverage levels, although this effect was abrogated by removing low-quality reads before variant identification. Finally, we demonstrate the utility of high-sensitivity variant callers with data from 15 clinical lung cancers.

Next-generation sequencing (NGS) has emerged as a powerful technique for the detection of genetic variants in the clinical laboratory. NGS can be performed using DNA from FFPE tissue, but it is unknown whether such specimens are truly equivalent to unfixed tissue for NGS applications. To address this question, we performed hybridization-capture enrichment and multiplexed Illumina NGS for 27 cancer-related genes using DNA from 16 paired fresh-frozen and routine FFPE lung adenocarcinoma specimens and conducted extensive comparisons between the sequence data from each sample type. This analysis revealed small but detectable differences between FFPE and frozen samples. Compared with frozen samples, NGS data from FFPE samples had smaller library insert sizes, greater coverage variability, and an increase in C to T transitions that was most pronounced at CpG dinucleotides, suggesting interplay between DNA methylation and formalin-induced changes; however, the error rate, library complexity, enrichment performance, and coverage statistics were not significantly different. Comparison of base calls between paired samples demonstrated concordances of 99.99%, with 96.8% agreement in the single-nucleotide variants detected and 98% accuracy of NGS data when compared with genotypes from an orthogonal single-nucleotide polymorphism array platform. This study demonstrates that routine processing of FFPE samples has a detectable but negligible effect on NGS data and that these samples can be a reliable substrate for clinical NGS testing.

A recurrent somatic mutation frequently found in cytogenetically normal acute myeloid leukemia (AML) is internal tandem duplication (ITD) in the fms-related tyrosine kinase 3 gene (FLT3). This mutation is generally detected in the clinical laboratory by PCR and electrophoresis-based product sizing. As the number of clinically relevant somatic mutations in AML increases, it becomes increasingly attractive to incorporate FLT3ITD testing into multiplex assays for many somatic mutations simultaneously, using next-generation sequencing (NGS). However, the performance of most NGS analysis tools for identifying medium-size insertions such as FLT3ITD mutations is largely unknown. We used a multigene, targeted NGS assay to obtain deep sequence coverage (1000-fold) of FLT3and 26 other genes from 22 FLT3ITD-positive and 29 ITD-negative specimens to examine the performance of several commonly used NGS analysis tools for identifying FLT3ITD mutations. ITD mutations were present in hybridization-capture sequencing data, and Pindel was the only tool out of the seven tested that reliably detected these insertions. Pindel had 100% sensitivity (95% CI = 83% to 100%) and 100% specificity (95% CI = 88% to 100%) in our samples; Pindel provided accurate ITD insertion sizes and was able to detect ITD alleles present at estimated frequencies as low as 1%. These data demonstrate that FLT3ITDs can be reliably detected in panel-based, next-generation sequencing assays.

Leukemias are currently subclassified based on the presence of recurrent cytogenetic abnormalities and gene mutations. These molecular findings are the basis for risk-adapted therapy; however, such data are generally obtained by disparate methods in the clinical laboratory, and often rely on low-resolution techniques such as fluorescent in situ hybridization. Using targeted next generation sequencing, we demonstrate that the full spectrum of prognostically significant gene mutations including translocations, single nucleotide variants (SNVs), and insertions/deletions (indels) can be identified simultaneously in multiplexed sequence data. As proof of concept, we performed hybrid capture using a panel of 20 genes implicated in leukemia prognosis (covering a total of 1 Mbp) from five leukemia cell lines including K562, NB4, OCI-AML3, kasumi-1, and MV4–11. Captured DNA was then sequenced in multiplex on an Illumina HiSeq. Using an analysis pipeline based on freely available software we correctly identified DNA-level translocations in three of the three cell lines where translocations were covered by our capture probes. Furthermore, we found all published gene mutations in commonly tested genes including NPM1, FLT3, and KIT. The same methodology was applied to DNA extracted from the bone marrow of a patient with acute myeloid leukemia, and identified a t(9;11) translocation with single base accuracy as well other gene mutations. These results indicate that targeted next generation sequencing can be successfully applied in the clinical laboratory to identify a full spectrum of DNA mutations ranging from SNVs and indels to translocations. Such methods have the potential to both greatly streamline and improve the accuracy of DNA-based diagnostics.

Leukemias are currently subclassified based on the presence of recurrent cytogenetic abnormalities and gene mutations. These molecular findings are the basis for risk-adapted therapy; however, such data are generally obtained by disparate methods in the clinical laboratory, and often rely on low-resolution techniques such as fluorescent in situhybridization. Using targeted next generation sequencing, we demonstrate that the full spectrum of prognostically significant gene mutations including translocations, single nucleotide variants (SNVs), and insertions/deletions (indels) can be identified simultaneously in multiplexed sequence data. As proof of concept, we performed hybrid capture using a panel of 20 genes implicated in leukemia prognosis (covering a total of 1 Mbp) from five leukemia cell lines including K562, NB4, OCI-AML3, kasumi-1, and MV4–11. Captured DNA was then sequenced in multiplex on an Illumina HiSeq. Using an analysis pipeline based on freely available software we correctly identified DNA-level translocations in three of the three cell lines where translocations were covered by our capture probes. Furthermore, we found all published gene mutations in commonly tested genes including NPM1, FLT3, and KIT. The same methodology was applied to DNA extracted from the bone marrow of a patient with acute myeloid leukemia, and identified a t(9;11) translocation with single base accuracy as well other gene mutations. These results indicate that targeted next generation sequencing can be successfully applied in the clinical laboratory to identify a full spectrum of DNA mutations ranging from SNVs and indels to translocations. Such methods have the potential to both greatly streamline and improve the accuracy of DNA-based diagnostics.

Although next-generation sequencing (NGS) has been the domain of large genome centers, it is quickly becoming more accessible to general pathology laboratories. In addition to finding single-base changes, NGS allows for the detection of larger structural variants, including insertions/deletions, translocations, and viral insertions. We describe the use of targeted NGS on DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissue, and show that the short read lengths of NGS are ideally suited to fragmented DNA obtained from FFPE tissue. Further, we describe a novel method for performing hybrid-capture target enrichment using PCR-generated capture probes. As a model, we captured the 5.3-kb Merkel cell polyomavirus (MCPyV) genome in FFPE cases of Merkel cell carcinoma using inexpensive, PCR-derived capture probes, and achieved up to 37,000-fold coverage of the MCPyV genome without prior virus-specific PCR amplification. This depth of coverage made it possible to reproducibly detect viral genome deletions and insertion sites anywhere within the human genome. Out of four cases sequenced, we identified the 5′ insertion sites in four of four cases and the 3′ sites in three of four cases. These findings demonstrate the potential for an inexpensive gene targeting and NGS method that can be easily adapted for use with FFPE tissue to identify large structural rearrangements, opening up the possibility for further discovery from archival tissue.

Luminal cytokeratin (CK) expression in breast papillary lesions, and its potential diagnostic utility among other markers in distinguishing between papillomas and papillary carcinomas, has not been previously evaluated. Such expression was determined in 42 papillary lesions (18 papillary carcinomas and 24 papillomas) by immunostaining with a CK5/p63/CK8/18 antibody cocktail. The mean CK8/18 intensity score and percentage of positive cells were significantly higher in papillary carcinomas (227 and 95%, respectively, vs86 and 42% in papillomas; both P-values <0.0001), whereas the mean CK5 intensity score and percentage of positive cells were significantly lower (7 and 5%, respectively, vs107 and 58% in papillomas; both P-values <0.0001). Half (9/18) of the papillary carcinomas expressed p63 vsall (24/24) of the papillomas (P=0.0001). P63 expression in papillary carcinoma was always (9/9; 100%) focal/limited in nature (expression in <10% of cells), whereas focal expression was seen in only four (17%) papillomas (P<0.0001). Both differential CK (CK8/18 and CK5) expression and p63 were equally sensitive (100%) for the diagnosis of papillary carcinoma, but differential CK expression was more specific (96 vs83%), resulting in a greater accuracy. However, the best discriminatory power in the distinction from papilloma was achieved when all three markers were used in combination, resulting in 100% sensitivity and specificity values. It is concluded that breast papillary lesions have differential CK expression profiles that, especially in combination with p63, can be useful for their stratification, potentially also in needle biopsy material, in which more accurate and reproducible characterization is needed.

Luminal cytokeratin (CK) expression in breast papillary lesions, and its potential diagnostic utility among other markers in distinguishing between papillomas and papillary carcinomas, has not been previously evaluated. Such expression was determined in 42 papillary lesions (18 papillary carcinomas and 24 papillomas) by immunostaining with a CK5/p63/CK8/18 antibody cocktail. The mean CK8/18 intensity score and percentage of positive cells were significantly higher in papillary carcinomas (227 and 95%, respectively, vs 86 and 42% in papillomas; both P-values <0.0001), whereas the mean CK5 intensity score and percentage of positive cells were significantly lower (7 and 5%, respectively, vs 107 and 58% in papillomas; both P-values <0.0001). Half (9/18) of the papillary carcinomas expressed p63 vs all (24/24) of the papillomas (P=0.0001). P63 expression in papillary carcinoma was always (9/9; 100%) focal/limited in nature (expression in <10% of cells), whereas focal expression was seen in only four (17%) papillomas (P<0.0001). Both differential CK (CK8/18 and CK5) expression and p63 were equally sensitive (100%) for the diagnosis of papillary carcinoma, but differential CK expression was more specific (96 vs 83%), resulting in a greater accuracy. However, the best discriminatory power in the distinction from papilloma was achieved when all three markers were used in combination, resulting in 100% sensitivity and specificity values. It is concluded that breast papillary lesions have differential CK expression profiles that, especially in combination with p63, can be useful for their stratification, potentially also in needle biopsy material, in which more accurate and reproducible characterization is needed.

Extramedullary hematopoiesis (EMH) represents abnormal development and growth of hematopoietic tissue outside the bone marrow. Recent studies have shown its association with myelofibrosis and myeloid metaplasia, chronic myeloproliferative disorders, and other hematologic malignancies in up to two-thirds of the cases. Eleven cases of uterine EMH (UEMH) have been reported earlier; of these half had a concurrent, or subsequently developed a clinically significant hematologic disorder. We studied a larger group of patients with UEMH to understand the relationship with hematologic disorders.

Rare breast neoplasms resembling the tall-cell variant of papillary thyroid carcinoma have been reported. In addition, papillary carcinoma of the breast occasionally displays nuclear features reminiscent of papillary thyroid carcinoma. In this study, we evaluated 33 intraductal/intracystic papillary carcinomas of the breast for the presence and extent of nuclear overlap, grooves, clearing, and inclusions, as well as features of the tall-cell or columnar-cell variants of papillary thyroid carcinoma. RET rearrangements were assessed in a subset of these cases. Paired probes localizing to the centromeric and telomeric ends of the RET gene on chromosome 10 were used for FISH using a break-apart approach. Single round and nested PCR was performed to detect RET/PTC1, RET/PTC2, RET/PTC3 and ELKS-RET fusion transcripts. Nuclear overlap, grooves, stratification, and clearing were identified in 24 (73%), 14 (42%), 11 (33%), and 9 (27%) cases respectively, whereas nuclear inclusions and ‘tall-cell’ features were each seen in only one (3%) and two (6%) cases, respectively. Four of 19 tested cases displayed split FISH signals in a low percentage of cells and were considered borderline for RET rearrangement. All 19 tested cases with amplifiable RNA were negative for the four RET fusion transcripts evaluated by RT-PCR. Although papillary carcinomas of breast often display one or more cytoarchitectural features of papillary thyroid carcinoma, there is no evidence that RET rearrangements are involved.

Rare breast neoplasms resembling the tall-cell variant of papillary thyroid carcinoma have been reported. In addition, papillary carcinoma of the breast occasionally displays nuclear features reminiscent of papillary thyroid carcinoma. In this study, we evaluated 33 intraductal/intracystic papillary carcinomas of the breast for the presence and extent of nuclear overlap, grooves, clearing, and inclusions, as well as features of the tall-cell or columnar-cell variants of papillary thyroid carcinoma. RETrearrangements were assessed in a subset of these cases. Paired probes localizing to the centromeric and telomeric ends of the RETgene on chromosome 10 were used for FISH using a break-apart approach. Single round and nested PCR was performed to detect RET/PTC1, RET/PTC2, RET/PTC3and ELKS-RETfusion transcripts. Nuclear overlap, grooves, stratification, and clearing were identified in 24 (73%), 14 (42%), 11 (33%), and 9 (27%) cases respectively, whereas nuclear inclusions and ‘tall-cell’ features were each seen in only one (3%) and two (6%) cases, respectively. Four of 19 tested cases displayed split FISH signals in a low percentage of cells and were considered borderline for RETrearrangement. All 19 tested cases with amplifiable RNA were negative for the four RETfusion transcripts evaluated by RT-PCR. Although papillary carcinomas of breast often display one or more cytoarchitectural features of papillary thyroid carcinoma, there is no evidence that RETrearrangements are involved.

It has recently been shown that Merkel cell carcinoma, a rare and often lethal cutaneous malignancy, frequently harbors a novel clonally integrated polyomavirus aptly named Merkel cell polyomavirus. We aimed to study the prevalence of Merkel cell polyomavirus in cases of Merkel cell carcinoma, using specimens from formalin-fixed, paraffin-embedded tissue blocks. In our archives we identified 41 cases of Merkel cell carcinoma (from 29 different patients). Of these, 20 cases were primary cutaneous tumors, 4 were local recurrences, and 17 were metastases. PCR using two previously published primer sets, LT1 (440 bp amplicon) and LT3 (308 bp amplicon), as well as a novel primer set MCVPS1 (109 bp amplicon), was performed on all cases. Selected PCR products were sequenced to confirm amplicon identity. In addition, the MCVPS1 products were digested with BamH1, yielding an 83 bp product. Amplifiable DNA was recovered in all 41 study cases. The detection rate of Merkel cell polyomavirus for each of the three primer sets was 22 of 29 patients (76%) for MCVPS1, 12 of 29 (41%) for LT3, and 8 of 29 (28%) for LT1. The variation between primer set detection rates was largely due to poor DNA quality, as supported by poor amplification of the higher molecular weight markers in size control ladder products and the fact that all cases that were positive by LT1 and LT3 were positive by MCVPS1. Our findings provide further evidence to link Merkel cell polyomavirus with a possible role in the oncogenesis of Merkel cell carcinoma. On a more practical level, our paraffin-optimized primer set may be used as an ancillary test to confirm the diagnosis of Merkel cell carcinoma in the clinical setting or for screening other rare tumor types for the causative virus, especially those tumor types that are underrepresented in frozen tissue repositories.

A case of primary alveolar soft part sarcoma (ASPS) of the heart is reported in an 11-year-old female as 1 of 16 cases of ASPS presenting in the first 2 decades of life in our institutional 17-year review period. The classic alveolar or organoid pattern was inconspicuous as compared to a more diffuse or formless pattern consisting of a population of uniform round cells with abundant eosinophilic cytoplasm, but in addition there was a second, minor population of gigantiform tumor cells with a variety of unusual shapes. Scattered tumor cells contained dense eosinophilic condensations in the cytoplasm. Other unusual features for ASPS in our case included a lymphohistocytic reaction and zonal necrosis. Immunohistochemistry revealed nuclear reactivity for TFE3, and the ASPL-TFE3fusion transcript was identified by reverse-transcriptase polymerase chain reaction. The only other examples of ASPS involving the heart were 3 cases in the literature of metastatic disease from tumors arising in the soft tissues. This initial case of primary cardiac ASPS joins the list of other types of sarcomas in children that have been reported as primary neoplasms of the heart.

Mucinous cystic neoplasms (MCNs) make up a morphologic family of similar appearing tumors arising in the ovary and various extraovarian sites, including the pancreas, hepatobiliary tract, paratesticular soft tissues, and mesentery. Other than the uncommon mucinous cystadenoma of the ovary presenting in adolescence, MCNs are rarely seen by the pediatric pathologist. The present case is a 5-year-old boy with an abdominal mass appearing to arise in the mesentery of the small intestine. Because of its unresect-ability, a generous biopsy was performed and disclosed a MCN with focal complex papillary architecture in the absence of appreciable cytologic atypia or invasion into the wall. Like other MCNs, this tumor had an inhibin-positive, ovarian-like stroma that was nonreactive for estrogen and progesterone receptors. Only 1 other case of a mesenteric MCN has been reported to date in a child and none in a male. The MCN of the mesentery joins other, somewhat more common cystic lesions of the omentum and mesentery presenting in childhood.

Ewing sarcoma/peripheral neuroectodermal tumor (EWS/PNET), since its characterization immunophenotypically and cytogenetically, has emerged as one of most common sarcomas of childhood. Currently, it is recognized that EWS/PNET can occur in any number of extraosseous sites and is one of several distinctive tumor types with an EWS translocation. In the past, the pathologic diagnosis of EWS/PNET relied on an open biopsy with the application of various ancillary studies, ranging from periodic acid‐Schiff stain to molecular testing, but the tumor increasingly is diagnosed on the basis of cytologic specimens alone.The authors report 3 cases of EWS/PNET in patients aged 11 years to 53 years. These 3 patients had tumors that involved the parotid gland, cervical soft tissue, and pelvis, and were diagnosed by cytologic evaluation of fine‐needle aspiration (FNA) biopsy material alone. The FNA materials also were evaluated prospectively by fluorescence in situ hybridization and/or reverse transcriptase‐polymerase chain reaction.The results emphasized the diagnostic utility of FNA biopsy material for morphologic and molecular analysis without compromising conventional cytologic and immunocytochemical analysis, and that prospective molecular testing of FNA specimens has utility in routine practice although it is subject to many of the same limitations that impact molecular analysis when applied to conventional tissue biopsy specimens.The current results demonstrated that molecular genetic techniques can provide clinically useful ancillary information for FNA specimens when cytologic features and/or immunophenotype are equivocal on the basis of limited sampling or secondary changes, such as hemorrhage and/or necrosis. Cancer (Cancer Cytopathol) 2007. © 2007 by the American Cancer Society.

Microarray-based analysis of global gene expression patterns defines groups of genes that correlate with specific tumor types and prognosis, but the identified genes may not all be of equal clinical utility due to technical factors that affect the precision of their measurement. To analyze how technical variability in measured expression levels may impact microarray-based analysis in a clinical setting, we used Ewing sarcoma/peripheral neuroectodermal tumor (EWS/PNET) in a model system that replicates the clinical scenario in which microarray-based analysis of gene expression will likely occur, namely analysis of a fresh tumor sample by a single chip. By comparing variability of measured expression due to purely technical factors with variability due to biologic factors, we confirm that variability is dependent on the level of gene expression. We also demonstrate that the variability in expression level from either cell line or tumor samples is significantly higher than can be attributed to specific probe sets that have an intrinsically poor performance. These results have significant impact on the application of cDNA microarray chip for molecular analysis performed in a clinical setting.

Due to its small size, rapid generation time, powerful genetic systems, and genomic resources, the zebrafish has emerged as an important model of vertebrate development and human disease. Its well-developed adaptive and innate cellular immune systems make the zebrafish an ideal model for the study of infectious diseases. With a natural and important pathogen of fish, Streptococcus iniae, we have established a streptococcus- zebrafish model of bacterial pathogenesis. Following injection into the dorsal muscle, zebrafish developed a lethal infection, with a 50% lethal dose of 10(3) CFU, and died within 2 to 3 days. The pathogenesis of infection resembled that of S. iniae in farmed fish populations and that of several important human streptococcal diseases and was characterized by an initial focal necrotic lesion that rapidly progressed to invasion of the pathogen into all major organ systems, including the brain. Zebrafish were also susceptible to infection by the human pathogen Streptococcus pyogenes. However, disease was characterized by a marked absence of inflammation, large numbers of extracellular streptococci in the dorsal muscle, and extensive myonecrosis that occurred far in advance of any systemic invasion. The genetic systems available for streptococci, including a novel method of mutagenesis which targets genes whose products are exported, were used to identify several mutants attenuated for virulence in zebrafish. This combination of a genetically amenable pathogen with a well-defined vertebrate host makes the streptococcus-zebrafish model of bacterial pathogenesis a powerful model for analysis of infectious disease.

ABSTRACTDue to its small size, rapid generation time, powerful genetic systems, and genomic resources, the zebrafish has emerged as an important model of vertebrate development and human disease. Its well-developed adaptive and innate cellular immune systems make the zebrafish an ideal model for the study of infectious diseases. With a natural and important pathogen of fish, Streptococcus iniae, we have established a streptococcus- zebrafish model of bacterial pathogenesis. Following injection into the dorsal muscle, zebrafish developed a lethal infection, with a 50% lethal dose of 103CFU, and died within 2 to 3 days. The pathogenesis of infection resembled that of S. iniaein farmed fish populations and that of several important human streptococcal diseases and was characterized by an initial focal necrotic lesion that rapidly progressed to invasion of the pathogen into all major organ systems, including the brain. Zebrafish were also susceptible to infection by the human pathogen Streptococcus pyogenes. However, disease was characterized by a marked absence of inflammation, large numbers of extracellular streptococci in the dorsal muscle, and extensive myonecrosis that occurred far in advance of any systemic invasion. The genetic systems available for streptococci, including a novel method of mutagenesis which targets genes whose products are exported, were used to identify several mutants attenuated for virulence in zebrafish. This combination of a genetically amenable pathogen with a well-defined vertebrate host makes the streptococcus-zebrafish model of bacterial pathogenesis a powerful model for analysis of infectious disease.

Abstract: Primitive neuroectodermal tumor (PNET) is a prototypic malignant small round cell tumor of childhood that is characterized in most cases by t(11;22) resulting in an EWS-FLI1 gene fusion. Once thought to be uncommon, PNET now accounts for almost 20% of malignant soft tissue tumors in children. Increased recognition of PNET is partly due to advances in immunohistochemistry and molecular diagnostics, which have led to the identification of the tumor in non-classical sites. We report the clinical, histologic, immunohistochemical, and molecular findings of two visceral PNETs of the digestive system—one involving the small intestine and the other involving the hepatic duct. Histologically, each tumor was composed of malignant small cells growing in sheets, nests, and lobules; the tumor cells of both cases showed characteristic immunoreactivity for vimentin and O13 (CD99). Reverse transcription-polymerase chain reaction (RT-PCR) analysis for t(11;22) using nested primers was performed with RNA extracted from paraffin-embedded, formalin-fixed tissue and demonstrated an EWS exon 7 to FLI1 exon 5 fusion in both cases, confirmed by Southern blot hybridization and DNA sequence analysis. These results illustrate the expanded clinicopathologic profile of PNET, and demonstrate that visceral PNETs, despite their unusual sites of presentation, maintain the characteristic immunohistochemical and genetic features of PNETs at more conventional sites.

• Two cases of endogenous Pseudallescheria boydii endophthalmitis are presented. One patient had severe pulmonary fibrosis but no history of ocular trauma and no clinical or laboratory evidence of immunocompromise. Despite therapy with repeated intravitreal miconazole nitrate injections and systemic fluconazole, enucleation of the globe was required, and the patient eventually died with disseminated pseudallescheriasis. The other patient was an immunosuppressed cardiac transplant recipient who also received systemic fluconazole therapy. The infected eye eventually required evisceration, but there was no evidence of disseminated pseudallescheriasis before his death of unrelated causes.