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Most spermatocytic tumors (STs) have an excellent prognosis. In rare instances, metastatic disease has been documented. However, it is unclear if aggressive tumors have specific molecular alterations. Herein, we have studied primary STs with (n = 4) and without (n = 3) anaplastic features, including single-nucleotide polymorphism microarrays for 5 ST (nonanaplastic: 3; anaplastic: 2). The mean age at orchiectomy and tumor size was 49 years and 6.5 cm, respectively. Lymphovascular invasion and necrosis were identified in 3 (of 4, 75%) anaplastic STs, including one with clinically metastatic disease and one with locally aggressive disease. None of the cases in this study exhibited sarcomatoid change. The mean mitotic count was higher in anaplastic tumors (59/10 versus 10/10 high-power fields). All STs in this study were positive for SALL4 and CD117 and negative for OCT3/4 and CD30 (7/7, 100%). SSX-C positivity was identified in all but the locally aggressive anaplastic ST (5 of 6, 83%). All STs showed a consistent gain of chromosome 9 including the locus for the DMRT1 gene (5 of 5 cases, 100%), while gains of chromosome 12p were only seen in 2 (of 2) anaplastic variants. Gains of 12p in anaplastic STs may represent a biomarker of transformation into more aggressive tumors. Alternatively, STs with gain of 12p may represent an intermediate state between type II and type III germ cell tumors. Future studies are needed to validate whether gain of 12p is a consistent feature of STs with anaplastic morphology and its association with aggressive clinical behavior.

Introduction: While non-mosaic genome-wide paternal uniparental disomy (patUPD) is consistent with complete hydatidiform mole, the prenatal presentation of mosaic genome-wide patUPD is not well defined. This report adds another case to the small cohort of patients with the rare genetic disorder of mosaic genome-wide patUPD and provides one of the few examples of a prenatal presentation of this disease. We discuss ultrasound findings and prenatal analysis to review predominant genetic and clinical features associated with mosaic genome-wide patUPD. Case Presentation: A 30-year-old gravida 1 para 0 woman was referred at 10 weeks gestation due to an abnormal first-trimester ultrasound suggesting a partial molar pregnancy. The patient undertook genetic counseling and reviewed possible genetic etiologies and testing options. Karyotype analysis demonstrated a female fetus (46, XX). The BWS methylation pattern suggested the absence of maternally derived copies of IC1 (H19) and IC2 (LIT1) critical regions, which could result from patUPD of chromosome 11. CMA of cultured amniocytes was significant for arr(1–22,X)x2 hmz, consistent with genome-wide absence of heterozygosity (shown in Fig. 3). Discussion/Conclusion: This case report is intended to add to the limited knowledge regarding prenatal diagnosis of mosaic genome-wide patUPD by highlighting the ultrasound findings, the genetic testing performed, and fetal outcome. The fetal karyotype was normal. CMA was consistent with a molecular diagnosis of GWUPD. Low-level mosaicism in our sample was inferred given the clinical presentation of a developing fetus. Methylation studies were consistent with a diagnosis of BWS. The diagnosis of genome-wide patUPD using CMA provides further knowledge of UPD and its functional relevance. In a prenatal setting, a CMA profile without heterozygosity is typical of a complete molar pregnancy. However, in the presence of a fetus, it likely represents mosaic GWUPD, a rare condition that is usually of paternal origin.

Summary Low-grade oncocytic tumor of the kidney (LOT) is characterized by cytoplasmic eosinophilia and a CK7-positive/CD117-negative immunophenotype. Morphologically, they exhibit overlapping features with oncocytoma and chromophobe renal cell carcinoma. Our aim was to obtain long-term clinical follow-up data, clinicopathological and molecular characteristics, and incidence of LOT. Tissue microarrays were constructed from 574 tumors historically diagnosed as oncocytoma and surgically treated at Mayo Clinic between 1970 and 2012, and immunostained for CK7 and CD117. An extended immunophenotype was obtained on whole slide sections, along with FISH for CCND1 rearrangement status and chromosomal microarray for copy number status. In addition, two cases were retrospectively identified in a set of tuberous sclerosis complex (TSC)-associated neoplasms and three more cases diagnosed on needle core biopsies were obtained during routine clinical practice. Twenty-four cases of LOT were identified among 574 consecutive tumors diagnosed as oncocytoma and treated with partial or radical nephrectomy, corresponding to an incidence of 4.18% of tumors historically diagnosed as oncocytomas, and 0.35% of 6944 nephrectomies performed between 1970 and 2012. Overall, 29 cases of LOT were identified in three clinical settings: sporadic, TSC-associated, and end-stage renal disease (ESRD). Multifocality was seen only in the setting of TSC and ESRD. No metastases attributable to LOT were identified (median follow-up 9.6 years). There were no recurrent arm level copy number changes detected by chromosomal microarray and all tested cases were negative for CCND1 rearrangement by FISH. LOT is an uncommon eosinophilic renal neoplasm with an indolent prognosis that constitutes ∼4% of tumors historically diagnosed as oncocytoma. The morphologic, immunophenotypic, and molecular features of this neoplasm suggest it is a distinct entity of renal neoplasia.

Inflammatory leiomyosarcoma (ILMS), defined as “a malignant neoplasm showing smooth muscle differentiation, a prominent inflammatory infiltrate, and near-haploidization”, is a very rare soft tissue tumor with a generally favorable prognosis. The morphologic features of “histiocyte-rich rhabdomyoblastic tumor” (HRRMT) are similar to those of ILMS, although this lesion shows by definition a skeletal muscle phenotype. Recent gene expression profiling and immunohistochemical studies have also suggested that ILMS and HRRMT may be related. We studied the clinicopathologic, immunohistochemical and genetic features of four cases previously classified as ILMS and nine classified as HRRMT. Tumors from both groups tended to occur in the deep soft tissues of the extremities of young to middle-aged males and exhibited indolent behavior. Morphologically, all were well-circumscribed, often encapsulated, and showed a striking histiocyte-rich inflammatory infiltrate admixed with variably pleomorphic tumor cells showing spindled and epithelioid to rhabdoid morphology, eosinophilic cytoplasm, and prominent nucleoli, but few, if any, mitotic figures. Immunohistochemically, the tumor cells expressed desmin, alpha-smooth muscle actin, and the rhabdomyoblastic markers PAX7, MyoD1, and myogenin. H-caldesmon expression was absent in all cases, using the specific h-CD antibody. Karyotypic study (1 HRRMT) and genome-wide copy number analysis (7 HRRMT, OncoScan SNP assay), revealed near-haploidization in four cases, with subsequent genome doubling in one, an identical phenotype to that seen in ILMS. We propose reclassification of ILMS and HRRMT as “inflammatory rhabdomyoblastic tumor”, a name which accurately describes the salient morphologic and immunohistochemical features of this distinctive tumor, as well as its intermediate (rarely metastasizing) clinical behavior.

PURPOSE Copy-number variants (CNVs) of uncertain clinical significance are routinely reported in a clinical setting only when exceeding predetermined reporting thresholds, typically based on CNV size. Given that very few genes are associated with triplosensitive phenotypes, it is not surprising that many interstitial duplications

OBJECTIVE: We aimed to test for an association between the amount of circulating fetal cell-free DNA and trisomy, and whether NIPS failure due to low fetal fraction indicates trisomy risk. METHOD: Maternal BMI, maternal age, fetal sex, gestational age, fetal cfDNA fraction, and NIPS results was collected on 2,374 pregnancies. Additional clinical information was available for 1,180 research consented patients. We investigated associations between fetal fraction and available variables and determined the success rate of repeat NIPS testing. RESULTS: Fetal trisomy was marginally associated with decreased fetal fraction (p = 0.067). However, the proportions of trisomy events were not significantly increased in women who had failed NIPS due to low fetal fraction (< 4%) (OR = 1.37 [0.3 – 7.4]; p = 0.714). 66% of repeated NIPS after a second blood draw were successful. CONCLUSION: Failure to meet the clinical cutoff of 4% fetal fraction established for NIPS accuracy did not suggest increased risk for trisomy in our cohort. Because repeat testing was successful in the majority of cases and most failures were explained by high BMI and low gestational age, a redraw may be an appropriate next step before invasive screening due to concerns for trisomic pregnancies.

The TCF3/PBX1 gene fusion is a recurrent genetic abnormality in pediatric B-lymphoblastic leukemia/lymphoma (B-ALL/LBL). While dual-color, dual-fusion fluorescence in situ hybridization (D-FISH) probes can detect TCF3/PBX1 fusions, further characterization of atypical TCF3 FISH patterns as indicated by additional or diminished TCF3 signals is currently limited. Herein we describe the use of a next-generation sequencing assay, mate-pair sequencing (MPseq), to characterize typical and cryptic TCF3/PBX1 fusions and to identify TCF3 translocation partners based on results obtained from our laboratory-developed TCF3/PBX1 D-FISH probe set. MPseq was performed on 21 cases of pediatric B-ALL/LBL with either TCF3/PBX1 fusion, or no TCF3/PBX1 fusion but with additional or diminished TCF3 signals obtained by our PBX1/TCF3 D-FISH probe set. In addition, MPseq was performed on one pediatric B-ALL/LBL case with an apparently normal karyotype and abnormal TCF3 break-apart probe results. Of 22 specimens successfully evaluated by MPseq, 13 cases (59%) demonstrated TCF3/PBX1 fusion, including three cases with previously undescribed insertional rearrangements. The remaining nine cases (41%) harbored various TCF3 partners, including six cases with TCF3/ZNF384, and one case each with TCF3/HLF, TCF3/FLI1 and TCF3/TEF. Our results illustrate the power of MPseq to characterize TCF3 rearrangements with increased precision and accuracy over traditional cytogenetic methodologies.

Inflammatory leiomyosarcoma (ILMS), defined as "a malignant neoplasm showing smooth muscle differentiation, a prominent inflammatory infiltrate, and near-haploidization", is a very rare soft tissue tumor with a generally favorable prognosis. The morphologic features of "histiocyte-rich rhabdomyoblastic tumor" (HRRMT) are similar to those of ILMS, although this lesion shows by definition a skeletal muscle phenotype. Recent gene expression profiling and immunohistochemical studies have also suggested that ILMS and HRRMT may be related. We studied the clinicopathologic, immunohistochemical and genetic features of four cases previously classified as ILMS and nine classified as HRRMT. Tumors from both groups tended to occur in the deep soft tissues of the extremities of young to middle-aged males and exhibited indolent behavior. Morphologically, all were well-circumscribed, often encapsulated, and showed a striking histiocyte-rich inflammatory infiltrate admixed with variably pleomorphic tumor cells showing spindled and epithelioid to rhabdoid morphology, eosinophilic cytoplasm, and prominent nucleoli, but few, if any, mitotic figures. Immunohistochemically, the tumor cells expressed desmin, alpha-smooth muscle actin, and the rhabdomyoblastic markers PAX7, MyoD1, and myogenin. H-caldesmon expression was absent in all cases, using the specific h-CD antibody. Karyotypic study (1 HRRMT) and genome-wide copy number analysis (7 HRRMT, OncoScan SNP assay), revealed near-haploidization in four cases, with subsequent genome doubling in one, an identical phenotype to that seen in ILMS. We propose reclassification of ILMS and HRRMT as "inflammatory rhabdomyoblastic tumor", a name which accurately describes the salient morphologic and immunohistochemical features of this distinctive tumor, as well as its intermediate (rarely metastasizing) clinical behavior.

Copy-number variants (CNVs) of uncertain clinical significance are routinely reported in a clinical setting only when exceeding predetermined reporting thresholds, typically based on CNV size. Given that very few genes are associated with triplosensitive phenotypes, it is not surprising that many interstitial duplications1 Mb are found to be inherited and anticipated to be of limited or no clinical significance.In an effort to further refine our reporting criteria to maximize diagnostic yield while minimizing the return of uncertain variants, we performed a retrospective analysis of all clinical microarray cases reported in a 10-year window. A total of 1112 reported duplications had parental follow-up, and these were compared by size, RefSeq gene content, and inheritance pattern. De novo origin was used as a rough proxy for pathogenicity.Approximately 6% of duplications 500 kb-1 Mb were de novo observations, compared with approximately 14% for 1-2 Mb duplications (p = 0.0005). On average, de novo duplications had higher gene counts than inherited duplications.Our data reveal limited diagnostic utility for duplications of uncertain significance1 Mb. Considerations for revised reporting criteria are discussed and are applicable to CNVs detected by any genome-wide exploratory methodology, including exome/genome sequencing.

Whole-genome sequencing (WGS) has shown promise in becoming a first-tier diagnostic test for patients with rare genetic disorders; however, standards addressing the definition and deployment practice of a best-in-class test are lacking. To address these gaps, the Medical Genome Initiative, a consortium of leading healthcare and research organizations in the US and Canada, was formed to expand access to high-quality clinical WGS by publishing best practices. Here, we present consensus recommendations on clinical WGS analytical validation for the diagnosis of individuals with suspected germline disease with a focus on test development, upfront considerations for test design, test validation practices, and metrics to monitor test performance. This work also provides insight into the current state of WGS testing at each member institution, including the utilization of reference and other standards across sites. Importantly, members of this initiative strongly believe that clinical WGS is an appropriate first-tier test for patients with rare genetic disorders, and at minimum is ready to replace chromosomal microarray analysis and whole-exome sequencing. The recommendations presented here should reduce the burden on laboratories introducing WGS into clinical practice, and support safe and effective WGS testing for diagnosis of germline disease.

Shallow-depth whole-genome sequencing (WGS) of circulating cell-free DNA (ccfDNA) is a popular approach for non-invasive genomic screening assays, including liquid biopsy for early detection of invasive tumors as well as non-invasive prenatal screening (NIPS) for common fetal trisomies. In contrast to nuclear DNA WGS, ccfDNA WGS exhibits extensive inter- and intra-sample coverage variability that is not fully explained by typical sources of variation in WGS, such as GC content. This variability may inflate false positive and false negative screening rates of copy-number alterations and aneuploidy, particularly if these features are present at a relatively low proportion of total sequenced content. Herein, we propose an empirically-driven coverage correction strategy that leverages prior annotation information in a multi-distance learning context to improve within-sample coverage profile correction. Specifically, we train a weighted k-nearest neighbors-style method on non-pregnant female donor ccfDNA WGS samples, and apply it to NIPS samples to evaluate coverage profile variability reduction. We additionally characterize improvement in the discrimination of positive fetal trisomy cases relative to normal controls, and compare our results against a more traditional regression-based approach to profile coverage correction based on GC content and mappability. Under cross-validation, performance measures indicated benefit to combining the two feature sets relative to either in isolation. We also observed substantial improvement in coverage profile variability reduction in leave-out clinical NIPS samples, with variability reduced by 26.5-53.5% relative to the standard regression-based method as quantified by median absolute deviation. Finally, we observed improvement discrimination for screening positive trisomy cases reducing ccfDNA WGS coverage variability while additionally improving NIPS trisomy screening assay performance. Overall, our results indicate that machine learning approaches can substantially improve ccfDNA WGS coverage profile correction and downstream analyses.

The basis of cancer biology is built upon two fundamental processes that result in uncontrolled cell proliferation and tumor formation: loss of tumor suppressor gene function and gain of oncogene function. Somatic DNA copy number variants (CNVs), which generally range in size from kilobases to entire chromosomes, facilitate gains and losses of chromosomal material incorporating oncogenes and tumor suppressor genes, respectively. In fact, many cancer types are characterized by DNA copy number changes and relatively few single nucleotide mutations (Ciriello et al. Nat Genet 45:1127-1133, 2013). Currently, the optimal method to detect such somatic copy number changes across the cancer genome is whole-genome single nucleotide polymorphism (SNP) microarray analysis.

The recent advances in next-generation sequencing (NGS) technologies have enabled the development of effective high-throughput noninvasive prenatal screening (NIPS) assays for fetal genetic abnormalities using maternal circulating cell-free DNA (ccfDNA). An important NIPS quality assurance is quantifying the fetal proportion of the sampled ccfDNA. For methods using allelic read count ratios from targeted sequencing of single-nucleotide polymorphisms (SNPs), systematic biases and errors may reduce accuracy and diminish assay performance. We collected ccfDNA NIPS MiSeq sequencing data from an amplicon-based 92 SNP panel along with complementary low-depth whole-genome sequencing (WGS) on 243 normal male fetus pregnancies along with additional 144 nonpregnant female donor samples. Using fetal fraction estimates based on X and Y chromosome WGS coverage as gold standard, we compared an existing SNP-based approach, FetalQuant, to a more flexible Bayesian hierarchical modeling strategy that borrows information across interrogated SNPs to character SNP-level error rates and biases to improve fetal fraction estimates. Posterior distributions for SNP-level model parameters indicate most SNPs exhibited modest to moderate extrabinomial variation and a consistent underrepresentation of fetal alleles, with some extreme outliers in both regards. Fetal fraction estimates using FetalQuant, naive to these SNP properties, were relatively poor (R

Mate-pair sequencing (MPseq), using long-insert, paired-end genomic libraries, is a powerful next-generation sequencing-based approach for the detection of genomic structural variants. SVAtools is a set of algorithms to detect both chromosomal rearrangements and large (>10 kb) copy number variants (CNVs) in genome-wide MPseq data. SVAtools can also predict gene disruptions and gene fusions, and characterize the genomic structure of complex rearrangements. To illustrate the power of SVAtools' junction detection methods to provide comprehensive molecular karyotypes, MPseq data were compared against a set of samples previously characterized by traditional cytogenetic methods. Karyotype, FISH and chromosomal microarray (CMA), performed for 29 patients in a clinical laboratory setting, collectively revealed 285 breakpoints in 87 rearrangements. The junction detection methods of SVAtools detected 87% of these breakpoints compared to 48%, 42% and 57% for karyotype, FISH and CMA respectively. Breakpoint resolution was also reported to 1 kb or less and additional genomic rearrangement complexities not appreciable by standard cytogenetic techniques were revealed. For example, 63% of CNVs detected by CMA were shown by SVAtools' junction detection to occur secondary to a rearrangement other than a simple deletion or tandem duplication. SVAtools with MPseq provides comprehensive and accurate whole-genome junction detection with improved breakpoint resolution, compared to karyotype, FISH, and CMA combined. This approach to molecular karyotyping offers considerable diagnostic potential for the simultaneous detection of both novel and recurrent genomic rearrangements in hereditary and neoplastic disorders.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic neoplasm involving the bone marrow and blood that accounts for ∼15% of childhood and 25% of adult ALL. Whereas multiple, recurrent genetic abnormalities have been described in T-ALL, their clinical significance is unclear or controversial. Importantly, ABL1 rearrangements, most commonly described in BCR/ABL1-positive B-ALL and BCR-ABL1-like B-ALL, have been observed in T-ALL and may respond to tyrosine kinase inhibitor (TKI) therapy. We describe a newly diagnosed case of pediatric T-ALL with a fluorescence in situ hybridization abnormality suggesting a partial ABL1 deletion by a BCR/ABL1 dual-color dual-fusion probe but that demonstrated a normal result using an ABL1 break-apart probe. Mate-pair sequencing (MPseq), a next-generation sequencing (NGS)-based technology utilized to detect copy number and structural abnormalities with high resolution and precision throughout the genome, was performed and revealed a NUP214/ABL1 gene fusion that has been demonstrated to be sensitive to TKI therapy. This case demonstrates the power of MPseq to resolve chromosomal abnormalities unappreciable by traditional cytogenetic methodologies and highlights the clinical value of this novel NGS-based technology.

Rearrangements involving TCF3 (E2A) at 19p13.3 are a common cytogenetic abnormality in B-lymphoblastic leukemia/lymphoma (B-LL) across all age groups accounting for at least 5% of B-LL. The most common translocation partner, PBX1 at 1q23, is associated with an unbalanced der(19) or a balanced t(1;19) in 80% vs. 20% patients, respectively, and results in a PBX1/TCF3 fusion transcriptional activator. Several other rearrangement partners have been described, and as such TCF3 is approaching the dubious status of a "promiscuous" gene partner. Clinically, our laboratory uses a dual-color, dual fusion PBX1/TCF3 FISH probe to characterize the most common gene fusion; however, this FISH strategy also allows for the identification of atypical disruptions of the TCF3 gene region, and may suggest a gene partner based on abnormal metaphases/metaphase-FISH. Interrogation of our pediatric leukemia database (ages 0-30) identified 219 patients with abnormal TCF3 FISH results. We selected 23 samples for mate-pair evaluation to test the methodology, characterize novel rearrangements and identify new TCF3 fusion partners. In total, 19 patient samples were successfully sequenced by Mate-Pair, including 13 samples with variants of the 1;19 translocation. Of the samples with TCF3/PBX1 fusion, 3 patients demonstrated novel insertional translocation events characterized by mate-pair. A total of 6 samples with undefined/unknown TCF3 partner genes were evaluated. The mate-pair results identified 5 samples with a t(12;19)(p13.31;p13.3), resulting in TCF3/ZNF384 fusion. Interestingly, two of the ZNF384 rearrangements were observed in the unbalanced form, suggesting a similar mechanism related to the der(19)t(1;19) regularly observed in the common PBX1-TCF3 rearrangement. From a karyotype perspective, since these rearrangements result in only a small portion of chromosome 12p translocated onto distal 19p, this rearrangement would "cryptic" by conventional karyotyping. One patient sample demonstrated a new TCF3 gene fusion partner (TEF), associated with a t(19;22)(p13.3;q13.2). Overall, our results demonstrate the utility of mate-pair evaluation as an adjunct methodology in the identification of novel TCF3 rearrangements in B-LL. We have successfully expanded the application of mate-pair to characterize other novel/cryptic abnormalities in various hematologic and oncologic neoplasms encountered in our laboratory. Disclosures No relevant conflicts of interest to declare.

second degree consanguinity. This is at a much higher frequency than expected, and while this finding may be connected to an underlying recessive condition, this finding was not reported to patients. Overall this work indicates that there are a variety of different types of incidental findings. These studies demonstrate that it is important to develop protocols for reporting the findings that facilitate appropriate medical follow-up in the necessary cases.