Genomic analysis of spermatocytic tumors demonstrates recurrent molecular alterations in cases with malignant clinical behavior.

Spermatocytic tumor (ST) is a rare type of germ cell tumor that occurs exclusively in the postpubertal testis and typically affects elderly men. Most STs are benign, but rare cases exhibit aggressive clinical behavior, often in association with transition to sarcomatoid histology. Limited molecular analyses have been performed on STs; therefore, their genomic and epigenomic features remain incompletely described. Twenty-seven samples from 25 individual patients were analyzed with a combination of DNA sequencing panels, genomic methylation profiling, SNP array, isochromosome (12p) [i(12p)] FISH, and immunohistochemistry. The series included five metastasizing tumors (three with sarcomatoid transformation, one anaplastic, and one conventional) and 20 non-metastasizing tumors (14 anaplastic and six conventional). Anaplastic tumors comprised a monomorphic population of intermediate-sized neoplastic cells, as previously described. Multiomic analyses demonstrated that there were two genomic subgroups of STs: one with diploid genomes and hotspot RAS/RAF variants and the other with global ploidy shift and absence of recurrent mutations. Relative gain of chromosome 9 was a consistent finding in both subgroups. A comparison of metastasizing and non-metastasizing cases demonstrated that aggressive behavior was associated with the acquisition of pathogenic TP53 mutations and/or relative gains of 12p/i(12p). In cases with sarcomatoid transformation, TP53 mutations seem to underlie the transition to sarcomatoid histology. Genomic methylation analysis demonstrated that aggressive cases with gains of 12p cluster closer to pure seminomas than to STs without gains of 12p. In conclusion, STs include two genomic subgroups, characterized by global ploidy shifts without recurrent mutations and diploid genomes with RAS/RAF hotspot mutations, respectively. Biologic progression was associated with relative gains of 12p and TP53 mutations. The findings in STs with relative gains of 12p suggest that they may exhibit biologic characteristics akin to those seen in germ cell neoplasia in situ-related germ cell tumors rather than non-germ cell neoplasia in situ-derived STs. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

A multi-center analysis of individuals with a 47,XXY/46,XX karyotype.

INTRODUCTION: Klinefelter syndrome (KS), a sex chromosome aneuploidy, is associated with a 47,XXY chromosomal complement and is diagnosed in ∼1:600 live male births. Individuals with a 46,XX cell line in addition to 47,XXY are less common with a limited number of published case reports. METHODOLOGY: To better understand the implications of a 47,XXY/46,XX karyotype, we conducted a retrospective, multi-center analysis of the cytogenetic findings and associated clinical records of 34 patients diagnosed with this SCA across 14 institutions. RESULTS: Presence of the XX cell line ranged from 5-98% in patient specimens. Phenotypes also exhibited significant heterogeneity with some reporting a single reason for referral and others presenting with a constellation of symptoms, including ambiguous genitalia and ovotestes. Ovotestes were present in 12% of individuals in this cohort, who had a significantly higher percentage of XX cells. Notably, two patients were assigned female sex at birth DISCUSSION: These findings highlight the variability of the clinical phenotypes associated with this SCA as well as the challenges of clinical management for this population. Karyotype or FISH analysis, which offer single-cell resolution, rather than chromosomal microarray or molecular testing, is the ideal test strategy in these instances as mosaicism can occur at low levels.

Uniparental disomy of multiple chromosomes in two cases with a complex phenotype.

Uniparental disomy (UPD) is the inheritance of both chromosomal homologs from one parent. Depending on the chromosome involved and the parental origin, UPD may result in phenotypic abnormalities due to aberrant methylation patterns or unmasking recessive conditions in isodisomic regions. UPD primarily originates from somatic rescue of a single meiotically-derived aneuploidy, most commonly a trisomy. Double UPD is exceedingly rare and triple UPD has not been previously described. Here, we report two unrelated clinical cases with UPD of multiple chromosomes; an 8-month-old male with maternal isodisomy of chromosome 7 and paternal isodisomy of chromosome 9, and a 4-week-old female with mixed paternal UPD for chromosomes 4, 10, and 14. These cases also demonstrate that although extremely rare, the detection of AOH on two or more chromosomes may warrant additional clinical and laboratory investigation such as methylation and STR marker analysis, especially when involving chromosomes known to be associated with imprinting disorders.

Prenatal Diagnosis and Fetal Outcome with Mosaic Genome-Wide Uniparental Disomy.

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.

"Inflammatory Leiomyosarcoma" and "Histiocyte-rich Rhabdomyoblastic Tumor": a clinicopathological, immunohistochemical and genetic study of 13 cases, with a proposal for reclassification as "Inflammatory Rhabdomyoblastic Tumor".

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.

Limited diagnostic impact of duplications <1 Mb of uncertain clinical significance: a 10-year retrospective analysis of reporting practices at the Mayo Clinic.

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 <1 Mb are found to be inherited and anticipated to be of limited or no clinical significance. METHODS: 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. RESULTS: 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. CONCLUSION: Our data reveal limited diagnostic utility for duplications of uncertain significance <1 Mb. Considerations for revised reporting criteria are discussed and are applicable to CNVs detected by any genome-wide exploratory methodology, including exome/genome sequencing.

Most noninvasive prenatal screens failing due to inadequate fetal cell free DNA are negative for trisomy when repeated.

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 2374 pregnancies. Additional clinical information was available for 1180 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 = .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 = .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.

Clinical Validation of Tagmentation-Based Genome Sequencing for Germline Disorders.

Genome sequencing (GS) is a powerful clinical tool used for the comprehensive diagnosis of germline disorders. GS library preparation typically involves mechanical DNA fragmentation, end repair, and bead-based library size selection followed by adapter ligation, which can require a large amount of input genomic DNA. Tagmentation using bead-linked transposomes can simplify the library preparation process and reduce the DNA input requirement. Here we describe the clinical validation of tagmentation-based PCR-free GS as a clinical test for rare germline disorders. Compared with the Genome-in-a-Bottle Consortium benchmark variant sets, GS had a recall >99.7% and a precision of 99.8% for single nucleotide variants and small insertion-deletions. GS also exhibited 100% sensitivity for clinically reported sequence variants and the copy number variants examined. Furthermore, GS detected mitochondrial sequence variants above 5% heteroplasmy and showed reliable detection of disease-relevant repeat expansions and SMN1 homozygous loss. Our results indicate that while lowering DNA input requirements and reducing library preparation time, GS enables uniform coverage across the genome as well as robust detection of various types of genetic alterations. With the advantage of comprehensive profiling of multiple types of genetic alterations, GS is positioned as an ideal first-tier diagnostic test for germline disorders.

Cytogenetics of spermatocytic tumors with a discussion of gain of chromosome 12p in anaplastic variants.

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.

Single-Nucleotide Polymorphism Array for Histologically Ambiguous Melanocytic Tumors.

Genome-wide copy number profiling by single-nucleotide polymorphism (SNP) array is increasingly employed in the clinical diagnostic workup of melanocytic tumors. We present our SNP array results on 675 melanocytic tumors, including 615 histologically ambiguous tumors evaluated by our institution's dermatopathology consultation service and a separate validation cohort of 26 known benign nevi and 34 known malignant melanomas. The total number of somatic copy number abnormalities, sub-chromosomal copy number abnormalities, regions of homozygosity, and abnormalities at disease-associated regions was significantly associated with a diagnosis of malignancy across disease categories. In our study, the number of copy number abnormalities was the factor that best discriminated between benign versus malignant diagnoses, confirming recent published research. Histologically ambiguous tumors had a range and spectrum of abnormalities, including recurrent 11p gains, copy state transitions over kinase genes, and 3p deletions overlapping BAP1 in neoplasms with Spitzoid morphology. Our data suggest that histologically ambiguous melanocytic neoplasms and early primary melanomas have a range of abnormalities that is intermediate between unambiguous benign or malignant melanocytic neoplasms. Careful technical review and an integrated diagnostic approach are essential for the accurate interpretation of SNP array results on histologically ambiguous melanocytic tumors.

Characterization of TCF3 rearrangements in pediatric B-lymphoblastic leukemia/lymphoma by mate-pair sequencing (MPseq) identifies complex genomic rearrangements and a novel TCF3/TEF gene fusion.

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.

Detection of a cryptic NUP214/ABL1 gene fusion by mate-pair sequencing (MPseq) in a newly diagnosed case of pediatric T-lymphoblastic leukemia.

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.

Improving Single-Nucleotide Polymorphism-Based Fetal Fraction Estimation of Maternal Plasma Circulating Cell-Free DNA Using Bayesian Hierarchical Models.

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 (R2 = 0.14, root mean squared error [RMSE] = 0.050), particularly when the true fetal fraction was low (<5%). In contrast, by quantifying SNP-level biases and error rates, our proposed approach demonstrated improved performance by reducing the bias and variability in fetal fraction estimates (R2 = 0.794, RMSE = 0.025). Using high-depth targeted SNP sequencing data, we identified a high degree of variability in distributional properties across SNP allelic read counts. These results highlight the benefits of leveraging hierarchical modeling for SNP-based fetal quantification assays (FQAs) and the need to properly calibrate FQAs dependent on NGS allelic ratio data.

SVAtools for junction detection of genome-wide chromosomal rearrangements by mate-pair sequencing (MPseq).

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.

A specific family of interspersed repeats (SINEs) facilitates meiotic synapsis in mammals.

BACKGROUND: Errors during meiosis that affect synapsis and recombination between homologous chromosomes contribute to aneuploidy and infertility in humans. Despite the clinical relevance of these defects, we know very little about the mechanisms by which homologous chromosomes interact with one another during mammalian meiotic prophase. Further, we remain ignorant of the way in which chromosomal DNA complexes with the meiosis-specific structure that tethers homologs, the synaptonemal complex (SC), and whether specific DNA elements are necessary for this interaction. RESULTS: In the present study we utilized chromatin immunoprecipitation (ChIP) and DNA sequencing to demonstrate that the axial elements of the mammalian SC are markedly enriched for a specific family of interspersed repeats, short interspersed elements (SINEs). Further, we refine the role of the repeats to specific sub-families of SINEs, B1 in mouse and AluY in old world monkey (Macaca mulatta). CONCLUSIONS: Because B1 and AluY elements are the most actively retrotransposing SINEs in mice and rhesus monkeys, respectively, our observations imply that they may serve a dual function in axial element binding; i.e., as the anchoring point for the SC but possibly also as a suppressor/regulator of retrotransposition.