Expression of Potential Biomarker Targets by Immunohistochemistry in Cervical Carcinomas.

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 situ hybridization), and 1 had equivocal (2+) staining (negative for amplification by fluorescence in situ hybridization). 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.

Cancer of the Vulva: A Review.

BACKGROUND: Vulvar cancers, although rare, are becoming an increasingly serious threat to women's health. Cancer of the vulva accounted for 0.3% of all new cancers in the United States in 2019, with 6,070 newly diagnosed cases. This review details the epidemiology, pathogenesis, diagnosis, staging, and treatment of vulvar malignancies. OBJECTIVE: To review cancer entities of the vulva, including vulvar intraepithelial neoplasms, squamous cell carcinoma (SCC), malignant melanoma, basal cell carcinoma, neuroendocrine tumors, and adenocarcinomas. MATERIALS AND METHODS: Literature review using PubMed search for articles related to cancer of the vulva. RESULTS: Vulvar intraepithelial neoplasms represent premalignant precursors to SCC of the vulva. There are several different histopathologic subtypes of SCC, and treatment is dependent on characteristics of primary tumor and lymph node involvement. Melanoma is the second most common cancer to affect the vulva, and staging is based on tumor, node, and metastatic spread. CONCLUSION: Vulvar malignancies are rare, and diagnosis is dependent on biopsy and pathologic evaluation. Treatment for vulvar malignancies depends on histopathologic diagnosis but ranges from wide local excision with or without lymph node biopsy or dissection to radiation therapy with chemo- or immunotherapy. Overall survival varies by diagnosis.

Identity determination in diagnostic surgical pathology.

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.

Pitfalls in molecular diagnostics.

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.

Serum squamous cell carcinoma antigen as an early indicator of response during therapy of cervical cancer.

BACKGROUND: Pretreatment serum squamous cell carcinoma antigen (SCCA) is a prognostic biomarker in women with cervical cancer. SCCA has not been evaluated as an early indicator of response to chemoradiation therapy (CRT). The molecular role of the two SCCA isoforms, SCCA1 (SERPINB3) and SCCA2 (SERPINB4), in cervical cancer is unknown. We hypothesised that changes in serum SCCA during definitive CRT predicts treatment response, and that SCCA1 mediates radiation resistance. METHODS: Patients treated with definitive CRT for cervical squamous carcinoma with serum SCCA measured were included. SCCA immunohistochemistry was performed on tumour biopsies. Post-treatment FDG-PET/CT, recurrence, and overall survival were recorded. Radiation response of cervical tumour cell lines after SCCA1 expression or CRISPR/Cas9 knockout was evaluated by clonogenic survival assay. RESULTS: Persistently elevated serum SCCA during definitive CRT was an independent predictor of positive post-therapy FDG-PET/CT (P=0.043), recurrence (P=0.0046) and death (P=0.015). An SCCA1-expressing vector increased radioresistance, while SCCA knock out increased radiosensitivity of cervical tumour cell lines in vitro. CONCLUSIONS: Early response assessment with serum SCCA is a powerful prognostic tool. These findings suggest that escalation of therapy in patients with elevated or sustained serum SCCA and molecular targeting of SCCA1 should be considered.

Genomic heterogeneity of ALK fusion breakpoints in non-small-cell lung cancer.

In lung adenocarcinoma, canonical EML4-ALK inversion results in a fusion protein with a constitutively active ALK kinase domain. Evidence of ALK rearrangement 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 ALK genomic rearrangement detected by fluorescence in situ hybridization (FISH) of interphase cells in formalin-fixed, paraffin-embedded tissue sections. At the genomic level, however, ALK rearrangements 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-ALK rearrangements, and those without (20.6 months vs 5.4 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.

Soft Tissue Sarcoma, Version 2.2018, NCCN Clinical Practice Guidelines in Oncology.

Soft tissue sarcomas (STS) are rare solid tumors of mesenchymal cell origin that display a heterogenous mix of clinical and pathologic characteristics. STS can develop from fat, muscle, nerves, blood vessels, and other connective tissues. The evaluation and treatment of patients with STS requires a multidisciplinary team with demonstrated expertise in the management of these tumors. The complete NCCN Guidelines for STS provide recommendations for the diagnosis, evaluation, and treatment of extremity/superficial trunk/head and neck STS, as well as intra-abdominal/retroperitoneal STS, gastrointestinal stromal tumors, desmoid tumors, and rhabdomyosarcoma. This portion of the NCCN Guidelines discusses general principles for the diagnosis, staging, and treatment of STS of the extremities, superficial trunk, or head and neck; outlines treatment recommendations by disease stage; and reviews the evidence to support the guidelines recommendations.

Routine use of clinical exome-based next-generation sequencing for evaluation of patients with thrombotic microangiopathies.

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.

Spectrum of mutations in leiomyosarcomas identified by clinical targeted next-generation sequencing.

Recurrent genomic mutations in uterine and non-uterine leiomyosarcomas have not been well established. Using a next generation sequencing (NGS) panel of common cancer-associated genes, 25 leiomyosarcomas arising from multiple sites were examined to explore genetic alterations, including single nucleotide variants (SNV), small insertions/deletions (indels), and copy number alterations (CNA). Sequencing showed 86 non-synonymous, coding region somatic variants within 151 gene targets in 21 cases, with a mean of 4.1 variants per case; 4 cases had no putative mutations in the panel of genes assayed. The most frequently altered genes were TP53 (36%), ATM and ATRX (16%), and EGFR and RB1 (12%). CNA were identified in 85% of cases, with the most frequent copy number losses observed in chromosomes 10 and 13 including PTEN and RB1; the most frequent gains were seen in chromosomes 7 and 17. Our data show that deletions in canonical cancer-related genes are common in leiomyosarcomas. Further, the spectrum of gene mutations observed shows that defects in DNA repair and chromosomal maintenance are central to the biology of leiomyosarcomas, and that activating mutations observed in other common cancer types are rare in leiomyosarcomas.

Association of Li-Fraumeni Syndrome With Small Cell Carcinoma of the Ovary, Hypercalcemic Type and Concurrent Pleomorphic Liposarcoma of the Cervix.

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 TP53 mutations (p.R196*), and wild-type SMARCA4 in 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 FGFR1 in both tumors, and amplification of MYC in 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 TP53 mutation, confirming the diagnosis of Li-Fraumeni syndrome. These findings are novel and for the first time associate SCCOHT with Li-Fraumeni syndrome.

Soft Tissue Sarcoma, Version 2.2016, NCCN Clinical Practice Guidelines in Oncology.

Soft tissue sarcomas (STS) are rare solid tumors of mesenchymal cell origin that display a heterogenous mix of clinical and pathologic characteristics. STS can develop from fat, muscle, nerves, blood vessels, and other connective tissues. The evaluation and treatment of patients with STS requires a multidisciplinary team with demonstrated expertise in the management of these tumors. The complete NCCN Guidelines for Soft Tissue Sarcoma (available at NCCN.org) provide recommendations for the diagnosis, evaluation, and treatment of extremity/superficial trunk/head and neck STS, as well as intra-abdominal/retroperitoneal STS, gastrointestinal stromal tumor, desmoid tumors, and rhabdomyosarcoma. This manuscript discusses guiding principles for the diagnosis and staging of STS and evidence for treatment modalities that include surgery, radiation, chemoradiation, chemotherapy, and targeted therapy.

Clinical next-generation sequencing in patients with non-small cell lung cancer.

BACKGROUND: A clinical assay was implemented to perform next-generation sequencing (NGS) of genes commonly mutated in multiple cancer types. This report describes the feasibility and diagnostic yield of this assay in 381 consecutive patients with non-small cell lung cancer (NSCLC). METHODS: Clinical targeted sequencing of 23 genes was performed with DNA from formalin-fixed, paraffin-embedded (FFPE) tumor tissue. The assay used Agilent SureSelect hybrid capture followed by Illumina HiSeq 2000, MiSeq, or HiSeq 2500 sequencing in a College of American Pathologists-accredited, Clinical Laboratory Improvement Amendments-certified laboratory. Single-nucleotide variants and insertion/deletion events were reported. This assay was performed before methods were developed to detect rearrangements by NGS. RESULTS: Two hundred nine of all requisitioned samples (55%) were successfully sequenced. The most common reason for not performing the sequencing was an insufficient quantity of tissue available in the blocks (29%). Excisional, endoscopic, and core biopsy specimens were sufficient for testing in 95%, 66%, and 40% of the cases, respectively. The median turnaround time (TAT) in the pathology laboratory was 21 days, and there was a trend of an improved TAT with more rapid sequencing platforms. Sequencing yielded a mean coverage of 1318×. Potentially actionable mutations (ie, predictive or prognostic) were identified in 46% of 209 samples and were most commonly found in KRAS (28%), epidermal growth factor receptor (14%), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (4%), phosphatase and tensin homolog (1%), and BRAF (1%). Five percent of the samples had multiple actionable mutations. A targeted therapy was instituted on the basis of NGS in 11% of the sequenced patients or in 6% of all patients. CONCLUSIONS: NGS-based diagnostics are feasible in NSCLC and provide clinically relevant information from readily available FFPE tissue. The sample type is associated with the probability of successful testing.

ATHLATES: accurate typing of human leukocyte antigen through exome sequencing.

Human leukocyte antigen (HLA) typing at the allelic level can in theory be achieved using whole exome sequencing (exome-seq) data with no added cost but has been hindered by its computational challenge. We developed ATHLATES, a program that applies assembly, allele identification and allelic pair inference to short read sequences, and applied it to data from Illumina platforms. In 15 data sets with adequate coverage for HLA-A, -B, -C, -DRB1 and -DQB1 genes, ATHLATES correctly reported 74 out of 75 allelic pairs with an overall concordance rate of 99% compared with conventional typing. This novel approach should be broadly applicable to research and clinical laboratories.

Retinoblastoma gene mutations detected by whole exome sequencing of Merkel cell carcinoma.

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.

Gastrointestinal stromal tumors, version 2.2014.

Gastrointestinal stromal tumors (GIST) are the most common soft tissue sarcoma of the gastrointestinal tract, resulting most commonly from KIT or platelet-derived growth factor receptor α (PDGFRα)-activating mutations. These NCCN Guideline Insights highlight the important updates to the NCCN Guidelines for Soft Tissue Sarcoma specific to the management of patients with GIST experiencing disease progression while on imatinib and/or sunitinib.

Soft tissue sarcoma, version 2.2014.

These NCCN Guidelines Insights highlight the important updates to the NCCN Guidelines for Soft Tissue Sarcoma (STS) specific to the role of radiation therapy in the management of patients with retroperitoneal/intra-abdominal STS. The guidelines have also included recommendations for genetic testing and counseling for patients with a clinical and/or family history of genetic cancer syndromes associated with a predisposition for the development of STS.

Lymphovascular space invasion in microcystic elongated and fragmented (MELF)-pattern well-differentiated endometrioid adenocarcinoma is associated with a higher rate of lymph node metastasis.

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.

BRAF V600E mutational status in pediatric thyroid cancer.

BACKGROUND: Clinical outcome of papillary thyroid carcinoma (PTC) in children differs significantly from that of adults. There is no clear explanation of this difference although previous studies have demonstrated a lower prevalence of the BRAF(V600E) mutation in PTC of children. However, data are limited due to the rarity of this diagnosis. BRAF(V600E) mutation prevalence and its relationship with outcome in pediatric PTC remain unclear. PROCEDURE: BRAF(V600E) mutational status was determined in 27 PTC patients less than 22 years of age using restriction fragment length polymorphism (RFLP) analysis. The relationship between BRAF(V600E) mutation status, patient and tumor characteristics as well as progression-free survival (PFS) were analyzed. RESULTS: BRAF(V600E) was present in 63% of patients and occurred more often in male patients versus females (P = 0.033). Presence of the mutation did not correlate with any difference in extent of disease at diagnosis, tumor size, capsular invasion, vascular invasion, soft tissue invasion, or margin status. At 10 years, PFS for BRAF(V600E) positive versus negative patients was 55.5% versus 70.0%, respectively (P = 0.48). Overall survival was 100% and median follow-up was 13.9 years. CONCLUSIONS: This study of pediatric PTC demonstrates that BRAF(V600E) mutations occur in children at a rate comparable to adults. We found a correlation of BRAF(V600E) with the male gender, but no evidence that the mutation correlates with more extensive or aggressive disease. This analysis suggests that differences in disease course of PTC in children versus adults are not strongly dependent upon the presence of the BRAF(V600E) mutation.

Transient receptor potential cation channel, subfamily C, member 5 (TRPC5) is a cold-transducer in the peripheral nervous system.

Detection and adaptation to cold temperature is crucial to survival. Cold sensing in the innocuous range of cold (>10-15 °C) in the mammalian peripheral nervous system is thought to rely primarily on transient receptor potential (TRP) ion channels, most notably the menthol receptor, TRPM8. Here we report that TRP cation channel, subfamily C member 5 (TRPC5), but not TRPC1/TRPC5 heteromeric channels, are highly cold sensitive in the temperature range 37-25 °C. We found that TRPC5 is present in mouse and human sensory neurons of dorsal root ganglia, a substantial number of peripheral nerves including intraepithelial endings, and in the dorsal lamina of the spinal cord that receives sensory input from the skin, consistent with a potential TRPC5 function as an innocuous cold transducer in nociceptive and thermosensory nerve endings. Although deletion of TRPC5 in 129S1/SvImJ mice resulted in no temperature-sensitive behavioral changes, TRPM8 and/or other menthol-sensitive channels appear to underpin a much larger component of noxious cold sensing after TRPC5 deletion and a shift in mechanosensitive C-fiber subtypes. These findings demonstrate that highly cold-sensitive TRPC5 channels are a molecular component for detection and regional adaptation to cold temperatures in the peripheral nervous system that is distinct from noxious cold sensing.

Characterization of translocations in mesenchymal hamartoma and undifferentiated embryonal sarcoma of the liver.

BACKGROUND: Mesenchymal hamartoma of the liver (MHL) is an uncommon benign primary liver tumor that typically occurs in the pediatric population, although cases have been described in adults. MHL is sometimes associated with the highly malignant undifferentiated embryonal sarcoma (UES), and the synchronous or metachronous occurrence of MHL and UES suggests they share a common genetic link. Although the exact mechanism of tumorigenesis has not been identified, MHL cases harbor recurring chromosomal rearrangements involving 19q13. DESIGN: In order to provide more details on the genetic events of MHL tumorigenesis, capture-based next generation sequencing (NGS) targeted to loci recently shown to be involved in a translocation in a case of UES arising in MHL (specifically, the MALAT1 gene on chromosome 11 and a gene poor region termed MHLB1 on chromosome 19) was performed on formalin fixed paraffin embedded tissue from seven cases of MHL. RESULTS: Chromosome rearrangements involving the MHLB1 locus were identified in three of the seven cases, including the translocation t(11,19)(q13.1;q13.42) involving the MALAT1 gene; the translocation t(2,19)(q31.1;q13.42) involving AK023515, an uncharacterized noncoding gene; and the inversion inv(19,19)(q13.42;q13.43) involving the PEG3 gene encoding a Kruppel-type zinc-finger protein. Rearrangements were exclusively identified in pediatric tumors. In each case, the presence of the rearrangement was confirmed by PCR and interphase FISH. Interphase FISH also demonstrated that the arrangements occur within the spindle cell component but not within the epithelial components of the tumor. CONCLUSIONS: Since the MHLB1 locus contains a CpG-rich region whose methylation regulates C19MC miRNA genes, rearrangements that disrupt this region may contribute to MHL development through alteration of miRNA expression. The demonstration that the loose stromal cells harbor the rearrangements indicates that (some cases of) MHL are a neoplastic process due to a somatic genetic change and not a germline abnormality.