Functional Enhancers Shape Extrachromosomal Oncogene Amplifications.

Non-coding regions amplified beyond oncogene borders have largely been ignored. Using a computational approach, we find signatures of significant co-amplification of non-coding DNA beyond the boundaries of amplified oncogenes across five cancer types. In glioblastoma, EGFR is preferentially co-amplified with its two endogenous enhancer elements active in the cell type of origin. These regulatory elements, their contacts, and their contribution to cell fitness are preserved on high-level circular extrachromosomal DNA amplifications. Interrogating the locus with a CRISPR interference screening approach reveals a diversity of additional elements that impact cell fitness. The pattern of fitness dependencies mirrors the rearrangement of regulatory elements and accompanying rewiring of the chromatin topology on the extrachromosomal amplicon. Our studies indicate that oncogene amplifications are shaped by regulatory dependencies in the non-coding genome.

IGJ and SPATS2L immunohistochemistry sensitively and specifically identify BCR::ABL1+ and BCR::ABL1-like B-acute lymphoblastic leukaemia.

Therapeutic management and prognostication for patients with B-acute lymphoblastic leukaemia (B-ALL) require appropriate disease subclassification. BCR::ABL1-like B-ALL is unique in that it is defined by a gene expression profile similar to BCR::ABL1+ B-ALL rather than a unifying recurrent translocation. Current molecular/cytogenetic techniques to identify this subtype are expensive, not widely accessible, have long turnaround times and/or require an adequate liquid biopsy. We have studied a total of 118 B-ALL cases from three institutions in two laboratories to identify surrogates for BCR::ABL1+/like B-ALL. We report that immunoglobulin joining chain (IGJ) and spermatogenesis associated serine-rich 2-like (SPATS2L) immunohistochemistry (IHC) sensitively and specifically identify BCR::ABL1+/like B-ALL. IGJ IHC positivity has a sensitivity of 83%, a specificity of 95%, a positive predictive value (PPV) of 89% and a negative predictive value (NPV) of 90%. SPATS2L staining has similar sensitivity and NPV but lower specificity (85%) and PPV (70%). The presence of either IGJ or SPATS2L staining augments the sensitivity (93%) and NPV (95%). While these findings would need to be validated in larger studies, they suggest that IGJ and/or SPATS2L IHC may be utilized in identifying BCR::ABL1-like B-ALL or in selecting B-ALL cases for confirmatory molecular/genetic testing, particularly in resource-limited settings.

A novel subtype of sporadic Creutzfeldt-Jakob disease with PRNP codon 129MM genotype and PrP plaques.

The presence of amyloid kuru plaques is a pathological hallmark of sporadic Creutzfeldt-Jakob disease (sCJD) of the MV2K subtype. Recently, PrP plaques (p) have been described in the white matter of a small group of CJD (p-CJD) cases with the 129MM genotype and carrying resPrPD type 1 (T1). Despite the different histopathological phenotype, the gel mobility and molecular features of p-CJD resPrPD T1 mimic those of sCJDMM1, the most common human prion disease. Here, we describe the clinical features, histopathology, and molecular properties of two distinct PrP plaque phenotypes affecting the gray matter (pGM) or the white matter (pWM) of sCJD cases with the PrP 129MM genotype (sCJDMM). Prevalence of pGM- and pWM-CJD proved comparable and was estimated to be ~ 0.6% among sporadic prion diseases and ~ 1.1% among the sCJDMM group. Mean age at onset (61 and 68 years) and disease duration (~ 7 months) of pWM- and pGM-CJD did not differ significantly. PrP plaques were mostly confined to the cerebellar cortex in pGM-CJD, but were ubiquitous in pWM-CJD. Typing of resPrPD T1 showed an unglycosylated fragment of ~ 20 kDa (T120) in pGM-CJD and sCJDMM1 patients, while a doublet of ~ 21-20 kDa (T121-20) was a molecular signature of pWM-CJD in subcortical regions. In addition, conformational characteristics of pWM-CJD resPrPD T1 differed from those of pGM-CJD and sCJDMM1. Inoculation of pWM-CJD and sCJDMM1 brain extracts to transgenic mice expressing human PrP reproduced the histotype with PrP plaques only in mice challenged with pWM-CJD. Furthermore, T120 of pWM-CJD, but not T121, was propagated in mice. These data suggest that T121 and T120 of pWM-CJD, and T120 of sCJDMM1 are distinct prion strains. Further studies are required to shed light on the etiology of p-CJD cases, particularly those of T120 of the novel pGM-CJD subtype.

Impact of an alternative chromosome 17 probe and the 2013 American Society of Clinical Oncology and College of American Pathologists guidelines on fluorescence in situ hybridization for the determination of HER2 gene amplification in breast cancer.

BACKGROUND: The dual-probe fluorescence in situ hybridization (FISH) assay for human epidermal growth factor receptor 2 (HER2) gene amplification in breast cancer provides an HER2:CEP17 (centromere enumeration probe for chromosome 17) ratio. Copy number alteration (CNA) in CEP17 may skew this ratio. The authors analyzed the impact of the 2013 American Society of Oncology/College of American Pathologists (ASCO/CAP) guidelines and an alternative chromosome 17 probe on HER2 status in tumor specimens with CEP17 CNA. METHODS: Specimens with CEP17 CNA (n = 310) were selected from 3048 tumor samples that were received from January 2013 to June 2015 for testing with the alternative chromosome 17 probe D17S122. Reclassification of HER2 status was assessed using the 2007 and 2013 ASCO/CAP guidelines. RESULTS: The alternative chromosome 17 probe reclassified 82 of 310 (26.5%) and 87 of 310 (28.1%) tumors using the 2007 and 2013 guidelines, respectively. Of the 41 of 310 tumors (13.2%) that were reclassified from nonamplified to amplified according to 2007 guidelines, 28 of 41 (68.3%) had an average HER2 copy number ≥4.0 and <6.0. The 39 of 310 tumors (12.6%) that were reclassified from equivocal to amplified according to 2013 guidelines had a mean HER2 copy number between ≥4.0 and <6.0. Most of these patients had stage I, hormone receptor-positive, lymph node-negative tumors, which is an unusual clinicopathologic profile for HER2-amplified tumors, and most received HER2-targeted therapy in addition to endocrine therapy. CONCLUSIONS: Reflex testing with an alternative chromosome 17 probe using the 2013 ASCO/CAP guidelines reclassified 28.1% of tumor samples that had CEP17 CNA, converting nearly one-half from equivocal to amplified. The benefit of HER2-targeted therapy in this patient population requires further study. Cancer 2017;123:2230-2239. © 2017 American Cancer Society.

Transient abnormal myelopoiesis of a newborn not associated with chromosome 21 abnormalities or GATA1 mutations.

Transient abnormal myelopoiesis (TAM) is a disorder of Down syndrome newborns characterized by megakaryocytic blasts indistinguishable from acute myeloid leukemia (AML), which undergoes spontaneous remission. Acquired GATA1 mutations are present in blasts of both TAM and the subsequent AML which sometimes develops. We present a unique case of a newborn with leukemic megakaryoblasts indistinguishable from those of TAM who had neither extra material from chromosome 21 in the germline or blasts, nor evidence of GATA1 mutations. These findings suggest there are other genetic abnormalities that can lead to TAM besides GATA1 mutation in the setting of trisomy 21. Pediatr Blood Cancer 2015;62:353-355. © 2014 Wiley Periodicals, Inc.

Subjectivity in chromosome band-level estimation: a multicenter study.

PURPOSE: Chromosome band level is the primary quality indicator for G-banded metaphase chromosome analysis. Although current professional guidelines address the minimum necessary band level for constitutional studies, there is no study documenting the comparative performance of different band-level estimation methods. METHODS: This study compared 5 band-level estimation methods (Stallard, Vancouver, Welborn, United Kingdom External Quality Assurance Scheme, and Ford) in a multicenter study in which 82 readers from 7 different clinical cytogenetics laboratories evaluated the same 10 karyotypes (5 from amniotic fluid and 5 from peripheral blood) by each method. RESULTS: There was a 94% correlation between the five band-level estimation methods. The Welborn method yielded significantly lower scores for amniotic fluid karyotypes (P < 0.01) but not for peripheral blood karyotypes (P = 0.75). The distribution of scores obtained from different readers suggests a high level of subjectivity in chromosome band-level assessment. The variation in band-level estimation did not correlate with reader experience or study center, except for readers from one laboratory, for which the distribution of scores was significantly lower (P < 0.01). CONCLUSION: The results from this study suggest that the consistent use of one method is more important than the actual method employed for monitoring karyotype quality.

Aggressive Lymphoplasmacytic Neoplasm With an Unusual In-frame Deletion of MYD88 Associated With TRAF3 and TP53 Mutations and Complex Karyotype.

Lymphoplasmacytic lymphoma often needs to be differentiated from other B-cell lymphomas with plasmacytic differentiation, especially marginal zone cell lymphoma. Molecular detection of MYD88 p.L265P hotspot mutation supports the diagnosis of lymphoplasmacytic lymphoma since it is seen in about 90% of such lymphoma, which is much higher than other B-cell lymphomas. MYD88 p.L265P is a gain-of-function mutation that enhances the activity of the NF-κB signaling pathway and therefore drives lymphomagenesis. Other mutations in MYD88 are rarely reported. This study aims to report an unusual MYD88 in-frame deletion in an aggressive lymphoplasmacytic neoplasm. This is an IgM-positive, CD5- and CD10-negative mature B-cell lymphoma with prominent plasmacytic differentiation and aggressive features. The clinical and pathologic findings were most consistent with lymphoplasmacytic lymphoma. Next-generation sequencing identified an unusual MYD88 in-frame deletion in the absence of the hotpot p.L265P mutation. Other concurrent pathogenic mutations also include truncating mutations of TRAF3, which is a negative regulator of the NF-κB signaling pathway, and a missense mutation of TP53. Karyotype analysis showed complex karyotypes, including chromosome 6q deletion. By searching literature and online cancer databases, we identified only 8 other mature B-cell lymphomas with MYD88 in-frame deletions, but none of them was diagnosed with lymphoplasmacytic lymphoma. Recognizing such in-frame deletions is necessary to help understand the mutational spectrum of MYD88 in B-cell lymphomas. It remains to be further investigated whether such MYD88 in-frame deletions are also overrepresented in lymphoplasmacytic lymphoma among other B-cell lymphomas.

Philadelphia chromosome-positive T-cell acute lymphoblastic leukemia: a case report.

Philadelphia chromosome-positive (Ph+) T-cell acute lymphoblastic leukemia (T-ALL) is a rare and aggressive type of acute leukemia. The Philadelphia chromosome is the hallmark of chronic myeloid leukemia (CML). The differentiation between Ph+ T-ALL and T-cell lymphoblastic crisis of CML may be problematic in some cases. Here, we report a rare case of de novo Ph+ T-ALL that presented a diagnostic challenge. The overall clinical, immunophenotypic, cytogenetic, and xenotransplantation results suggest a diagnosis of Ph+ T-ALL. The patient was treated with induction chemotherapy including imatinib followed by haploidentical stem cell transplantation and achieved complete remission.

The challenges and opportunities of offering and integrating training in clinical molecular genetics and clinical cytogenetics: A survey of LGG Fellowship Program Directors.

Purpose: The specialty of Laboratory Genetics and Genomics (LGG) was created in 2017 in an effort to reflect the increasing convergence in technologies and approaches between clinical molecular genetics and clinical cytogenetics. However, there has not yet been any formal evaluation of the merging of these disciplines and the challenges faced by Program Directors (PDs) tasked with ensuring the successful training of laboratory geneticists under the new model. Methods: An electronic multi-question Qualtrics survey was created and was sent to the PD for each of the Accreditation Council for Graduate Medical Education-accredited LGG fellowship programs at the time. The data were collected, and the responses were aggregated for each question. Results: All of the responding PDs had started training at least 1 LGG fellow. PDs noted challenges with funding, staff shortages, molecular/cytogenetics content integration, limited total training time, increased remote work, increased sendout testing, and a lack of prior cytogenetics knowledge among incoming fellows. Conclusion: This survey attempted to assess the challenges that LGG PDs have been facing in offering and integrating clinical molecular genetics and clinical cytogenetics fellowship training. Common challenges between programs were noted, and a set of 6 concluding comments are provided to facilitate future discussion.

Mucin 4 protein is expressed in B-acute lymphoblastic leukemia and is restricted to BCR::ABL1-positive and BCR::ABL-like subtypes.

Mucin 4 (MUC4) is a transmembrane mucin that, like most mucins, is not expressed in normal hematopoietic cells, but little is known about its expression in malignant hematopoiesis. B-acute lymphoblastic leukemia (B-ALL) consists of genetically distinct disease subtypes with similarities and differences in gene expression most frequently studied at the mRNA level, which is less amenable to widespread routine clinical use. Here, we demonstrate using immunohistochemistry (IHC) that MUC4 protein is expressed in less than 10% of B-ALL, with expression restricted to BCR::ABL1+ and BCR::ABL1-like (CRLF2 rearranged) subtypes of B-ALL (4/13, 31%). None (0/36, 0%) of the remaining B-ALL subtypes expressed MUC4. We compare clinical and pathologic features of MUC4+ and MUC4- BCR::ABL1+/like cases and most significantly report a possible shorter time to relapse for MUC4+ BCR::ABL1 B-ALL that would need to be validated in larger studies. In conclusion, MUC4 is a specific, albeit insensitive, marker for these high-risk subtypes of B-ALL. We propose that MUC4 IHC may be used diagnostically to rapidly identify these B-ALL subtypes, particularly in resource-limited settings or when an aspirate sample is not available for ancillary genetic studies.

Genetic aspects of human prion diseases.

Human prion diseases are rapidly progressive and fatal neurodegenerative conditions caused by a disease-causing isoform of the native prion protein. The prion protein gene (PRNP) encodes for the cellular prion protein, which is the biological substrate for prion disease transmission and neurotoxicity. Human prion diseases have three etiologies: sporadic, genetic, and acquired. PRNP polymorphisms and pathogenic variants play a large role in the frequency, age at onset, and clinicopathologic phenotype of prion diseases. Genetic prion diseases will be covered in detail and information necessary for clinical care, predictive genetic testing, and genetic counseling will be reviewed. Because the prion protein is necessary for transmission and neurotoxicity, many experimental treatments targeting its production are being investigated and hold potential promise as a disease modifying treatment for all forms of prion disease, including asymptomatic mutation carriers. This article will review genetic aspects of human prion disease and their influence on epidemiology, clinicopathologic phenotype, diagnostics, clinical management, and potential treatment approaches.

Identification of a Cancer-Predisposing Germline POT1 p.Ile49Metfs*7 Variant by Targeted Sequencing of a Splenic Marginal Zone Lymphoma.

Germline disruptive variants in Protection of Telomeres 1 (POT1) predispose to a wide variety of cancers, including melanoma, chronic lymphocytic leukemia (CLL), Hodgkin lymphoma, myeloproliferative neoplasms, and glioma. We report the first case of splenic marginal zone lymphoma (SMZL) arising in a patient with a germline POT1 variant: a 65-year-old male with an extensive history of cancer, including melanoma and papillary thyroid carcinoma, who presented with circulating atypical lymphocytosis. Bone marrow biopsy revealed 20% involvement by a CD5-CD10- B-cell lymphoma that was difficult to classify. During the clinical workup of his low-grade lymphoma, targeted next-generation sequencing (NGS) identified POT1 p.I49Mfs*7 (NM_015450:c. 147delT) at a variant allele frequency (VAF) of 51%. NGS of skin fibroblasts confirmed the POT1 variant was germline. This likely pathogenic POT1 loss-of-function variant has only been reported once before as a germline variant in a patient with glioma and likely represents one of the most deleterious germline POT1 variants ever linked to familial cancer. The spectrum of cancers associated with germline pathogenic POT1 variants (i.e., autosomal dominant POT1 tumor predisposition syndrome) should potentially be expanded to include SMZL, a disease often associated with the loss of chromosome 7q: the location of the POT1 genetic locus (7q31.33).

An evidence-based approach to establish the functional and clinical significance of copy number variants in intellectual and developmental disabilities.

PURPOSE: Copy number variants have emerged as a major cause of human disease such as autism and intellectual disabilities. Because copy number variants are common in normal individuals, determining the functional and clinical significance of rare copy number variants in patients remains challenging. The adoption of whole-genome chromosomal microarray analysis as a first-tier diagnostic test for individuals with unexplained developmental disabilities provides a unique opportunity to obtain large copy number variant datasets generated through routine patient care. METHODS: A consortium of diagnostic laboratories was established (the International Standards for Cytogenomic Arrays consortium) to share copy number variant and phenotypic data in a central, public database. We present the largest copy number variant case-control study to date comprising 15,749 International Standards for Cytogenomic Arrays cases and 10,118 published controls, focusing our initial analysis on recurrent deletions and duplications involving 14 copy number variant regions. RESULTS: Compared with controls, 14 deletions and seven duplications were significantly overrepresented in cases, providing a clinical diagnosis as pathogenic. CONCLUSION: Given the rapid expansion of clinical chromosomal microarray analysis testing, very large datasets will be available to determine the functional significance of increasingly rare copy number variants. This data will provide an evidence-based guide to clinicians across many disciplines involved in the diagnosis, management, and care of these patients and their families.

An unusual case of cutaneous blastic plasmacytoid dendritic cell neoplasm with concomitant B-cell lymphoproliferative disorder.

Blastic plasmacytoid dendritic cell neoplasm is a rare hematologic malignancy characterized by aggressive clinical behavior and frequent cutaneous involvement. We describe a case of a 64-year-old man with a rapidly enlarging subcutaneous forearm mass. Histologic examination of the excisional biopsy specimen revealed a diffuse proliferation of atypical hematolymphoid cells in the dermis extending to the deep subcutaneous soft tissues. Occasional aggregates of small lymphocytes were noted to be distributed within the mass. The tumor cells expressed CD4, CD45, CD56, CD123, and terminal deoxynucleotidyl transferase (TdT) but not CD3, CD20, or CD34. A diagnosis of blastic plasmacytoid dendritic cell neoplasm was rendered. Chromosome analysis revealed a 45 X, -Y karyotype. In addition, flow cytometry identified a small population of monoclonal B cells. A staging bone marrow aspirate and biopsy was performed, which showed normal cytogenetics and no evidence of involvement by blastic plasmacytoid dendritic cell neoplasm. Flow cytometric evaluation of the bone marrow revealed a CD5-negative, CD10-negative monoclonal B-cell population consistent with a B-cell lymphoproliferative disorder. This is a very unusual example of cutaneous blastic plasmacytoid dendritic cell neoplasm with a novel cytogenetic finding and concomitant B-cell lymphoproliferative disorder. Although previously not reported, our case shows that blastic plasmacytoid dendritic cell neoplasm may be associated with lymphoid malignancy. The relationship between the 2 neoplasms, however, is unclear. A high degree of suspicion and bone marrow examination in patients with a new diagnosis of blastic plasmacytoid dendritic cell neoplasm is required to avoid this potential diagnostic pitfall.