JAK2 Mutations Are Rare and Diverse in Myelodysplastic Syndromes: Case Series and Review of the Literature.

OBJECTIVES: To investigate and characterize JAK2 mutations in myelodysplastic syndrome (MDS), we present three cases with diverse JAK2 mutations and review the literature. METHODS: The institutional SoftPath software was used to find MDS cases between January 2020 and April 2022. The cases with a diagnosis of a myelodysplastic/myeloproliferative overlap syndrome including MDS/MPN with ring sideroblasts and thrombocytosis were excluded. The cases with molecular data by next generation sequencing looking for gene aberrations commonly seen in myeloid neoplasms were reviewed for the detection of JAK2 mutations including variants. A literature review on the identification, characterization, and significance of JAK2 mutations in MDS was performed. RESULTS: Among 107 cases of the MDS reviewed, a JAK2 mutation was present in three cases, representing 2.8% of the overall cases. A JAK2 V617F mutation was found in one case representing slightly less than 1% of all the MDS cases. In addition, we found JAK2 R564L and JAK2 I670V point mutation variants to be associated with a myelodysplastic phenotype. CONCLUSIONS: JAK2 mutations in MDS are rare and represent less than 3% of cases. It appears that JAK2 variant mutations in MDS are diverse and further studies are needed to understand their role in the phenotype and prognosis of the disease.

Cytogenetic and Molecular Characterization of IDH-Wildtype Glioblastomas and Grade 4 IDH-Mutant Astrocytomas with Unusual Histology.

Glioblastoma (GBM) is a highly heterogenous tumor. Though several well-defined histological patterns of GBMs are known, these are infrequent, and the molecular correlates of several of these histological patterns are not well understood. We identified 31 adult-type infiltrating grade 4 gliomas with unusual histology in our institutional archives from 2016 to 2020, including tumors with a preponderant component of giant cell (n = 15), gemistocytes (n = 6), spindle cells (n = 5), small cells (n = 3), and ependymoma-like features (n = 2). We performed molecular and cytogenetic profiles of IDH-wildtype GBMs with unusual histology and compared to 48 tumors with conventional histology. We found that the majority (85%) of giant cell GBM had increased numbers of whole chromosome loss and genomic haploidization compared to conventional GBMs and other variants. Furthermore, we identified a genetically confirmed GBM with prominent ependymal features, indicating that glial tumors with ependymal features should be considered in the differential diagnosis of GBM. We also identified 6 IDH-mutant grade 4 astrocytomas with unusual histology and similar molecular and cytogenetic profiles to conventional appearing grade 4 IDH-mutant astrocytomas. These findings emphasize the role of molecular/cytogenetic analyses in the diagnostic clarification of GBMs with unusual histological patterns, refine the classification of unusual GBMs, and potentially pave the way for personalized therapies.

Cytogenetically cryptic PML::RARA fusion in acute promyelocytic leukemia: Testing strategies in the modern era.

Acute promyelocytic leukemia (APL) is a unique leukemia that is characterized by the PML::RARA fusion. This fusion is often detected by conventional karyotype and fluorescence in situ hybridization (FISH); however, rare cases are cryptic and require molecular techniques to identify the PML::RARA fusion. Furthermore, as the incidence of these cases is rare, analysis by a targeted next-generation sequencing (NGS) panel of myeloid associated genes has never been reported. Herein, a clinical APL case is reported where the PML::RARA fusion was detected only by reverse transcriptase-polymerase chain reaction (RT-PCR), thus underscoring the necessity of utilizing complementary techniques when suspicion for APL is present.

Multiple Whole Chromosomal Gains Define Angiomatous Meningiomas and Are Absent From the Tumor Vasculature.

Angiomatous meningioma is a variant with prominent vascularity that can mimic other highly vascularized tumors and present diagnostic challenges. Unlike most meningioma variants, where NF2 gene loss on chromosome 22 is the most common genetic abnormality, angiomatous meningiomas are unique in having multiple whole chromosome gains (polysomies). We analyzed 38 meningiomas, 9 angiomatous (including 2 atypical and 1 anaplastic), and 29 nonangiomatous meningiomas, using array comparative genomic hybridization (aCGH). Angiomatous meningiomas showed multiple chromosomal alterations including polysomies and copy neutral loss of heterozygosity in comparison to nonangiomatous variants. The most frequent gains were of chromosomes 5 and 20 (100% and 89% of cases, respectively); none showed chromosome 22 loss. Furthermore, using fluorescence in situ hybridization we show that the vasculature lacked chromosomal polysomy. While generally benign, we present 2 grade II and the first cytogenetically confirmed grade III angiomatous meningioma, demonstrating their potentially aggressive behavior. Thus, multiple polysomies define angiomatous meningioma and aCGH can distinguish this variant from nonangiomatous meningiomas and other histological mimics in diagnostically challenging cases. Furthermore, the prominent vasculature is not neoplastic and likely induced by angiogenic factors. Together, these findings suggest a distinct tumorigenic pathway in angiomatous meningiomas.

A high-grade glioma with SOS1 amplification.

The molecular alterations underlying progression of low-grade glial/glioneuronal tumors remain to be elucidated. We present a case of a 56-year-old male with an enhancing left temporal lobe tumor. Histology revealed a high-grade glioma adjacent to a low-grade glioneuronal component with abundant Rosenthal fibers, focal eosinophilic granular bodies, and CD34-positive neurons. The tumor was negative for IDH1 (R132H), BRAF-V600E, and the KIAA1549-BRAF fusion. Comparative genomic hybridization detected a large amplification (> 15 copies) of the Son of Sevenless 1 (SOS1) gene, a component of the MAPK pathway. Although activating mutations in the MAPK pathway occur frequently in gliomas and glioneuronal tumors, SOS1 gene amplification has not been reported previously. This case indicates another potential mechanism for MAPK activation in glial tumors.

Characteristics of myeloproliferative neoplasms in patients exposed to ionizing radiation following the Chernobyl nuclear accident.

Myeloproliferative neoplasms (MPNs) driver mutations are usually found in JAK2, MPL, and CALR genes; however, 10%-15% of cases are triple negative (TN). A previous study showed lower rate of JAK2 V617F in primary myelofibrosis patients exposed to low doses of ionizing radiation (IR) from Chernobyl accident. To examine distinct driver mutations, we enrolled 281 Ukrainian IR-exposed and unexposed MPN patients. Genomic DNA was obtained from peripheral blood leukocytes. JAK2 V617F, MPL W515, types 1- and 2-like CALR mutations were identified by Sanger Sequencing and real time polymerase chain reaction. Chromosomal alterations were assessed by oligo-SNP microarray platform. Additional genetic variants were identified by whole exome and targeted sequencing. Statistical significance was evaluated by Fisher's exact test and Wilcoxon's rank sum test (R, version 3.4.2). IR-exposed MPN patients exhibited a different genetic profile vs unexposed: lower rate of JAK2 V617F (58.4% vs 75.4%, P = .0077), higher rate of type 1-like CALR mutation (12.2% vs 3.1%, P = .0056), higher rate of TN cases (27.8% vs 16.2%, P = .0366), higher rate of potentially pathogenic sequence variants (mean numbers: 4.8 vs 3.1, P = .0242). Furthermore, we identified several potential drivers specific to IR-exposed TN MPN patients: ATM p.S1691R with copy-neutral loss of heterozygosity at 11q; EZH2 p.D659G at 7q and SUZ12 p.V71 M at 17q with copy number loss. Thus, IR-exposed MPN patients represent a group with distinct genomic characteristics worthy of further study.

Assessing copy number aberrations and copy neutral loss of heterozygosity across the genome as best practice: An evidence based review of clinical utility from the cancer genomics consortium (CGC) working group for myelodysplastic syndrome, myelodysplastic/myeloproliferative and myeloproliferative neoplasms.

Multiple studies have demonstrated the utility of chromosomal microarray (CMA) testing to identify clinically significant copy number alterations (CNAs) and copy-neutral loss-of-heterozygosity (CN-LOH) in myeloid malignancies. However, guidelines for integrating CMA as a standard practice for diagnostic evaluation, assessment of prognosis and predicting treatment response are still lacking. CMA has not been recommended for clinical work-up of myeloid malignancies by the WHO 2016 or the NCCN 2017 guidelines but is a suggested test by the European LeukaemiaNet 2013 for the diagnosis of primary myelodysplastic syndrome (MDS). The Cancer Genomics Consortium (CGC) Working Group for Myeloid Neoplasms systematically reviewed peer-reviewed literature to determine the power of CMA in (1) improving diagnostic yield, (2) refining risk stratification, and (3) providing additional genomic information to guide therapy. In this manuscript, we summarize the evidence base for the clinical utility of array testing in the workup of MDS, myelodysplastic/myeloproliferative neoplasms (MDS/MPN) and myeloproliferative neoplasms (MPN). This review provides a list of recurrent CNAs and CN-LOH noted in this disease spectrum and describes the clinical significance of the aberrations and how they complement gene mutation findings by sequencing. Furthermore, for new or suspected diagnosis of MDS or MPN, we present suggestions for integrating genomic testing methods (CMA and mutation testing by next generation sequencing) into the current standard-of-care clinical laboratory testing (karyotype, FISH, morphology, and flow).

Assessing copy number abnormalities and copy-neutral loss-of-heterozygosity across the genome as best practice in diagnostic evaluation of acute myeloid leukemia: An evidence-based review from the cancer genomics consortium (CGC) myeloid neoplasms working group.

Structural genomic abnormalities, including balanced chromosomal rearrangements, copy number gains and losses and copy-neutral loss-of-heterozygosity (CN-LOH) represent an important category of diagnostic, prognostic and therapeutic markers in acute myeloid leukemia (AML). Genome-wide evaluation for copy number abnormalities (CNAs) is at present performed by karyotype analysis which has low resolution and is unobtainable in a subset of cases. Furthermore, examination for possible CN-LOH in leukemia cells is at present not routinely performed in the clinical setting. Chromosomal microarray (CMA) analysis is a widely available assay for CNAs and CN-LOH in diagnostic laboratories, but there are currently no guidelines how to best incorporate this technology into clinical testing algorithms for neoplastic diseases including AML. The Cancer Genomics Consortium Working Group for Myeloid Neoplasms performed an extensive review of peer-reviewed publications focused on CMA analysis in AML. Here we summarize evidence regarding clinical utility of CMA analysis in AML extracted from published data, and provide recommendations for optimal utilization of CMA testing in the diagnostic workup. In addition, we provide a list of CNAs and CN-LOH regions which have documented clinical significance in diagnosis, prognosis and treatment decisions in AML.

EBV-positive, HHV8-negative large B-cell lymphoma with an unusual germinotropic growth pattern in an immunocompetent patient.

Clinicopathologic and cytogenetic findings of an unusual EBV+ve, HHV8-ve germinotropic lymphoma, with a nongerminal center immunophenotype occurring in an immunocompetent individual, are presented. A comprehensive literature search revealed a single report of three similar cases. These may represent a unique subset of EBV-positive large B-cell lymphomas in immunocompetent individuals.

Vemurafenib-resistance via de novo RBM genes mutations and chromosome 5 aberrations is overcome by combined therapy with palbociclib in thyroid carcinoma with BRAFV600E.

PURPOSE: Papillary thyroid carcinoma (PTC) is the most frequent endocrine tumor. BRAFV600E represents the PTC hallmark and is targeted with selective inhibitors (e.g. vemurafenib). Although there have been promising results in clinical trials using these inhibitors, most patients develop resistance and progress. Tumor clonal diversity is proposed as one mechanism underlying drug resistance. Here we have investigated mechanisms of primary and secondary resistance to vemurafenib in BRAFWT/V600E-positive PTC patient-derived cells with P16-/- (CDKN2A-/-). EXPERIMENTAL DESIGN: Following treatment with vemurafenib, we expanded a sub-population of cells with primary resistance and characterized them genetically and cytogenetically. We have used exome sequencing, metaphase chromosome analysis, FISH and oligonucleotide SNP-microarray assays to assess clonal evolution of vemurafenib-resistant cells. Furthermore, we have validated our findings by networks and pathways analyses using PTC clinical samples. RESULTS: Vemurafenib-resistant cells grow similarly to naïve cells but are refractory to apoptosis upon treatment with vemurafenib, and accumulate in G2-M phase. We find that vemurafenib-resistant cells show amplification of chromosome 5 and de novo mutations in the RBM (RNA-binding motifs) genes family (i.e. RBMX, RBM10). RBMX knockdown in naïve-cells contributes to tetraploidization, including expansion of clones with chromosome 5 aberrations (e.g. isochromosome 5p). RBMX elicits gene regulatory networks with chromosome 5q cancer-associated genes and pathways for G2-M and DNA damage-response checkpoint regulation in BRAFWT/V600E-PTC. Importantly, combined therapy with vemurafenib plus palbociclib (inhibitor of CDK4/6, mimicking P16 functions) synergistically induces stronger apoptosis than single agents in resistant-cells and in anaplastic thyroid tumor cells harboring the heterozygous BRAFWT/V600E mutation. CONCLUSIONS: Critically, our findings suggest for the first time that targeting BRAFWT/V600E and CDK4/6 represents a novel therapeutic strategy to treat vemurafenib-resistant or vemurafenib-naïve radioiodine-refractory BRAFWT/V600E-PTC. This combined therapy could prevent selection and expansion of aggressive PTC cell sub-clones with intrinsic resistance, targeting tumor cells either with primary or secondary resistance to BRAFV600E inhibitor.

Chromothriptic cure of WHIM syndrome.

Chromothripsis is a catastrophic cellular event recently described in cancer in which chromosomes undergo massive deletion and rearrangement. Here, we report a case in which chromothripsis spontaneously cured a patient with WHIM syndrome, an autosomal dominant combined immunodeficiency disease caused by gain-of-function mutation of the chemokine receptor CXCR4. In this patient, deletion of the disease allele, CXCR4(R334X), as well as 163 other genes from one copy of chromosome 2 occurred in a hematopoietic stem cell (HSC) that repopulated the myeloid but not the lymphoid lineage. In competitive mouse bone marrow (BM) transplantation experiments, Cxcr4 haploinsufficiency was sufficient to confer a strong long-term engraftment advantage of donor BM over BM from either wild-type or WHIM syndrome model mice, suggesting a potential mechanism for the patient's cure. Our findings suggest that partial inactivation of CXCR4 may have general utility as a strategy to promote HSC engraftment in transplantation.

Incidence and patterns of ALK FISH abnormalities seen in a large unselected series of lung carcinomas.

BACKGROUND: Anaplastic lymphoma receptor tyrosine kinase (ALK) gene rearrangements have been reported in 2-13% of patients with non-small cell lung cancer (NSCLC). Patients with ALK rearrangements do not respond to EGFR-specific tyrosine kinase inhibitors (TKIs); however, they do benefit from small molecule inhibitors targeting ALK. RESULTS: In this study, fluorescence in situ hybridization (FISH) using a break-apart probe for the ALK gene was performed on formalin fixed paraffin-embedded tissue to determine the incidence of ALK rearrangements and hybridization patterns in a large unselected cohort of 1387 patients with a referred diagnosis of non-small cell lung cancer (1011 of these patients had a histologic diagnosis of adenocarcinoma). The abnormal FISH signal patterns varied from a single split signal to complex patterns. Among 49 abnormal samples (49/1387, 3.5%), 32 had 1 to 3 split signals. Fifteen samples had deletions of the green 5' end of the ALK signal, and 1 of these 15 samples showed amplification of the orange 3' end of the ALK signal. Two patients showed a deletion of the 3'ALK signal. Thirty eight of these 49 samples (38/1011, 3.7%) were among the 1011 patients with confirmed adenocarcinoma. Five of 8 patients with ALK rearrangements detected by FISH were confirmed to have EML4-ALK fusions by multiplex RT-PCR. Among the 45 ALK-rearranged samples tested, only 1 EGFR mutation (T790M) was detected. Two KRAS mutations were detected among 24 ALK-rearranged samples tested. CONCLUSIONS: In a large unselected series, the frequency of ALK gene rearrangement detected by FISH was approximately 3.5% of lung carcinoma, and 3.7% of patients with lung adenocarcinoma, with variant signal patterns frequently detected. Rare cases with coexisting KRAS and EGFR mutations were seen.

Clinical and molecular characterization of overlapping interstitial Xp21-p22 duplications in two unrelated individuals.

Development and implementation of high-density DNA arrays demonstrated the important role of copy number changes on the X chromosome in the etiology of developmental delay and mental retardation (MR). We describe two unrelated patients with developmental delay due to similar interstitial duplications at Xp21-p22. The first patient is a 6-month-old male with multiple affected family members including many females. The second patient is a 5-year-old adopted female. In both patients, chromosome analysis and array comparative genomic hybridization (aCGH) showed duplications of overlapping regions at Xp21-p22. The duplicated segments contain numerous genes associated with MR, including AP1S2, NHS, CDKL5, RPS6KA3, SMS, and ARX. Except for developmental delay, there is little phenotypic overlap between the male and the female patient. Additionally, the female patient and affected female relatives of the male patient have variable severities of cognitive impairment, likely due to different X-inactivation patterns and effects of other, nonduplicated genes important for normal development. These cases illustrate that increased gene dosage of X-linked MR genes lead to cognitive impairment. Precise delineation of chromosome rearrangements by aCGH and identification of genes within duplicated segments helped in establishing genotype-phenotype correlations for each of our patients, in comparing them to each other, as well as with previously reported cases of Xp21-p22 duplications. However, we show that even with detailed molecular characterization, phenotype prediction remains challenging in patients with structural abnormalities of the X chromosome.

Myeloproliferative disorder with eosinophilia and ETV6-ABL gene rearrangement: efficacy of second-generation tyrosine kinase inhibitors.

ETV6/ABL is a rare gene rearrangement that has rarely been detected in Philadelphia-negative chronic myeloproliferative disorders (C-MPD) and found to have tyrosine kinase activity similar to the BCR/ABL fusion protein. We describe a case of a 61-year-old female with a C-MPD associated with an ETV6/ABL gene rearrangement. She achieved complete cytogenetic remission on imatinib 400mg daily for 17 months, but then developed morphologic and cytogenetic relapse. After starting nilotinib 400mg orally twice daily, she achieved CCyR at 3, 6, and 11 months, suggesting that second-generation TKIs can result in favorable responses in patients with ETV6/ABL rearrangement who relapse after imatinib.

Marfan syndrome type II: there is more to Marfan syndrome than fibrillin 1.

Marfan syndrome is a well-described autosomal dominant syndrome with widely variable clinical manifestations. Cardiovascular complications include mitral valve prolapse with or without associated mitral valve insufficiency, aortic root dilatation, and most importantly the occasional development of aortic aneurysms or rupture. Given the inconsistent phenotype along with the potentially life-threatening implications, clinicians are increasingly turning to genetic testing for definitive diagnostic confirmation. It has been well established that mutations in the FBN1 gene encoding the structural protein Fibrillin 1 is the molecular etiology of Marfan syndrome. However, there are numerous patients who meet the Ghent clinical diagnostic criteria for Marfan syndrome who do not have identifiable FBN1 mutations. Recently, mutations in TGFBR1 and TGFBR2 (transforming growth factor beta receptors 1 and 2, respectively) have been shown to result in Loeys-Dietz syndrome, a connective tissue disorder with significant phenotypic overlap with Marfan syndrome. Individuals with this Marfanoid disorder lack the ocular findings of Marfan syndrome and often have dysmorphic features such as unusual facies, cleft palate, and contractures. In addition, Loeys-Dietz syndrome patients often present in childhood with significant cardiovascular problems. This article serves to report an illustrative case of Loeys-Dietz syndrome and reviews the phenotypic consequences of FBN1 and TGFBR1 and TGFBR2 gene mutations.