A novel heptasomy 21 associated with complete loss of heterozygosity and loss of function RUNX1 mutation in acute myeloid leukemia.

Chromosomal aberrations are among the most important prognostic parameters in AML, and conventional cytogenetic analysis remains essential for risk stratification. In this report, we describe an adult male patient with a high percentage of circulating blasts, pathologically confirmed as AML with maturation. Cytogenetic analysis of a bone marrow sample revealed heptasomy 21 and trisomy 13 within a complex karyotype of 52,XY,der(2)t(2;13)(q33.3;q32.1),+13,+21,+21,+21,+21,+21 in all 20 cells examined, which was confirmed by metaphase FISH. Chromosomal microarray analysis (CMA) revealed complete loss of heterozygosity (LOH) of chromosome 21, supporting a common origin. In addition, LOH of chromosome 1p, trisomy 13, and partial tetrasomy of 13q and partial monosomy of 2q as a result of an unbalanced translocation between chromosomes 2 and 13 were observed. Molecular analysis identified two pathogenic missense variants: RUNX1 p.D198Y and SRSF2 p.P95R. The clonal allele ratio of RUNX1 p.D198Y was consistent with all copies of chromosome 21 in the leukemic clone carrying the mutation. Within the medical literature, there are no reports of heptasomy 21 for comparison; however, there are reports of AML with either polysomy 21 or trisomy 13. Our results suggest that even relatively 'common' AML aneuploidies may be associated with much more complex genomic changes, including loss of heterozygosity, which impact prognosis.

Adult acute myeloid leukemia patients with NUP98 rearrangement have frequent cryptic translocations and unfavorable outcome.

Acute myeloid leukemia (AML) with NUP98 rearrangement (AML-NUP98) has been uncommonly reported in adults, and its incidence in our institution is ∼2.5%. There were four men and five women with a median age of 49 years, among which six cases were de novo AML and three were therapy-related. Five cases were AML with minimal differentiation or without maturation, followed by four with monocytic differentiation. NUP98 rearrangement was confirmed in all cases by FISH, and five cases showed cryptic translocations. The median overall survival (OS) was 13 months, shorter than that of AML-NPM1 (p < 0.05), and similar to that in AML-KMT2A patients in our institution. The unfavorable OS was further confirmed by comparing to AML patients in TCGA database. In conclusion, adult AML-NUP98 is associated with cryptic translocations and an unfavorable outcome. Our study suggests that incorporating the NUP98 probe into AML FISH panels are warranted to improve clinical management.

Differentiation of leukemic blasts is not completely blocked in acute myeloid leukemia.

Hematopoiesis, the formation of blood cells, involves the hierarchical differentiation of immature blast cells into mature, functional cell types and lineages of the immune system. Hematopoietic stem cells precisely regulate self-renewal versus differentiation to balance the production of blood cells and maintenance of the stem cell pool. The canonical view of acute myeloid leukemia (AML) is that it results from a combination of molecular events in a hematopoietic stem cell that block differentiation and drive proliferation. These events result in the accumulation of primitive hematopoietic blast cells in the blood and bone marrow. We used mathematical modeling to determine the impact of varying differentiation rates on myeloblastic accumulation. Our model shows that, instead of the commonly held belief that AML results from a complete block of differentiation of the hematopoietic stem cell, even a slight skewing of the fraction of cells that differentiate would produce an accumulation of blasts. We confirmed this model by interphase fluorescent in situ hybridization (FISH) and sequencing of purified cell populations from patients with AML, which showed that different leukemia-causing molecular abnormalities typically thought to block differentiation were consistently present in mature myeloid cells such as neutrophils and monocytes at similar levels to those in immature myeloid cells. These findings suggest reduced or skewed, rather than blocked, differentiation is responsible for the development of AML. Approaches that restore normal regulation of hematopoiesis could be effective treatment strategies.

A case of recurrent epilepsy-associated rosette-forming glioneuronal tumor with anaplastic transformation in the absence of therapy.

Rosette-forming glioneuronal tumor (RGNT) most commonly occurs adjacent to the fourth ventricle and therefore rarely presents with epilepsy. Recent reports describe RGNT occurrence in other anatomical locations with considerable morphologic and genetic overlap with the epilepsy-associated dysembryoplastic neuroepithelial tumor (DNET). Examples of RGNT or DNET with anaplastic change are rare, and typically occur in the setting of radiation treatment. We present the case of a 5-year-old girl with seizures, who underwent near total resection of a cystic temporal lobe lesion. Pathology showed morphologic and immunohistochemical features of RGNT, albeit with focally overlapping DNET-like patterns. Resections of residual or recurrent tumor were performed 1 year and 5 years after the initial resection, but no adjuvant radiation or chemotherapy was given. Ten years after the initial resection, surveillance imaging identified new and enhancing nodules, leading to another gross total resection. This specimen showed areas similar to the original tumor, but also high-grade foci with oligodendroglial morphology, increased cellularity, palisading necrosis, microvascular proliferation, and up to 13 mitotic figures per 10 high power fields. Ancillary studies the status by sequencing showed wild-type of the isocitrate dehydrogenase 1 (IDH1), IDH2, and human histone 3.3 (H3F3A) genes, and BRAF studies were negative for mutation or rearrangement. Fluorescence in situ hybridization (FISH) showed codeletion of 1p and 19q limited to the high-grade regions. By immunohistochemistry there was loss of nuclear alpha-thalassemia mental retardation syndrome, X-linked (ATRX) expression only in the high-grade region. Next-generation sequencing showed an fibroblast growth factor receptor receptor 1 (FGFR1) kinase domain internal tandem duplication in three resection specimens. ATRX mutation in the high-grade tumor was confirmed by sequencing which showed a frameshift mutation (p.R1427fs), while the apparent 1p/19q-codeletion by FISH was due to loss of chromosome arm 1p and only partial loss of 19q. Exceptional features of this case include the temporal lobe location, 1p/19q loss by FISH without true whole-arm codeletion, and anaplastic transformation associated with ATRX mutation without radiation or chemotherapy.

Two myeloid leukemia cases with rare FLT3 fusions.

Genetic rearrangements involving FLT3 are rare and only recently have been detected in myeloid/lymphoid neoplasms associated with eosinophilia (MLN-eos) and chronic myeloproliferative disorders. Here we report two cases with FLT3 fusions in patients demonstrating mixed features of myelodysplastic/myeloproliferative neoplasms. In the first case, FLT3 was fused with a new fusion partner MYO18A in a patient with marrow features most consistent with atypical chronic myeloid leukemia; the second case involving ETV6-FLT3 fusion was observed in a case with bone marrow features most consistent with chronic myelomonocytic leukemia. Notably, we observed that samples from both patients demonstrated FLT3 inhibitor (quizartinib and sorafenib) sensitivity in ex vivo drug screening assay.

MYC immunohistochemical and cytogenetic analysis are required for identification of clinically relevant aggressive B cell lymphoma subtypes.

Accurate subclassification of aggressive B cell lymphomas (ABCLs) requires integration of morphologic, immunohistochemical (IHC), and cytogenetic information. Optimal strategies have not been well defined for diagnosis of high grade B cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBLwR) and double expressor lymphomas with MYC and BCL2 protein overexpression. One hundred and eighty seven ABCLs were investigated with complete IHC and FISH analysis. Morphologic and IHC analysis was insufficient to identify clinically relevant HGBLwR. Approximately, 75% of cases classified as HGBLwR showed conventional DLBCL morphologic features. Fourteen percent of MYC-rearranged cases were negative by IHC. Conversely, 60% of cases positive for MYC by IHC did not demonstrate a MYC rearrangement. Analysis by FISH without MYC and BCL2 IHC would miss 41 cases of double expressor lymphoma. Complete IHC and FISH analysis is recommended in the evaluation of all ABCLs.

Androgen receptor amplification is concordant between circulating tumor cells and biopsies from men undergoing treatment for metastatic castration resistant prostate cancer.

Increased AR activity has been shown to be preserved in spatially distinct metastatic tumors from the same patient suggesting the requirement for lineage-specific dependencies for metastatic castration resistant prostate cancer (mCRPC). Amplification of the AR gene is a common mechanism by which mCRPC increase AR activity. To determine whether AR amplification in circulating tumor cells (CTC) could complement metastatic tissue biopsies in men undergoing treatment for mCRPC, we developed a novel two-step assay to isolate CTCs and subsequently analyzed AR amplification status in CTCs and matched biopsy tissue from the same patient by fluorescence in situ hybridization (FISH). AR gene status in CTCs showed strong concordance with AR gene status in matched tissue samples in 24 of 25 patients (Correlation: 96%; Kappa: 0.83; Sensitivity: 100%, Specificity: 83%). Our work demonstrates that AR amplification is conserved between CTCs and biopsies and that CTCs can serve as non-invasive surrogate to document AR amplification in mCRPC.

MECOM (EVI1) Rearrangements: A Review and Case Report of Two MDS Patients with Complex 3q Inversion/Deletions.

Acute myelogeneous leukemia (AML) with inv(3)/t(3;3)(q13q25) is associated with aberrant expression of the stem-cell regulator MECOM (aka EVI1). Two bone marrow samples received in the OHSU Knight Diagnostic Laboratories (KDL) Cytogenetics Laboratory for chromosomes and FISH for a question of progression of myelodysplastic syndrome (MDS) to AML showed complex abnormalities including a deletion of chromosome 3q, one with del(3)(q13q25) and the other with del(3)(q22q25). In light of the prognostic importance of the activation of the MECOM oncogene and the concurrent inactivation of the GATA2 tumor suppressor that occurs with the classic inversion of chromosome 3q, fluorescence in situ hybridization (FISH) was performed using two different probe designs to better define the 3q deletions in the two cases. Using the Abbott Molecular Laboratories dual fusion MECOM/RPN1 probe, interphase and metaphase cells in both patients showed a variant single fusion (orange/green/fusion) signal pattern consistent with fusion and deletion. Using the three-color (red/green/aqua) Cytocell EVI1 probe, interphase cells in both cases showed a split red/green signal with the aqua signal remaining with the green signal. The distance between the split signals was generally less than is usually seen in the commonly described inverted chromosome 3. These findings are therefore consistent with a complex inversion and concurrent deletion/deletions of chromosome 3q. Thus, the deletion 3q seen in G-banded chromosomes from bone marrow from these two patients is most consistent with the activation of MECOM and the inactivation of GATA2.

Sponge Sampling with Fluorescent In Situ Hybridization as a Screening Tool for the Early Detection of Esophageal Cancer.

INTRODUCTION: Sponge cytology is a novel screening tool for esophageal cancer but has been unable to be validated for widespread use. Our aim was to apply fluorescent in situ hybridization to sponge cytology samples in order to evaluate the safety and efficacy of this modality in screening for esophageal cancer. MATERIALS AND METHODS: At a single, multidisciplinary, NCI-designated cancer center, patients completed sponge cytology sampling prior to upper endoscopy. Samples were analyzed by p53 fluorescent in situ hybridization, and results were compared to the endoscopic diagnosis. RESULTS: Fifty patients were enrolled (96 % Caucasian, 68 % male, median age of 67). All patients successfully swallowed the capsule. No complications (string breakage, bleeding, mucosal injury) occurred. Endoscopy revealed that 38 % had normal esophageal mucosa and 62 % had an esophageal mucosal abnormality. In total, six samples demonstrated p53 loss (94 % specificity for any abnormality). The sensitivity of the p53 fluorescent in situ hybridization probe was13.3 % for any abnormality, 10 % for intestinal metaplasia, and 0 % for dysplasia or esophageal cancer. DISCUSSION: Esophageal sponge cytology is a promising, safe, and tolerable method for collecting esophageal cell samples. However, our data suggest that p53 fluorescent in situ hybridization does not improve the sensitivity for detecting cancer in these samples.

Functional Human Oocytes Generated by Transfer of Polar Body Genomes.

Oocyte defects lie at the heart of some forms of infertility and could potentially be addressed therapeutically by alternative routes for oocyte formation. Here, we describe the generation of functional human oocytes following nuclear transfer of first polar body (PB1) genomes from metaphase II (MII) oocytes into enucleated donor MII cytoplasm (PBNT). The reconstructed oocytes supported the formation of de novo meiotic spindles and, after fertilization with sperm, meiosis completion and formation of normal diploid zygotes. While PBNT zygotes developed to blastocysts less frequently (42%) than controls (75%), genome-wide genetic, epigenetic, and transcriptional analyses of PBNT and control ESCs indicated comparable numbers of structural variations and markedly similar DNA methylation and transcriptome profiles. We conclude that rescue of PB1 genetic material via introduction into donor cytoplasm may offer a source of oocytes for infertility treatment or mitochondrial replacement therapy for mtDNA disease.

Metformin improves defective hematopoiesis and delays tumor formation in Fanconi anemia mice.

Fanconi anemia (FA) is an inherited bone marrow failure disorder associated with a high incidence of leukemia and solid tumors. Bone marrow transplantation is currently the only curative therapy for the hematopoietic complications of this disorder. However, long-term morbidity and mortality remain very high, and new therapeutics are badly needed. Here we show that the widely used diabetes drug metformin improves hematopoiesis and delays tumor formation in Fancd2-/- mice. Metformin is the first compound reported to improve both of these FA phenotypes. Importantly, the beneficial effects are specific to FA mice and are not seen in the wild-type controls. In this preclinical model of FA, metformin outperformed the current standard of care, oxymetholone, by improving peripheral blood counts in Fancd2-/- mice significantly faster. Metformin increased the size of the hematopoietic stem cell compartment and enhanced quiescence in hematopoietic stem and progenitor cells. In tumor-prone Fancd2-/-Trp53+/- mice, metformin delayed the onset of tumors and significantly extended the tumor-free survival time. In addition, we found that metformin and the structurally related compound aminoguanidine reduced DNA damage and ameliorated spontaneous chromosome breakage and radials in human FA patient-derived cells. Our results also indicate that aldehyde detoxification might be one of the mechanisms by which metformin reduces DNA damage in FA cells.

FANCD2 Facilitates Replication through Common Fragile Sites.

Common fragile sites (CFSs) are genomic regions that are unstable under conditions of replicative stress. Although the characteristics of CFSs that render them vulnerable to stress are associated mainly with replication, the cellular pathways that protect CFSs during replication remain unclear. Here, we identify and describe a role for FANCD2 as a trans-acting facilitator of CFS replication, in the absence of exogenous replicative stress. In the absence of FANCD2, replication forks stall within the AT-rich fragility core of CFS, leading to dormant origin activation. Furthermore, FANCD2 deficiency is associated with DNA:RNA hybrid formation at CFS-FRA16D, and inhibition of DNA:RNA hybrid formation suppresses replication perturbation. In addition, we also found that FANCD2 reduces the number of potential sites of replication initiation. Our data demonstrate that FANCD2 protein is required to ensure efficient CFS replication and provide mechanistic insight into how FANCD2 regulates CFS stability.

Fibersol-2 induces apoptosis of Apc-deficient colorectal Cancer (SW480) cells and decreases polyp formation in Apc MIN mice.

The consumption of dietary fibers has been implicated with a lowered risk of human colorectal cancer. Proposed mechanisms involve alterations in the stool consistency, transit time, and formation of short-chain fatty acid by dietary fiber fermentation, and the reorganization of gut microbiota. Here we show that Fibersol-2, a digest-resistant maltodextrin, not only inhibits proliferation of colorectal SW480 cancer cell lines by increasing reactive oxygen species (ROS), but decreases the numbers of the adenoma count in Multiple Intestinal Neoplasia (MIN) mice carrying a mutation in the Adenomatous Polyposis Coli gene by 84 d of age. These observations provide direct evidence that Fibersol-2 intrinsically contains anti-cancer activity, independent of the intestinal metabolism and any potential interactions with the microbiota.

Cytokine overproduction and crosslinker hypersensitivity are unlinked in Fanconi anemia macrophages.

The Fanconi anemia proteins participate in a canonical pathway that repairs cross-linking agent-induced DNA damage. Cells with inactivated Fanconi anemia genes are universally hypersensitive to such agents. Fanconi anemia-deficient hematopoietic stem cells are also hypersensitive to inflammatory cytokines, and, as importantly, Fanconi anemia macrophages overproduce such cytokines in response to TLR4 and TLR7/8 agonists. We questioned whether TLR-induced DNA damage is the primary cause of aberrantly regulated cytokine production in Fanconi anemia macrophages by quantifying TLR agonist-induced TNF-α production, DNA strand breaks, crosslinker-induced chromosomal breakage, and Fanconi anemia core complex function in Fanconi anemia complementation group C-deficient human and murine macrophages. Although both M1 and M2 polarized Fanconi anemia cells were predictably hypersensitive to mitomycin C, only M1 macrophages overproduced TNF-α in response to TLR-activating signals. DNA damaging agents alone did not induce TNF-α production in the absence of TLR agonists in wild-type or Fanconi anemia macrophages, and mitomycin C did not enhance TLR responses in either normal or Fanconi anemia cells. TLR4 and TLR7/8 activation induced cytokine overproduction in Fanconi anemia macrophages. Also, although TLR4 activation was associated with induced double strand breaks, TLR7/8 activation was not. That DNA strand breaks and chromosome breaks are neither necessary nor sufficient to account for the overproduction of inflammatory cytokines by Fanconi anemia cells suggests that noncanonical anti-inflammatory functions of Fanconi anemia complementation group C contribute to the aberrant macrophage phenotype and suggests that suppression of macrophage/TLR hyperreactivity might prevent cytokine-induced stem cell attrition in Fanconi anemia.

The New Equivocal: Changes to HER2 FISH Results When Applying the 2013 ASCO/CAP Guidelines.

OBJECTIVES: Human epidermal growth factor receptor 2 (HER2, ERBB2) testing is an important prognostic/predictive marker in breast cancer management, especially in selecting HER2-targeted treatment. American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines address HER2 status and were recently revised in 2013, replacing the 2007 version. For in situ hybridization interpretation, 2013 guidelines return to the prior threshold of a HER2/CEP17 ratio of 2.0 or greater for positive and eliminate 1.8 to 2.2 as the equivocal range. Also, the HER2 signal/nucleus ratio is accounted for, with 6.0 or greater for positive and 4.0 to less than 6.0 for equivocal, even in cases with a HER2/CEP17 ratio less than 2.0. METHODS: With institutional review board approval, we reviewed our 2006 to 2012 HER2 fluorescence in situ hybridization (FISH) results and classified them according to both the 2007 and 2013 guidelines as negative, positive, or equivocal. RESULTS: Of 717 HER2 FISH results, 55 (7.7%) changed category when reassessed by 2013 guidelines. Nineteen of 25 results in the 2007 equivocal category were reassigned as positive (n = 13) or negative (n = 6). Thirty-five previously negative cases became equivocal in the 2013 scheme, 12 of these with 1+ immunohistochemistry. The positive category increased from 71 to 85. CONCLUSIONS: The 2013 ASCO/CAP guidelines increased the number of HER2 FISH positive and equivocal results. The equivocal group is substantially different, posing a dilemma for clinical management.

Tumor suppression by resistant maltodextrin, Fibersol-2.

Resistant maltodextrin Fibersol-2 is a soluble and fermentable dietary fiber that is Generally Recognized As Safe (GRAS) in the United States. We tested whether Fibersol-2 contains anti-tumor activity. Human colorectal cancer cell line, HCT116, and its isogenic cells were treated with FIbersol-2. Tumor growth and tumorigenesis were studied in vitro and in vivo. Apoptotic pathway and generation of reactive oxygen species (ROS) were investigated. We discovered that Fibersol-2 significantly inhibits tumor growth of HCT116 cells by inducing apoptosis. Fibersol-2 strongly induces mitochondrial ROS and Bax-dependent cleavage of caspase 3 and 9, which is shown by isogenic HCT116 variants. Fibersol-2 induces phosphorylation of Akt, mTOR in parental HCT116 cells, but not in HCT116 deficient for Bax or p53. It prevents growth of tumor xenograft without any apparent signs of toxicity in vivo. These results identify Fibersol-2 as a mechanism-based dietary supplement agent that could prevent colorectal cancer development.

Acquisition of Relative Interstrand Crosslinker Resistance and PARP Inhibitor Sensitivity in Fanconi Anemia Head and Neck Cancers.

PURPOSE: Fanconi anemia is an inherited disorder associated with a constitutional defect in the Fanconi anemia DNA repair machinery that is essential for resolution of DNA interstrand crosslinks. Individuals with Fanconi anemia are predisposed to formation of head and neck squamous cell carcinomas (HNSCC) at a young age. Prognosis is poor, partly due to patient intolerance of chemotherapy and radiation requiring dose reduction, which may lead to early recurrence of disease. EXPERIMENTAL DESIGN: Using HNSCC cell lines derived from the tumors of patients with Fanconi anemia, and murine HNSCC cell lines derived from the tumors of wild-type and Fancc(-/-) mice, we sought to define Fanconi anemia-dependent chemosensitivity and DNA repair characteristics. We utilized DNA repair reporter assays to explore the preference of Fanconi anemia HNSCC cells for non-homologous end joining (NHEJ). RESULTS: Surprisingly, interstrand crosslinker (ICL) sensitivity was not necessarily Fanconi anemia-dependent in human or murine cell systems. Our results suggest that the increased Ku-dependent NHEJ that is expected in Fanconi anemia cells did not mediate relative ICL resistance. ICL exposure resulted in increased DNA damage sensing and repair by PARP in Fanconi anemia-deficient cells. Moreover, human and murine Fanconi anemia HNSCC cells were sensitive to PARP inhibition, and sensitivity of human cells was attenuated by Fanconi anemia gene complementation. CONCLUSIONS: The observed reliance upon PARP-mediated mechanisms reveals a means by which Fanconi anemia HNSCCs can acquire relative resistance to the ICL-based chemotherapy that is a foundation of HNSCC treatment, as well as a potential target for overcoming chemoresistance in the chemosensitive individual.

Comparison of chromosome breakage in non-mosaic and mosaic patients with Fanconi anemia, relatives, and patients with other inherited bone marrow failure syndromes.

Fanconi anemia (FA) is a rare inherited bone marrow failure syndrome (IBMFS). Affected individuals must be distinguished from relatives, patients with mosaicism must be identified, and patients with other IBMFS classified as non-FA. The diagnostic feature of FA is increased chromosomal breakage in blood lymphocytes cultured with diepoxybutane or mitomycin C. Here, we sought a method to uniquely identify patients with FA with mosaicism, using cells from participants in the National Cancer Institute IBMFS cohort. Lymphocytes were treated with diepoxybutane or mitomycin C, and metaphases scored for breaks and radials. Analyses included the percentage of cells with any aberration, breaks per cell, and breaks per aberrant cell. There were 26 patients with FA (4 mosaics), 46 FA relatives, and 62 patients with a non-FA IBMFS. By all analytic methods, patients with FA were abnormal compared with other groups. Those with FA mosaicism had more breakage than relatives or patients with non-FA IBMFS, but there was some individual overlap. The choices of clastogen are laboratory-dependent, but there was no method or analysis of lymphocytes that clearly distinguished all individuals mosaic for FA from relatives or patients with other IBMFS. Thus, genotyping remains the best method for providing absolute clarity.

Aberrations of MYC are a common event in B-cell prolymphocytic leukemia.

OBJECTIVES: B-cell prolymphocytic leukemia (B-PLL) remains a controversial entity, and its molecular pathogenesis is largely unknown. Patients are older, typically having marked lymphocytosis and splenomegaly in the absence of lymphadenopathy. It is defined as a mature B-cell leukemia with more than 55% circulating prolymphocytes. Leukemic mantle cell lymphoma and chronic lymphocytic leukemia in prolymphocytic transformation must be excluded. METHODS: Case archives were retrospectively reviewed for B-PLL in patients without a previous diagnosis of chronic lymphocytic leukemia or other B-cell neoplasm. RESULTS: We identified six cases of B-PLL with available cytogenetic data, five of which showed evidence of aberrations in MYC. Three cases showed additional signals for the MYC gene by fluorescence in situ hybridization (FISH), and two cases demonstrated t(8;14)MYC/IGH by karyotyping or FISH. High levels of MYC protein expression were detected in all cases tested with MYC aberrations. CONCLUSIONS: These results suggest that deregulation of MYC plays an important role in the pathogenesis of B-PLL and expands the spectrum of B-cell neoplasms associated with aberrations of MYC.