Clonal Abnormalities Research Articles

Whole Genome Sequencing and RNA Sequencing of 27 Patients with Persistent Polyclonal B-Cell Lymphocytosis Reveals a High Mutation Frequency/Overexpression of Lymphoma Associated Genes: Really a Benign Disorder?

Background: Persistent polyclonal B-cell lymphocytosis (PPBL) is a rare disorder, occurs almost exclusively in smoking women and is characterized by a chronic polyclonal lymphocytosis with circulating binucleated lymphocytes, clonal cytogenetic abnormalities involving chromosome 3, and chromosomal instability. Outcome of PPBL patients is mostly benign, but subsequent malignancies (non-Hodgkin´s lymphomas and solid tumors) were described. Potential molecular factors leading to their development are yet unclear.Aims: Detailed molecular genetic characterization of PPBL by whole genome sequencing (WGS) and RNA sequencing (RNAseq) in comparison to the well-characterized lymphoid malignancy CLL.Patient cohorts and methods: The total cohort comprised 27 PPBL (3 male, 24 female) and 250 CLL cases (163 male, 87 female). WGS was performed for all patients: 150bp paired-end reads where generated on Illumina HiseqX and NovaSeq 6000 machines (Illumina, San Diego, CA). A mixture genomic DNA from multiple anonymous donors was used as normal controls. To remove potential germline variants, each variant was queried against the gnomAD database, variants with global population frequencies >1% where excluded. Final analysis was performed only on protein-altering and splice-site variants. For further analysis, a virtual panel of 355 lymphoid genes was selected. All reported p-values are two-sided and were considered significant at p<0.05. For gene expression analysis, estimated gene counts were normalized applying Trimmed mean of M-values (TMM) normalization method and the resulting log2 counts per million (CPMs) were used as a proxy of gene expression in each sample. Genes were kept if they were expressed (> 5 CPM) in at least 66% of the samples. Genes with FDR (false discovery rate) < 0.05 and an absolute logFC > 1.5 were considered differentially expressed (DE).Results: Median age was 46 years for PPBL patients (range: 23-67 years) and 67 years for CLL patients (range: 39-94 years). Mean number of mutations per patient was 18 for PPBL and 20 for CLL. For both entities, the majority of mutations were missense mutations (88% in PPBL vs. 81% in CLL), followed by splice-site mutations (7% vs. 10%), other mutation types were only rarely detected. In PPBL, 42 genes were found to be mutated at a frequency of >15%, including ATM (22%), CREBBP (19%), NCOR2 (19%), AHNAK2 (15%), JAK3 (15%), NOTCH2 (15%) and TRAF1 (15%), all of which have been associated with a variety of cancers. Moreover, ATM, NOTCH2 and TRAF1 mutations were described before to be associated with lymphomas. In PPBL patients, mutations in TRAF1 and ATM as well as mutations in TRAF1 and NOTCH2 were found to be mutually exclusive. For CLL patients, 29 genes showed a mutation frequency of >15%, comprising ATM (26%), KMT2D (23%), NOTCH1 (23%), LRP1B (19%), TP53 (16%) and CREBBP (15%). Comparison of the mutation frequencies between the two entities revealed several genes with significant differences: whereas mutations in CKAP5 (11% vs. 2%, p=0.022), DNMT3A (11% vs. 3%, p=0.033), MAP2 (19% vs. 4%, p=0.009), ROBO1 (15% vs. 4%, p=0.046) and TRAF1 (15% vs. 2%, p=0.006) were found to be more frequent in PPBL cases compared to CLL cases, KMT2D (4% vs. 23%, p=0.014), TDRD6 (0% vs. 14%, p=0.032) and TP53 (4% vs. 16%, p=0.048) mutations were more abundantly detected in CLL patients. Moreover, NOTCH1 was mutated more frequently in CLL cases (7% vs. 23%, p=0.082), whereas mutated NOTCH2 (known to be frequently mutated in splenic marginal zone lymphoma), was more abundant in PPBL patients (15% vs. 6%, p=0.116), although both correlations were not statistically significant. Gene expression analyses by RNAseq revealed 337 genes to be differentially expressed between the entities. 207 genes were upregulated in PPBL, including PTPRK, CXCR1, BCL11B, CEPBA, CCR4 and MYC, whereas 130 genes were found to be upregulated in CLL cases, comprising ID3, BCL2, FGF2 and FLT1.Conclusions: 1) WGS analysis identifies high frequencies of cancer/lymphoma-associated gene mutations in PPBL, including mutated ATM, NOTCH2 and TRAF1. 2) Five genes showed a higher mutation frequency compared to CLL including TRAF1,DNMT3A, CKAP5 and MAP2. 3) Lymphoma associated genes (BCL11B and MYC) were overexpressed in PPBL vs CLL. 4) Taken together our results question PPBL as a benign entity and identify molecular markers that might contribute to development of subsequent malignancies. DisclosuresStengel:MLL Munich Leukemia Laboratory: Employment. Höllein:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Walter:MLL Munich Leukemia Laboratory: Employment. Hutter:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Bone Marrow T Cell Changes By Multiplex IHC after Treatment with Flotetuzumab, a CD123 x CD3 Bispecific Dart® Protein, in a Primary Refractory t-AML Patient

Therapy-related acute myelogenous leukemia (t-AML) is associated with adverse genetic lesions, complex karyotype, and TP53 mutation; it is challenging to treat and confers a poor prognosis. We describe a 74-year-old female patient with AML that developed 9 years after receiving 6 cycles of cytotoxic (FOLFOX) chemotherapy as adjuvant treatment of colorectal carcinoma. At diagnosis, the bone marrow (BM) biopsy revealed blast count of 35%, with normal karyotype, 5q loss and AML1 (RUNX1) locus (21q22) amplification by fluorescence in situ hybridization (FISH). Initial treatment with 5 cycles of azacitidine (AZA) failed to induce a response. The patient was subsequently treated on a Phase 1 trial of flotetuzumab (MGD006/S80880) (FLZ), a novel T-cell redirecting (CD123 x CD3) DART protein [NCT02152956]. Prior to FLZ, BM blasts demonstrated clonal evolution with a complex karyotype (92,XXXX, t(14;21)(q22;q22)) and new IDH1, and TET2 mutations.Patient received a total of 3 cycles (28 days/cycle) of FLZ. After one FLZ cycle, the patient achieved a (complete response) CR, with normal cytogenetics, and residual IDH1 and TET2 mutations. After 2 additional cycles of FLZ consolidation, the CR was maintained, with loss of the IDH1 mutation, but persistence of the TET2 mutation. CR was maintained with no additional therapy for approximately 7 months. Serial BM samples were evaluated for T cells (CD3, CD4, CD8, FOXP3 and PD-L1) using multiplex IHC (Fig 1). BM T cells were unchanged during AZA treatment. At the initial bone marrow on FLZ (CR), significant increases in CD3+, CD3+CD8-, CD3+ CD8+, and FOXP3-positive T cells and PD-L1 expression compared to baseline were noted. CD3+CD8+ T cells persisted in the BM 1 month beyond completion of the additional 2 cycles of consolidation with FLZ, while other T cells subsets and PD-L1 expression returned to baseline (Fig. 1) Three months after the last FLZ treatment, all T cell subsets had returned to baseline, with an early increase in leukemic blasts (8%), but normal cytogenetics. Five months after FLZ treatment blasts were unchanged, but a new abnormal cytogenetic clone, with additional mutations, accompanied by a rise in PD-L1 expression cells was observed. Seven months after FLZ treatment, frank leukemia with 50% blasts developed, and all T cell subsets and PD-L1 expression had returned to pre-treatment levels.Consistent with its proposed mechanism of action, these data highlight for the first time the induction of an increase in T-cell infiltration, which persist beyond FLZ, in the bone marrow microenvironment of an AML patient that also achieved a complete and durable anti-leukemic response after treatment with FLZ. [Display omitted] DisclosuresLelièvre:Servier: Employment. Wigginton:MacroGenics: Employment. Davidson-Moncada:MacroGenics: Employment. Fox:MacroGenics: Research Funding.

An Acquired High-Risk Chromosome Instability Phenotype in Multiple Myeloma: Jumping 1q Syndrome

IntroductionChromosome instability (CIN) is a driver of copy number aberrations (CNAs) in cancer, and is a major factor leading to tumor heterogeneity and resistance to therapy. By definition, CIN is an increased rate or ongoing acquisition and accumulation of CNAs and not simply the existence of structurally and numerically abnormal aneuploid clones. In multiple myeloma (MM), the most common whole-chromosome CNAs involve either hyperdiploid or non-hyperdiploid clones. Secondary segmental CNAs are associated with high-risk (HR) in MM and involve gains of 1q21 and deletions of 17p (del17p). These types of intra-chromosomal segmental CNAs are also found in the CIN phenotypes of the autosomal recessive (AR) chromosome instability syndromes. These syndromes include Fanconi anemia, Bloom's syndrome, and ICF syndrome (Immunodeficiency, Centromeric instability and Facial anomalies). These chromosome instability syndromes display a spectrum of aberrations characterized by higher rates of chromosomal breaks, chromatid exchanges, quadriradials, and pericentromeric aberrations. In particular, patients with ICF syndrome show a marked increase of 1q12 pericentromeric instability including 1q12 decondensation, triradials, multibranched chromosomes 1q, and 1q micronuclei. ICF patients also show transient 1q aberrations including isochromosome 1q (iso1q) and unbalanced translocations of 1q to 9q and 16q. In MM, we have previously reported increasing pericentromeric instability during tumor progression resulting in increasing CNAs of 1q21 by unbalanced jumping translocations of 1q12 (JT1q12). Strikingly, in a subset of MM patients with 1q21 CNAs of ≥ 5 a distinct cytogenetic phenotype emerges which demonstrates transient 1q12 aberrations including 1q12 decondensation, triradials, and multibranched chromosomes 1q morphologically identical to those seen in ICF patients. In MM this chromosome instability leads to a cascade of increasing clonal 1q21 duplications, iso 1qs, and unbalanced 1q translocations with 16q and 17p, resulting in losses in these receptor chromosomes (RC) and massive intra-clonal CNA heterogeneity.MethodsTo investigate the cytogenetic impact and progression of high CNAs of 1q21, we performed a comprehensive metaphase analysis of 50 patients showing segmental aneuploidies with 4 or more copies of 1q by G-banding. Locus specific FISH and spectral karyotyping were used to identify the key transient unstable and clonal structural aberrations of 1q12 resulting in segmental aneuploidies in the derivative RCs. Probe for 1q12 (Vysis) was used according to the manufacturer's protocol. Locus specific BAC clones for 1q21 (CKS1B) and 17p (TP53) were prepared and analyzed as previously described (Sawyer et al., Blood 123: 2014). IGH translocations were investigated with IGH break apart probes (Vysis).ResultsData for 50 patients including CNAs of 1q21 of ≥ 4, IGH translocations, del(17p), derivative RCs, are presented. The t(4;14) was found in 15 patients, del(17p) in 23, and both aberrations were found in 8 patients. All patients showed unbalanced gains of 1q and deletions of RCs, the most frequent being 7 patients with der(1;16) and 6 with iso1q. In four of the 23 patients with del(17p), the deletion was due to a JT1q12 to 17p. Seven patients with 1q21 CNAs of ≥ 5 showed profound instability involving the 1q12 satellite DNA, demonstrating both transient and clonal aberrations driving the 1q21 CNAs. These aberrations included unstable 1q21 triplications, JT1q12s, iso1q formation with intra-arm 1q12 CNAs, and region specific breakage-fusion-bridge cycle amplifications.ConclusionsAmong patients with ≥ 5 CNAs of 1q21, a subset develop an acquired HR chromosome instability phenotype with an elevated rate of 1q12 pericentromeric instability characterized by concomitant deletions in 16q, iso1q, del(17p), and intra-arm segmental instability. These patients show pronounced instability in the 1q12 satellite DNA, morphologically identical to ICF syndrome, suggesting hypomethylation of this region as a driver of both 1q21 CNAs and deletions in RCs. We hypothesize that region specific hypomethylation of 1q12 provides the genomic background for the onset of an acquired 1q12 chromosome instability phenotype in MM similar to that found in ICF syndrome. For myeloma patients demonstrating this 1q12 chromosome instability phenotype we propose the term “jumping 1q syndrome.” DisclosuresEpstein:University of Arkansas for Medical Sciences: Employment. Davies:Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; ASH: Honoraria; Abbvie: Consultancy; TRM Oncology: Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; MMRF: Honoraria; Janssen: Consultancy, Honoraria. Morgan:Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria.

Fully Human Bcma Targeted Chimeric Antigen Receptor T Cells Administered in a Defined Composition Demonstrate Potency at Low Doses in Advanced Stage High Risk Multiple Myeloma

Fully Human Bcma Targeted Chimeric Antigen Receptor T Cells Administered in a Defined Composition Demonstrate Potency at Low Doses in Advanced Stage High Risk Multiple Myeloma

Cegal Protocol : Evaluation of the Feasibility of a Chemogenomic Approach to Identify Personalized Therapy for Relapse or Refractory AML Patients

Introduction :Significant improvements of leukemia free survival have been obtained over the last 30 years in AML. This progress has resulted from optimization of the same chemotherapy-based “one-size-fits-all” approach. However, even though the majority of patients eligible for intensive chemotherapy achieve complete remission (60% to 80%), the majority still relapse and ultimately die (5-year overall survival being around 20%). Recent genomic studies have revealed the heterogeneity of AML at the molecular level, stressing the need for personalized therapeutic approaches. The fundamental idea in genomic medicine is that somatic genetic alterations can be identified and matched with drugs targeting those abnormalities for a patient's benefit. However, there are not that many potent, tolerated targeted agents in the clinic that can induce significant clinical responses. Interestingly, the majority of the most effective drugs do not function by exploiting genetic mutations in individual tumors. Thus, additional functional approaches are needed to identify new drugs and assign existing drugs to larger numbers of patients with AML.From this perspective, the“chemogenomic” approach realizes an integratedanalysis of the molecular clonal abnormalities for the leukemic patients, and compares it to a functional drug sensitivity testing (DST) which can test simultaneously dozens of drugsex vivo. This approach aims to identify correlations between genomic changes and sensibilities increased in certain categories of drugs. The CEGAL project is a prospectivemonocentric study aiming to evaluate the feasibility of this personalized approach for relapse or refractory AML patientsby determining the proportion of patients whosechemogenomic results are available in less than 21 days.Methods :Patients with relapsed and/or refractory AML were eligible if no alternative therapy was available. Written consent was obtained from all patients (Study CeGAL-IPC 2014-012, EUDRACT 2014-A01209-38). Ex vivo sensitivity profiling was performed on freshly isolated AML blasts derived from patient bone marrow aspirates and peripheral blood with a panel of 78 drugs representative of the main therapeutic classeskwown to be active in AML. After 48h of incubation, cellular viability was measured with the CellTiter-Glo© Luminescent cell viability assay (Promega) kit to obtain one EC50. Next generation sequencing (NGS) was performed on a 200 genes panel with Mi-Seq (Illumina) and genomic profils by cGH-array (Agilent4x180K) were established. Personalized treatment plans were then reviewed by an institutional personalized committee, which issued final treatment recommendations.Results: A total of 48 AML patients were included (refractory=8,relapse=40) with a median age of 64 years old (range 24-81) and a median number of prior therapies of 2. More than half of the patients presented an adverse prognostic according to the 2017 ELN risk stratification (n=29). The most frequent genomic alterations were TET2 in 32.5% of the patients, ASXL1 (27.5%), DNMT3A (25%), TP53 (22.5%), SRSF2 (20%) and RUNX1 (17.5%). The median number of mutations per patient was 5,6. The main objective was completed with chemogenomic results in less than 21 days for 62.5% of the patients (n=25) with a median time of 22.4 days. Twenty four patients (67,5%, n=27) had targetable mutations and for 36 patients (90%), there was at least one drug predicted efficient. In light of the chemogenomic analysis, we found a correlation between genomic alteration and drug sensitivy for 28 patients. Thirteen patients (32.5%) received a treatment guided by the analysis whatsoever chemotherapies or targeted therapies. The 27 others didn't receive a personalized treatment either because the molecule wasn't commercially available or because the physician decided so.Conclusion : In our experience, the chemogenomic is an innovative and promising approach, allowing to assign patient-specific treatment options according to their mutational profile and to in vitro drugs' response in a median time of 3 weeks. DisclosuresNo relevant conflicts of interest to declare.

Haploidentical Transplantation with Regulatory and Conventional T Cells Improves Outcome of Patients Affected By Acute Myeloid Leukemia with Complex Karyotype and/or Monosomy 7/Del(7q)

▪Acute myeloid leukemias (AML) with complex karyotype (≥ 3 abnormalities) and/or monosomy 7/del(7q) are a subset of AML with extremely poor prognosis. Hematopoietic stem cell transplantation (HSCT) is the only potentially curative treatment, but relapse rate is high (most often above 50%) and negatively impacts on patients' survival. (Ciurea et al. Cancer 2018) Such poor results are even questioning the role of HSCT and rise the need of new transplant procedures for these patients. To this end, we are performing a clinical trial of haploidentical HSCT with adoptive immunotherapy with regulatory T cells (Tregs) and conventional T cells (Tcons) in the absence of post-transplant pharmacologic immune suppression (Treg-Tcon haplo-HSCT). Such approach allows for an extremely low relapse rate in patients with high-risk acute leukemias transplanted in complete remission. (Martelli et al. Blood 2014)In this study we analyzed the outcome of all the patients that were referred to our center from January 1st 2007 to May 31st 2018 with a diagnosis of AML with complex karyotype and/or monosomy 7/del(7q) and that were fit for receiving an induction treatment.49 patients were included. Median age at diagnosis was 52; 22 were female, 27 were male. 20/49 had chromosome 7 abnormalities (isolated in 12 patients; in a complex karyotype in 8 patients). 11 patients received a hypomethylating agent as first line treatment, while the other 38 patients received high-dose chemotherapy. We performed 30 HSCT procedures in 25 patients; 5 patients received a second transplant (4 after disease relapse and 1 after rejection). 6 underwent a HLA-matched HSCT, 6 a T-depleted haploidentical HSCT, and 18 a Treg-Tcon haplo-HSCT. Median age at transplant was 42 with a median follow up of 40 months.Regarding disease status at transplant, 26 HSCT were performed in patients that were in 1st or 2nd hematological remission and 4 in patients with refractory disease. Cytogenetic analysis (karyotype and/or FISH analysis) revealed the presence of abnormal clones in 11 patients and multiparametric flow cytometry analysis detected minimal residual disease in 16 patients. Thus, most of the patients (17/30, 57%) were transplanted with detectable disease.15/49 patients were disease-free and alive at the time of the analysis, all of them after receiving HSCT (HSCT vs Non-HSCT, p<0.01, Fig. A). Treg-Tcon haplo-HSCT allowed for a markedly better disease free survival as compared to the other transplant procedures (p=0.01, Fig. B). Such survival advantage was only due to a reduced rate of disease relapse (31% vs 79%, p=0.02, Fig. C), as transplant related mortality was similar between the 2 groups (9% vs 9%). 17/18 Treg-Tcon haplo-HSCT patients engrafted and only 2/17 developed acute GvHD grade ≥ 2. 4/17 patients relapsed and none of them received a transplant from a NK alloreactive donor. (Ruggeri et al. Science 2002) Multivariate analysis that included chromosome 7 involvement, presence of disease at multiparametric flow cytometry and/or cytogenetic analysis, and blast count at transplant confirmed that Treg-Tcon haplo-HSCT was the only predictor of a better survival.In conclusion, our results not only confirm that HSCT is the only potentially curative treatment for AML with complex karyotype and/or chromosome 7 involvement, but also demonstrate that Treg-Tcon haplo-HSCT allows for a strong antileukemic effect, the only needed for a long term control of such unfavorable AMLs with high disease burden at transplant. While it is necessary to confirm these results in a larger cohort of patients, Treg-Tcon haplo-HSCT could represent the best transplant option for this subset of very high-risk AML patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Clinical Significance of Clonal Cytogenetic Heterogeneity (CCH) at Diagnosis in Adult Patients with Acute Lymphoblastic Leukemia (ALL)

Background:Although cytogenetic abnormalities at diagnosis are recognized as one of the most potent prognostic factors in acute leukemia patients, CCH acquisition at diagnosis, which are considered as a result of clonal evolution of leukemia cells, is not taken into account in prognostic classifications. Recent studies reported that CCH acquisition was observed in 24 - 32% of adult AML patients with abnormal karyotype, was more likely to occur in patients with older age and complex karyotype, and showed adverse prognostic impact. However, the clinical significance of CCH acquisition has not been investigated in adult ALL patients to date. Patients and methods: Of the 238 adult ALL patients diagnosed between 1990 and 2016, 120 patients with abnormal karyotype at diagnosis, who underwent intensive chemotherapy, were included in this study. CCH was defined as presence of two or more cytogenetically abnormal clones. A defined ancestral clonal evolution included either mother-daughter and/or branched patterns. In the mother-daughter pattern, a daughter clone showed all cytogenetic abnormalities of a mother clone plus additional abnormality(s), which define a distinct subclone. In a branch pattern, all subclones possessed common cytogenetic abnormalities suggesting presence of a common ancestry, but each subclone acquires unique additional abnormality(s), which define them as distinctive subclones. Both patterns of cytogenetic clonal evolution were sometimes seen in a patient. Composite karyotypes were applied to patients where a common ancestry could not be clearly determined because of too complicated cytogenetic findings. Fisher's exact test was used to compare binary variables. The logistic regression model was used for multivariate analysis of predisposing factors. Overall survival (OS) was estimated with the Kaplan-Meier method and compared using the log-rank test. The Cox proportional hazard model was used for multivariate analysis of prognostic factors. Values of p < 0.05 were considered to indicate statistical significance. Results:Of the 120 patients included in this study, 64 patients were male, and 56 were female. The median age was 50 years (range, 16-79 years). Karyotypes at diagnosis were Philadelphia chromosome (Ph) in 56 patients, complex in 15, and t(8;14) in seven. According to the definition described above, 47 patients (39%) showed CCH at diagnosis, and two (4%) among them were categorized as composite karyotype. Of the 45 patients harboring a defined ancestral clonal evolution, numbers of subclones were two, three, and four in 32 patients (68%), 11 (24%), and 2 (4%), respectively. Mother-daughter pattern, branched pattern, and both were seen in 34 patients (76%), 5 (11%), and 6 (13%). In univariate analysis for predisposing factors of CCH acquisition, only younger age was significantly associated with CCH acquisition (48% in age <= 50 vs. 29% in age > 50; p = 0.04), but not karyotype. This statistical significance was confirmed with multivariate analysis (odds ratio = 0.44; p = 0.03). When investigating the prognostic impact of CCH acquisition, patients were divided into Ph-negative or Ph-positive ALL groups. In the 64 Ph-negative ALL patients, the CR rates were not significantly different between patients with or without CCH (78% vs. 78%, respectively; p = 1.00). The OS rates were similar between two groups (26% vs. 39% at five years, respectively; p = 0.56). Multivariate analysis for OS revealed that complex karyotype and t(8;14) were independent prognostic factors, but not CCH acquisition. Likewise, in the 56 Ph-positive ALL patients, CCH acquisition was not significantly associated with the CR rates (92% vs. 78%, respectively; p = 0.27), and the OS rates did not significantly differ between the two groups (34% vs. 40% at five years, respectively; p = 0.90). In multivariate analysis for OS, no independent prognostic factor was identified. Conclusion: Adult ALL patients with abnormal karyotype acquired CCH at diagnosis with a frequency comparable to that of AML patients. However, unlike AML patients, CCH acquisition was more frequently observed in younger population and did not show any prognostic impact in ALL patients. These findings suggested that biological backgrounds of CCH acquisition at diagnosis were possibly different between in patients with ALL and AML. So, to confirm these important findings, clinical studies with larger study subjects are warranted. DisclosuresHanda:Celgene: Honoraria, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Associations of complementation group, ALDH2 genotype, and clonal abnormalities with hematological outcome in Japanese patients with Fanconi anemia.

Fanconi anemia (FA) is a genetically and clinically heterogeneous disorder that predisposes patients to bone marrow failure (BMF), myelodysplastic syndromes (MDS), and acute myeloid leukemia (AML). To study which genetic and phenotypic factors predict clinical outcomes for Japanese FA patients, we examined the FA genes, bone marrow karyotype, and aldehyde dehydrogenase-2 (ALDH2) genotype; variants of which are associated with accelerated progression of BMF in FA. In 88 patients, we found morphologic MDS/AML in 33 patients, including refractory cytopenia in 16, refractory anemia with excess blasts (RAEB) in 7, and AML in 10. The major mutated FA genes observed in this study were FANCA (n = 52) and FANCG (n = 23). The distribution of the ALDH2 variant alleles did not differ significantly between patients with mutations in FANCA and FANCG. However, patients with FANCG mutations had inferior BMF-free survival and received hematopoietic stem cell transplantation (HSCT) at a younger age than those with FANCA mutations. In FANCA, patients with the c.2546delC mutation (n = 24) related to poorer MDS/AML-free survival and a younger age at HSCT than those without this mutation. All patients with RAEB/AML had an abnormal karyotype and poorer prognosis after HSCT; specifically, the presence of a structurally complex karyotype with a monosomy (n = 6) was associated with dismal prognosis. In conclusion, the best practice for a clinician may be to integrate the morphological, cytogenetic, and genetic data to optimize HSCT timing in Japanese FA patients.

Diagnostic utility of the lymphoid screening tube supplemented with CD34 for Ogata score calculation in patients with peripheral cytopenia

ABSTRACTObjectives: The diagnosis of myelodysplastic syndrome (MDS) is not always straightforward in the absence of objective markers such as ringed sideroblasts, an excess of blasts or clonal cytogenetic abnormalities. Moreover, the lack of specificity of morphological dysplasia makes the differentiation between MDS and other causes of peripheral cytopenia difficult. The WHO 2016 classification of MDS recognizes multiparameter flow cytometry (MFC) as an adjuvant tool for MDS diagnosis. An easily applicable MFC protocol based on CD34 and CD45 is proposed by Ogata et al. Furthermore, in the diagnostic workup of patients with peripheral cytopenia, the integration of MFC by means of a Lymphoid Screening Tube (LST) is recommended by the EuroFlow™ consortium. The aim of this study was to investigate whether the LST, supplemented with CD34, can be used to calculate the Ogata score, thereby obviating the need to run different flow cytometric tubes.Methods: Bone marrow samples from 108 patients with peripheral cytopenia were analyzed (MDS n = 32; non-MDS n = 76). The LST used in the present study was based on the tube designed by the EuroFlow™ consortium, but with addition of CD34 and without TCRγδ.Results: Rather low sensitivities of 55% in low-grade MDS patients and 80% in high-grade MDS patients were observed. However, a high specificity of 92% was found in the non-MDS group.Conclusion: Besides screening for clonal lymphocytes, plasma cells and blasts, an LST supplemented with CD34 allows the calculation of the Ogata score as an adjuvant tool in the diagnostic workup of cytopenic patients suspected of MDS.

From Eosinophilic Gastroenteritis to Chronic Eosinophilic Leukemia: A Case of Hypereosinophilia

Introduction Hypereosinophilia is generally defined as a peripheral blood eosinophil count greater than 1500/mm3 and can be associated with reactive (secondary) or hematologic (primary) disorders, which can lead to end-organ damage. We present a patient with gastrointestinal symptoms in the setting of absolute eosinophilia. Work-up was initially suggestive of eosinophilic gastroenteritis, a rare inflammatory disorder characterized by eosinophilic tissue infiltration with no known natural progression to date. However, additional studies revealed a clonal genetic abnormality on chromosomal analysis in the diagnosis of chronic eosinophilic leukemia, not otherwise specified (CEL, NOS). Case Description An 82-year-old female with history of psoriatic arthritis presented with a 2-week history of watery, nonbloody bowel movements (5-20 per day) along with abdominal cramping, nausea and worsening oral intolerance. Vital signs and physical exam were unremarkable. Laboratory studies were significant for a WBC of 25,000 (40% eosinophils) and elevated IgE level at 138IU/mL. CT of the abdomen was also unremarkable.2578_A Figure 1. Duodenal biopsy, increased eosinophils infiltrating epithelium and lamina propria.2578_B Figure 2. Bone marrow biopsy, hypercellular bone marrow with eosinophilia.An esophagogastroduodenoscopy was performed, demonstrating diffuse duodenal erythema, edema and villous blunting as well as erythematous and hypertrophied gastric folds. Random duodenal, gastric, esophageal, and colonic biopsies revealed increased eosinophils (60 to >100 eos/hpf). Bone marrow biopsy was notable for hypercellularity and marked eosinophilia. The chromosomal analysis detected a clonal cytogenetic abnormality, del(11q), satisfying the 2017 WHO diagnostic criteria for CEL, NOS. The patient was treated with corticosteroids with resolution of absolute eosinophilia and gastrointestinal symptoms. Discussion This case elucidates the importance of undergoing a complete evaluation for hypereosinophilia, beginning with assessing for secondary causes, followed by thorough hematologic diagnostic studies. The localization of the patient's symptoms in the gastrointestinal tract, coupled with biopsy-proven eosinophilic infiltration, pointed initially towards eosinophilic gastroenteritis. However, the abnormal chromosomal analysis proved the diagnosis of CEL, NOS. As more cases suggestive of eosinophilic gastroenteritis emerge, the hope is to better understand the pathogenesis and natural progression of this rare disease, as well as to compare the effectiveness of therapeutic strategies with that of the hematologydriven processes.

A Review on Leukemia and iPSC Technology: Application in Novel Treatment and Future

Leukemia is an uncontrollable growth of hematopoietic cells due to a mutation in DNA followed by cellular dysregulation and one or more chromosomal disorder that generally leads to a clonal abnormality. Theoretical and technical inability in early screening and distinguishing cancer, tumor tolerance to common treatment methods, repeated relapses of cancer after remission phase, heterogeneous chromosomal abnormality, and the side effects of current chemotherapies are some of challenges that we face with leukemia and other malignancies. Induced pluripotent stem cells (iPSC) opened a promising window to a wide range of diseases, including leukemia. Overcoming the barriers in leukemia is possible with iPSC technology. Induced hematopoietic stem cell transplantation (and gene therapy), induced cytotoxic T-lymphocytes and reprogrammed NK cells that strengthen the immune system, miRNAs, modeling approaches, and supportive cares are some aspects of the novel treatment based on iPSC technology.

Hemophagocytic Lymphohistiocytosis Masquerading as Alcoholic Hepatitis

Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of excessive immune activation. Timely diagnosis can be challenging and prompt treatment is the only hope for survival. A 35 year old male with history of alcohol dependence was sent to the gastroenterology clinic for evaluation of fatigue, bilateral lower extremity edema, and orange colored urine. Lab work showed abnormal liver function tests (AST 295, alkaline phosphatase (ALP) 505, ALT 230, bilirubin 3.4). Exam was significant for lower extremity edema and hepatosplenomegaly. Further testing revealed elevated ferritin (12799 ng/ml) and iron saturation (68%). Repeat LFTs one week later showed AST 356, ALP 569, ALT 155, bilirubin 9.5. The patient was admitted to the hospital for further evaluation. CT scan a month prior incidentally showed hepatosplenomegaly and diffuse lymphadenopathy. PET scan revealed hypermetabolic splenomegaly, mediastinal and retroperitoneal lymphadenopathy, and numerous bone marrow abnormalities. Bone marrow biopsy and endoscopic ultrasound (EUS) were then performed. EUS revealed many abnormal enlarged lymph nodes extending from the supraclavicular region to the liver. Biopsies taken were normal and bone marrow biopsy showed no cytogenetic evidence of an abnormal clone. The patient was started empirically on ganciclovir for possible CMV infection and dexamethasone for suspected HLH while cultures and biopsy results were pending. Viral cultures were positive for CMV IgG and IgM, and EBV IgG and nuclear antibody. A clinical diagnosis of HLH was made and he was started on etoposide, in addition to dexamethasone, but his clinical status deteriorated rapidly and he died of multiorgan failure seven days later. HLH is a rare aggressive disease that is more frequently observed in infants. Immune homeostasis disruption can be triggered by infection, the most common being Epstein-Barr virus. It presents as a febrile illness that mimics infections, fever of unknown origin, hepatitis, and encephalitis, making timely diagnosis especially difficult. Patients typically present with hepatomegaly, lymphadenopathy, cytopenias, and high ferritin levels. High ferritin in the absence of iron overload carries high sensitivity and specificity for this disease. It is important that gastroenterologists keep a high index of suspicion when a patient presents with liver failure, high ferritin, and cytopenia, as they can be the first to evaluate these patients when they present.2259 Figure 1. From: Hemophagocytic LymphohistiocytosisAn Update on Diagnosis and Pathogenesis Am J Clin Pathol. 2013;139(6):713-727. doi:10.1309/AJCP4ZDKJ4ICOUAT Am J Clin Pathol | © American Society for Clinical Pathology

Clonal architecture in patients with myelodysplastic syndromes and double or minor complex abnormalities: Detailed analysis of clonal composition, involved abnormalities, and prognostic significance.

The study analyzes the clonal architecture and the abnormalities involved in a series of 191 patients with myelodysplastic syndromes (MDS) and 2-3 clonal abnormalities. All patients were extracted from an international database. The patients were classified into six clonal subtypes (2A-3C) based on the number of abnormalities and the presentation of unrelated clones (UC) and/or a clonal evolution. UC were detected in 23/191 patients (12%). The composition of UC showed great variability. The only recurrent combination of abnormalities was del(5q) and + 8 in 8 of 23 patients (35%). In patients with clonal evolution, the clone size of the primary and secondary clone varied: Patients with -7 and + 8 in the primary clone showed a larger primary and a smaller secondary clone (-7: median 74% vs 10%; +8 73% vs 18%) while patients with del(5q) in the primary clone showed a smaller primary and a larger secondary clone (33% vs 61%). Univariate and multivariate analyses showed no significant differences regarding overall or AML-free survival between the clonal subtypes. Only the subtype 3C (3 abnormalities and clonal evolution) was an independent risk factor for developing AML (Hazard Ratio 5.5 as compared to subtype 2A, P < .05). Finally, our study confirms that the number of abnormalities clearly defines a significant risk factor for overall- as well as AML-free survival. Importantly, in patients with more than one clone, the calculation of the number of abnormalities in the entire sample instead of the number of abnormalities per clone allows a higher prognostic accuracy.

Inflammatory myofibroblastic tumour of gall bladder and liver: a case series & review of literature

Inflammatory myofibroblastic tumors are rare benign tumors that can mimic malignancy of unknown aetiology. It has spectrum of myofibroblastic proliferation along with varying amount of inflammatory infiltrate. Recently, the concept of this lesion being reactive has been challenged based on the clinical demonstration of recurrences and metastasis and cytogenetic evidence of acquired clonal chromosomal abnormalities. We hereby report a case series of three patients with inflammatory myofibroblastic tumour involving gallbladder, liver. Inflammatory myofibroblastic tumor (IMT) occurs more frequently in childhood and the most common involvement is seen in the lungs. Primary inflammatory myofibroblastic tumors of the gallbladder are rather infrequent. The present knowledge is based on case reports. Initiating factors such as reactive, infections, autoimmune and neoplastic processes, has been proposed but the etiology of most remains unknown. The most frequently involved organ in the abdomen is the liver, while primary gallbladder involvement is quite rare. Present knowledge about this entity is based on case reports in the literature (9). The clinical picture in cases with IMT depends on the organ of involvement and site of the organ; abdominal pain, jaundice and ascites might be the presenting symptoms, according to the localisation in the liver involvement. In our cases symptoms were abdominal pain and dyspepsia. CT demonstrated involvement of liver, gallbladder, which is rare. Hence, decision of extended cholecystectomy taken to do in these cases.

A transcription factor, MrMsn2, in the dimorphic fungus Metarhizium rileyi is essential for dimorphism transition, aggravated pigmentation, conidiation and microsclerotia formation.

SummaryMicrosclerotia (MS) are pseudoparenchymatous aggregations of hyphae of fungi that can be induced in liquid culture for biocontrol applications. Previously, we determined that the high‐osmolarity glycerol (HOG) signalling pathway was involved in regulating MS development in the dimorphic insect pathogen Metarhizium rileyi. To further investigate the mechanisms by which the signalling pathway is regulated, we characterized the transcriptional factor MrMsn2, a homologue of the yeast C2H2 transcriptional factor Msn2, which is predicted to function downstream of the HOG pathway in M. rileyi. Compared with wild‐type and complemented strains, disruption of MrMsn2 increased the yeast‐to‐hypha transition rate, enhanced conidiation capacity and aggravated pigmentation in M. rileyi. The ▵MrMsn2 mutants were sensitive to stress, produced morphologically abnormal clones and had significantly reduced MS formation and decreased virulence levels. Digital expression profiling revealed that genes involved in antioxidation, pigment biosynthesis and ion transport and storage were regulated by MrMsn2 during conidia and MS development. Taken together, our findings confirm that MrMsn2 controlled the yeast‐to‐hypha transition, conidia and MS formation, and virulence.

Hexavalent Chromium-Induced Chromosome Instability Drives Permanent and Heritable Numerical and Structural Changes and a DNA Repair-Deficient Phenotype.

A key hypothesis for how hexavalent chromium [Cr(VI)] causes cancer is that it drives chromosome instability (CIN), which leads to neoplastic transformation. Studies show chronic Cr(VI) can affect DNA repair and induce centrosome amplification, which can lead to structural and numerical CIN. However, no studies have considered whether these outcomes are transient or permanent. In this study, we exposed human lung cells to particulate Cr(VI) for three sequential 24-hour periods, each separated by about a month. After each treatment, cells were seeded at colony-forming density, cloned, expanded, and retreated, creating three generations of clonal cell lines. Each generation of clones was tested for chromium sensitivity, chromosome complement, DNA repair capacity, centrosome amplification, and the ability to grow in soft agar. After the first treatment, Cr(VI)-treated clones exhibited a normal chromosome complement, but some clones showed a repair-deficient phenotype and amplified centrosomes. After the second exposure, more than half of the treated clones acquired an abnormal karyotype including numerical and structural alterations, with many exhibiting deficient DNA double-strand break repair and amplified centrosomes. The third treatment produced new abnormal clones, with previously abnormal clones acquiring additional abnormalities and most clones exhibiting repair deficiency. CIN, repair deficiency, and amplified centrosomes were all permanent and heritable phenotypes of repeated Cr(VI) exposure. These outcomes support the hypothesis that CIN is a key mechanism of Cr(VI)-induced carcinogenesis.Significance: Chromium, a major public health concern and human lung carcinogen, causes fundamental changes in chromosomes and DNA repair in human lung cells. Cancer Res; 78(15); 4203-14. ©2018 AACR.

Abstract 3415: Cytogenetics of renal oncocytomas identify three distinct and mutually exclusive diagnostic classes of chromosome aberrations

Abstract Christopher B. Anderson1, Michael Lipsky1, Subhadra V. Nandula2,3, Christopher E. Freeman2, Thomas Matthews2, Catlin Walsh2, Gen Li1, Matthias Szabolcs2, Mahesh M. Mansukhani 2, James M. McKiernan1, Vundavalli V. Murty 2 1Departments of Urology, 2Pathology and Cell Biology, Columbia University Medical Center, New York, New York; 3Cancer Genetics Inc, Rutherford, New Jersey The cytogenetic alterations in renal oncocytoma (RO) are poorly understood. We analyzed 130 consecutive RO for karyotypic alterations. Clonal chromosome abnormalities were identified in 63 (48.5%) cases, which could be categorized into 3 classes of mutually exclusive cytogenetic categories. Class 1 RO had diploid karyotypes with 11q13 rearrangement in balanced translocations with ten or more different chromosome partners in all 20 (31.7%) cases. We identified recurrent translocation partners at 5q35, 6p21, 9p24, 11p13-14 and 11q23, and confirmed that CCND1 gene rearrangement at 11q13 utilizing fluorescence in situ hybridization (FISH). Class 2 RO exhibited hypodiploid karyotypes with loss of chromosome 1 and concurrent losses of Y in males and X in females in 25 (39.7%) cases. The class 3 tumors comprising of 18 (30.2%) cases showed diverse types of abnormalities with the involvement of 2 or more chromosomes exclusive of abnormalities seen in Class 1 and 2 tumors. Furthermore, karyotypically uninformative cases were subjected to FISH analysis to identify class 1 and 2 abnormalities. In this group we found similar frequencies of CCND1 rearrangement, loss of 1 or Y as with karyotypically abnormal cases. We validated our results against 91 tumors from the Mittleman database. Correlation of clinical data with all the 3 classes of RO showed no clear evidence of overall patient survival. Our findings support the hypothesis that RO exhibit 3 principal cytogenetic categories, which may have different roles in initiation and/or progression. These cytogenetic markers provide a key tool in the diagnostic evaluation of RO. Citation Format: Christopher B. Anderson, Michael Lipsky, Subhadra V. Nandula, Freeman E. Christopher, Matthews Thomas, Caitlin E. Walsh, Gen Li, Matthias Szabolcs, Mahesh M. Mansukhani, James M. McKiernan, Murty V. Vundavalli. Cytogenetics of renal oncocytomas identify three distinct and mutually exclusive diagnostic classes of chromosome aberrations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3415.

Abstract 3401: Comprehensive analysis of genetic alterations and intratumor heterogeneity in myxofibrosarcoma

Abstract Myxofibrosarcoma (MFS) is a rare subtype of soft tissue sarcomas (STSs) preferentially affecting elderly. Histologically, MFS is distinct from other STSs, in that it is characterized by the proliferation of pleomorphic spindle cells with varying degrees of myxoid components. However, the molecular pathogenesis of MSF is poorly understood. In this study, we conducted an integrated molecular analysis of 44 samples from primary MFS patients, in which samples were analyzed by whole-genome sequencing (WGS) (n=2), whole-exome sequencing (WES) (n=44), RNA sequencing (n=3), DNA methylation array (n=16), and immunohistochemistry (IHC, n=27). Copy number alterations (CNAs) were identified by sequence based copy number analysis. The obtained genomic data were combined with those from STS cases from the Cancer Genome Atlas (TCGA) cohort, including 17 MSF samples and compared to the data from other STS samples (n=189). To further investigate the genetic basis of mixed histological components and chronological changes in MFS, we performed analysis from multi-regional and/or multi-time-point samples from 8 MSF cases. A total of 4,613 mutations were identified by WES in 61 primary MFS samples with a median of 44.0 mutations/sample. Mutations were dominated by age-related C to T transitions at CpG sites. Most frequently mutated genes included TP53 (n=21, 34.4%), ATRX (n=9, 14.8%), and RB1 (n=3, 4.9%). A fusion gene associated with TRIO was detected by RNA sequencing in a single case. Among other STSs, undifferentiated pleomorphic sarcoma (UPS) harbored the most similar genetic abnormalities (most frequently mutated genes included TP53 (40.9%), ATRX (29.5%), and RB1 (11.3%)), suggesting that these two subtypes are genetically closely related. Also combined cases with CNAs (n=35) and strong staining in IHC (n=13), TP53 abnormality was found in most cases (n=56, 91.8%). Two MSF cases evaluated by WGS showed complex structural abnormalities, where 491 and 198 somatic structural variations were detected suggestive of increased genetic instability. Multi-regional sampling (n=5) disclosed a high level of intratumor heterogeneity with less than 29.0% of mutations being shared by different samples. Multi-time points sampling (n=6) revealed that the number of mutations was significantly higher in relapse samples (odds ratio 1.6, p = 0.03). While in all cases, TP53 lesions were observed at the initial time-point, others were subclonal and acquired during the clinical course. Finally, in methylation analysis, 3,817 differentially methylated regions were detected (Stouffer's p &amp;lt; 0.05), based on which MFS were clustered into two distinct subtypes. In summary, the genetic profile of MFS is characterized by frequent abnormalities in TP53, ATRX, and RB1 and closely related to other STSs, especially to UPS. Clonal TP53 abnormalities resulted in complex chromosomal structure and a high degree of intratumor heterogeneity. Citation Format: Yasuhide Takeuchi, Annegret Kunitz, Hiromichi Suzuki, Kenichi Yoshida, Nobuyuki Kakiuchi, Yusuke Shiozawa, Akira Yokoyama, Yoichi Fujii, Tetsuichi Yoshizato, Kosuke Aoki, Keisuke Kataoka, Yasuhito Nannya, Yuichi Shiraishi, Teppei Shimamura, Kenichi Chiba, Hiroko Tanaka, Hideki Makishima, Satoru Miyano, Hironori Haga, Frederik Damm, Seishi Ogawa. Comprehensive analysis of genetic alterations and intratumor heterogeneity in myxofibrosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3401.

Targeted sequencing analysis of hyper-eosinophilic syndrome and chronic eosinophilic leukemia

目的分析嗜酸性粒细胞增多症的分子学特征。方法对24例伴PDGFRA、PDGFRB或FGFR1重排的慢性嗜酸性粒细胞白血病（CEL）及62例高嗜酸性粒细胞综合征（HES）患者进行靶向测序，利用权威数据库进行变异注释及氨基酸突变分析，推测可能致病性突变。结果17例（71%）CEL患者检出37种克隆性异常，但未发现重现性突变位点及热点突变区域，19例伴PDGFRA重排患者未检出致病性突变，2例进展为急性髓系白血病的伴FGFR1重排患者及1例进展为T淋巴母细胞淋巴瘤的伴PDGFRB重排患者分别检出ASXL1、RUNX1和NRAS的致病性突变。49例（71%）HES患者检出102种克隆性异常，主要热点突变区域包括：CEBPA Exon1、TET2 Exon3、ASXL1 Exon12、IDH1 Y208C和FGFR3 L164V。其中2例经伊马替尼单药治疗获得血液学缓解的HES患者分别检出CRRLF2 P224L和PDGFRB R370C点突变。结论伴PDGFRA、PDGFRB或FGFR1重排的CEL致病因素单一，疾病进展可能有其他突变参与。HES可能由具有热点突变区域的多种基因参与致病，部分突变位点对酪氨酸激酶抑制剂治疗敏感。

Myelodysplastic Syndrome in Pakistan: Clinicohematological Characteristics, Cytogenetic Profile, and Risk Stratification.

Objective:Myelodysplastic syndrome (MDS) is a group of bone marrow diseases that not only have variable morphological presentation and heterogeneous clinical courses but also have a wide range of cytogenetic abnormalities. Clinicohematological parameters have a significant role in diagnosis and along with identification of cytogenetic abnormalities are important for prognostic scoring and risk stratification of patients to plan management and make treatment decisions. This study aimed to determine the clinicohematological characteristics, cytogenetic abnormalities, and risk stratification of newly diagnosed de novo MDS patients.Materials and Methods:This cross-sectional study was conducted in the Department of Hematology, Armed Forces Institute of Pathology, Rawalpindi, from January 2013 to January 2017. Patients were diagnosed on the basis of World Health Organization criteria for MDS, clinicohematological parameters were noted, and cytogenetic analysis was performed. Risk stratification was done using the Revised International Prognostic Scoring System.Results:A total of 178 cases of MDS were analyzed, including 119 males (66.9%) and 59 females (33.1%). The median age was 58 years. The most common presenting feature was anemia in 162 (91%) of the patients. MDS with multilineage dysplasia was the most common diagnosis, seen in 103 (57.9%) patients. A normal karyotype was seen in 95 (53.4%), while 83 (46.6%) showed clonal karyotypic abnormalities at diagnosis. Of these, the common abnormalities found were trisomy 8, complex karyotype, and del 5q. Risk stratification revealed low-risk disease in 73 (41%) patients.Conclusion:Cytogenetic analysis showed the normal karyotype to be the most common while risk stratification revealed a predominance of low-risk disease at the time of presentation.