A novel chimeric antigen receptor (CAR) system using an exogenous protease, in which activation of T cells is controlled by expression patterns of cell­surface proteins on target cells.

Anti­CD19 chimeric antigen receptor (CAR)­T cell therapy against refractory B­cell malignancies shows excellent therapeutic effects. However, there are some obstacles to be overcome in this treatment. Since current CAR­T cells target a single cell­surface protein on tumor cells, the CAR­T cells also attack normal cells expressing the protein. This is one of the major adverse effects of this therapy. To improve target­cell­specificity of this therapy, we established a novel CAR system, in which T­cell activation was controlled by expression patterns of proteins on target cells. Our novel CAR­T cells had two distinct CARs consisting of a 'Signal­CAR', recognizing a protein on tumor cells, and a 'Scissors­CAR', recognizing another protein on normal cells. The signal­CAR had a peptide sequence which was cleaved by the Scissors­CAR, and functional domains for cellular activation. The Scissors­CAR had a protease domain that cleaved its recognition peptide sequence in the Signal­CAR. When tumor cells expressed only the protein recognized by the Signal­CAR, the tumor cells were attacked. By contrast, normal cells expressing both the proteins induced inactivation of the Signal­CAR through cleavage of the recognition site when getting in contact with the CAR­T cells. To establish this system, we invented a Scissors­CAR that was dominantly localized on cell membranes and was activated only when the CAR­T cells were in contact with the normal cells. Using a T­cell line, Jurkat, and two proteins, CD19 and HER2, as target proteins, we showed that the anti­CD19­Signal­CAR was cleaved by the anti­HER2­Scissors­CAR when the CAR­T cells were co­cultivated with cells expressing both the proteins, CD19 and HER2. Furthermore, we demonstrated that primary CAR­T cells expressing both the CARs showed attenuated cytotoxicity againsT cells with both the target proteins. Our novel system would improve safety of the CAR­T cell therapy, leading to expansion of treatable diseases by this immunotherapy.

MPL overexpression induces a high level of mutant-CALR/MPL complex: a novel mechanism of ruxolitinib resistance in myeloproliferative neoplasms with CALR mutations.

Ruxolitinib (RUX), a JAK1/2-inhibitor, is effective for myeloproliferative neoplasm (MPN) with both JAK2V617 F and calreticulin (CALR) mutations. However, many MPN patients develop resistance to RUX. Although mechanisms of RUX-resistance in cells with JAK2V617 F have already been characterized, those in cells with CALR mutations remain to be elucidated. In this study, we established RUX-resistant human cell lines with CALR mutations and characterized mechanisms of RUX-resistance. Here, we found that RUX-resistant cells had high levels of MPL transcripts, overexpression of both MPL and JAK2, and increased phosphorylation of JAK2 and STAT5. We also found that mature MPL proteins were more stable in RUX-resistant cells. Knockdown of MPL in RUX-resistant cells by shRNAs decreased JAK/STAT signaling. Immunoprecipitation assays showed that binding of mutant CALR to MPL was increased in RUX-resistant cells. Reduction of mutated CALR decreased proliferation of the resistant cells. When resistant cells were cultured in the absence of RUX, the RUX-resistance was reversed, with reduction of the mutant-CALR/MPL complex. In conclusion, MPL overexpression induces higher levels of a mutant-CALR/MPL complex, which may cause RUX-resistance in cells with CALR mutations. This mechanism may be a new therapeutic target to overcome RUX-resistance.

Outcome of allogeneic hematopoietic stem cell transplantation for T-cell lymphoblastic leukemia/lymphoma: A single-center study.

Although the indications for allogeneic hematopoietic stem cell transplantation (allo-HSCT) as a treatment for T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LBL) and Philadelphia chromosome (Ph)-negative B-cell acute lymphoblastic leukemia (B-ALL) are similar, few studies have compared its outcomes for T-ALL/LBL and Ph-negative B-ALL. The clinical data of 28 patients with T-ALL, 16 with T-LBL, and 99 with Ph-negative B-ALL who underwent the first allo-HSCT from 2000 to 2019 were retrospectively analyzed. Complete remission (CR) rates at allo-HSCT were 79 %, 63 %, and 75 % for T-ALL, T-LBL, and B-ALL, respectively; the 3-year overall survival (OS) rates were 55.7 %, 56.2 %, and 58.6 %, respectively (p = 0.92). Univariate analysis revealed that disease subtypes were not significantly associated with OS (B-ALL vs. T-ALL: hazard ratio [HR]=0.89, p = 0.70; T-LBL vs. T-ALL: HR=0.87, p = 0.75), and CR at allo-HSCT was the only prognostic factor for OS (HR=0.25, p < 0.001). Multivariate analysis demonstrated that CR at allo-HSCT was the only predictor of OS (HR=0.24, p < 0.001). In all three disease subtypes, patients in CR at allo-HSCT tended to have a lower cumulative incidence of relapse than did those in non-CR (T-ALL: 13.6 % vs. 50.0 %, p = 0.10; T-LBL: 20.0 % vs. 50.0 %, p = 0.21; B-ALL: 10.0 % vs. 56.0 %, p < 0.01). Thus, the outcomes of allo-HSCT for T-ALL/LBL were comparable to those of Ph-negative B-ALL. Irrespective of the disease subtypes, achieving CR before allo-HSCT was associated with a favorable OS. Further advances in chemotherapy before allo-HSCT and defining the optimal timing of allo-HSCT would improve the prognosis of patients with T-ALL/LBL.

An Antibody-Drug Conjugate That Selectively Targets Human Monocyte Progenitors for Anti-Cancer Therapy.

As hematopoietic progenitors supply a large number of blood cells, therapeutic strategies targeting hematopoietic progenitors are potentially beneficial to eliminate unwanted blood cells, such as leukemic cells and immune cells causing diseases. However, due to their pluripotency, targeting those cells may impair the production of multiple cell lineages, leading to serious side effects such as anemia and increased susceptibility to infection. To minimize those side effects, it is important to identify monopotent progenitors that give rise to a particular cell lineage. Monocytes and monocyte-derived macrophages play important roles in the development of inflammatory diseases and tumors. Recently, we identified human monocyte-restricted progenitors, namely, common monocyte progenitors and pre-monocytes, both of which express high levels of CD64, a well-known monocyte marker. Here, we introduce a dimeric pyrrolobenzodiazepine (dPBD)-conjugated anti-CD64 antibody (anti-CD64-dPBD) that selectively induces the apoptosis of proliferating human monocyte-restricted progenitors but not non-proliferating mature monocytes. Treatment with anti-CD64-dPBD did not affect other types of hematopoietic cells including hematopoietic stem and progenitor cells, neutrophils, lymphocytes and platelets, suggesting that its off-target effects are negligible. In line with these findings, treatment with anti-CD64-dPBD directly killed proliferating monocytic leukemia cells and prevented monocytic leukemia cell generation from bone marrow progenitors of chronic myelomonocytic leukemia patients in a patient-derived xenograft model. Furthermore, by depleting the source of monocytes, treatment with anti-CD64-dPBD ultimately eliminated tumor-associated macrophages and significantly reduced tumor size in humanized mice bearing solid tumors. Given the selective action of anti-CD64-dPBD on proliferating monocyte progenitors and monocytic leukemia cells, it should be a promising tool to target cancers and other monocyte-related inflammatory disorders with minimal side effects on other cell lineages.

[Molecular analysis of the BCR-ABL1 fusion gene in a chronic myeloid leukemia patient with BCR-ABL1 International Scale-undetectable disease].

BCR-ABL1 International Scale (IS) measurement is an important technique used to monitor chronic myeloid leukemia (CML) when patients are treated with tyrosine kinase inhibitors. In very rare cases, the BCR-ABL1 IS measurement fails to detect BCR-ABL1 chimeric messages for unknown reasons. Such a situation was also experienced in our study; therefore, molecular analysis of BCR-ABL1 fusion gene was performed. A breakpoint in the ABL1 gene was identified in the middle of the a2 exon, leading to generation of chimeric messages lacking a nucleic acid sequence encoded by this exon (e14a3 chimeric message). The ABL1 a2 exon is critical for quantification of BCR-ABL1 chimeric messages with the measurement scales used in medical laboratories. Absence of this referred nucleic acid sequence was the reason why BCR-ABL1 IS measurement failed to detect the chimeric messages in our study. In the future, such rare cases of CML, with BCR-ABL1 chimeric messages not detected by standard IS measurements, in Japan should be collected and their molecular structures should be analyzed.

[Novel therapies for acute myeloid leukemia based on genomic aberrations].

Standard treatment for acute myeloid leukemia (AML) comprises (1) induction therapy with both cytarabine and anthracycline and (2) consolidation therapy that is modified according to patients' conditions, including prognostic factors. However, this strategy is not satisfactory, especially for elderly patients. Novel technologies have revealed several driver mutations of numerous critical genes in AML, which can be targeted by novel drugs; the discovery of such targetable genes and the development of novel drugs have evolved the treatment strategy for AML. We should always monitor these advances in hematology. In the United States, the FDA has already approved several new drugs for AML, including FLT3 inhibitors and IDH neoenzyme inhibitors. In Japan, gilteritinib, an FLT3 inhibitor, was also approved at the end of 2018. These promising drugs will facilitate performing "precision medicine" on patients with AML soon.

[Follicular lymphoma accompanied by paraneoplastic pemphigus and bronchiolitis obliterans: a case report].

A 54-year-old female complained of oral erosion. A flaccid blister appeared on the trunk 2 months after the onset. The high titer of the anti-desmoglein 1 antibody in the absence of Nikolsky's sign led to the diagnosis of pemphigus vulgaris. The lymphadenopathy in the mesenteric and para-aortic regions indicated the possibility of paraneoplastic pemphigus. The steroid pulse therapy and therapeutic plasma exchange were ineffective. As CT-guided intraperitoneal lymph node biopsy revealed follicular lymphoma, R-CHOP therapy was performed. Although partial remission was attained accompanied by an improvement in the skin and mucosal findings after four courses of R-CHOP therapy, an occlusive ventilatory disturbance, possibly attributed to bronchiolitis obliterans, appeared 4 months after the treatment initiation. Although the treatment with tacrolimus was attempted, it was not feasible to be continued because of opportunistic infection, and the patient died 9 months after the onset of the skin lesion. Although specific anti-plakin antibodies were negative, this case was diagnosed as paraneoplastic pemphigus due to follicular lymphoma and complicated by obstructive bronchiolitis based on the clinical findings. The accumulation of similar cases is needed to establish effective treatment strategies.

Copy number analysis identifies tumor suppressive lncRNAs in human osteosarcoma.

Osteosarcoma (OS) has a high degree of chromosomal instability and total copy number (CN) changes. We examined 58 human OS samples including 40 primary tumors, 11 explants, and 7 cell lines using single nucleotide polymorphism (SNP) arrays, and revealed that 70% of the samples had one or more recurrent CN-neutral loss of heterozygosity (CNN­LOH) also known as uniparental disomy (UPD). Importantly, 17% of the samples showed prominent homozygous deletion of 3q13.31, suggesting its role in tumorigenesis. We identified and characterized two novel lncRNAs, LOC285194 and BC040587, within this genomic locus, strongly suggesting their tumor suppressor activity. Frequent deletions and UPD suggest that OS often has mutant or non-expressed tumor suppressor genes including two lncRNAs.

Mutational Landscape of Pediatric Acute Lymphoblastic Leukemia.

Current standard of care for patients with pediatric acute lymphoblastic leukemia (ALL) is mainly effective, with high remission rates after treatment. However, the genetic perturbations that give rise to this disease remain largely undefined, limiting the ability to address resistant tumors or develop less toxic targeted therapies. Here, we report the use of next-generation sequencing to interrogate the genetic and pathogenic mechanisms of 240 pediatric ALL cases with their matched remission samples. Commonly mutated genes fell into several categories, including RAS/receptor tyrosine kinases, epigenetic regulators, transcription factors involved in lineage commitment, and the p53/cell-cycle pathway. Unique recurrent mutational hotspots were observed in epigenetic regulators CREBBP (R1446C/H), WHSC1 (E1099K), and the tyrosine kinase FLT3 (K663R, N676K). The mutant WHSC1 was established as a gain-of-function oncogene, while the epigenetic regulator ARID1A and transcription factor CTCF were functionally identified as potential tumor suppressors. Analysis of 28 diagnosis/relapse trio patients plus 10 relapse cases revealed four evolutionary paths and uncovered the ordering of acquisition of mutations in these patients. This study provides a detailed mutational portrait of pediatric ALL and gives insights into the molecular pathogenesis of this disease. Cancer Res; 77(2); 390-400. ©2016 AACR.

Variegated clonality and rapid emergence of new molecular lesions in xenografts of acute lymphoblastic leukemia are associated with drug resistance.

The use of genome-wide copy-number analysis and massive parallel sequencing has revolutionized the understanding of the clonal architecture of pediatric acute lymphoblastic leukemia (ALL) by demonstrating that this disease is composed of highly variable clonal ancestries following the rules of Darwinian selection. The current study aimed to analyze the molecular composition of childhood ALL biopsies and patient-derived xenografts with particular emphasis on mechanisms associated with acquired chemoresistance. Genomic DNA from seven primary pediatric ALL patient samples, 29 serially passaged xenografts, and six in vivo selected chemoresistant xenografts were analyzed with 250K single-nucleotide polymorphism arrays. Copy-number analysis of non-drug-selected xenografts confirmed a highly variable molecular pattern of variegated subclones. Whereas primary patient samples from initial diagnosis displayed a mean of 5.7 copy-number alterations per sample, serially passaged xenografts contained a mean of 8.2 and chemoresistant xenografts a mean of 10.5 copy-number alterations per sample, respectively. Resistance to cytarabine was explained by a new homozygous deletion of the DCK gene, whereas methotrexate resistance was associated with monoallelic deletion of FPGS and mutation of the remaining allele. This study demonstrates that selecting for chemoresistance in xenografted human ALL cells can reveal novel mechanisms associated with drug resistance.

Genetic differences between Asian and Caucasian chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a common hematological malignancy in Western countries. However, this disease is very rare in Asian countries. It is not clear whether the mechanisms of development of CLL in Caucasians and Asians are the same. We compared genetic abnormalities in Asian and Caucasian CLL using 250k GeneChip arrays. Both Asian and Caucasian CLL had four common genetic abnormalities: deletion of 13q14.3, trisomy 12, abnormalities of ATM (11q) and abnormalities of 17p. Interestingly, trisomy 12 and deletion of 13q14.3 were mutually exclusive in both groups. We also found that deletions of miR 34b/34c (11q), caspase 1/4/5 (11q), Rb1 (13q) and DLC1 (8p) are common in both ethnic groups. Asian CLL more frequently had gain of 3q and 18q. These suggest that classic genomic changes in the Asian and Caucasina CLL are same. Further, we found amplification of IRF4 and deletion of the SP140/SP100 genes; these genes have been reported as CLL-associated genes by previous genome-wide-association study. We have found classic genomic abnormalities in Asian CLL as well as novel genomic alteration in CLL.

SOX7 is down-regulated in lung cancer.

BACKGROUND: SOX7 is a transcription factor belonging to the SOX family. Its role in lung cancer is unknown. METHODS: In this study, whole genomic copy number analysis was performed on a series of non-small cell lung cancer (NSCLC) cell lines and samples from individuals with epidermal growth factor receptor (EGFR) mutations using a SNP-Chip platform. SOX7 was measured in NSCLC samples and cell lines, and forced expressed in one of these lines. RESULTS: A notable surprise was that the numerous copy number (CN) changes observed in samples of Asian, non-smoking EGFR mutant NSCLC were nearly the same as those CN alterations seen in a large collection of NSCLC from The Cancer Genome Atlas which is presumably composed of predominantly Caucasians who often smoked. However, four regions had CN changes fairly unique to the Asian EGFR mutant group. We also examined CN changes in NSCLC lines. The SOX7 gene was homozygously deleted in one (HCC2935) of 10 NSCLC cell lines and heterozygously deleted in two other NSCLC lines. Expression of SOX7 was significantly downregulated in NSCLC cell lines (8/10, 80%) and a large collection of NSCLC samples compared to matched normal lung (57/62, 92%, p= 0.0006). Forced-expression of SOX7 in NSCLC cell lines markedly reduced their cell growth and enhanced their apoptosis. CONCLUSION: These data suggest that SOX7 is a novel tumor suppressor gene silenced in the majority of NSCLC samples.

Allelic imbalance in sporadic parathyroid carcinoma and evidence for its de novo origins.

Parathyroid cancer is a rare, clinically aggressive cause of primary hyperparathyroidism, and whether these malignancies generally evolve from pre-existing benign adenomas or arise de novo is unclear. Furthermore, while inactivation of the CDC73 (HRPT2) tumor suppressor gene, encoding parafibromin, is a major contributor, other genes essential to parathyroid carcinogenesis remain unknown. We sought to identify genomic regions potentially harboring such oncogenes or tumor suppressor genes, and to gain insight into the origins and molecular relationship of malignant versus benign parathyroid tumors. We performed genome-wide copy-number and loss of heterozygosity analysis using Affymetrix 50K SNP mapping arrays and/or comparative genomic hybridization on 16 primary parathyroid carcinomas, local recurrences or distant metastases, and matched normal controls, from 10 individuals. Recurrent regions of allelic loss were observed on chromosomes 1p, 3, and 13q suggesting that key parathyroid tumor suppressor genes are located in these chromosomal locations. Recurrent allelic gains were seen on chromosomes 1q and 16, suggesting the presence of parathyroid oncogenes on these chromosomes. Importantly, the most common alteration in benign parathyroid adenomas, loss of 11q, was not found as a recurrent change in the malignant parathyroid tissues. Molecular allelotyping using highly polymorphic microsatellite markers provided further confirmation that the prevalence of 11q loss is markedly and significantly lower in carcinomas as compared with adenomas. Our observations provide molecular support for the concept that sporadic parathyroid cancer usually arises de novo, rather than evolving from a pre-existing typical benign adenoma. Furthermore, these results help direct future investigation to ultimately determine which of the candidate genes in these chromosomal locations make significant contributions to the molecular pathogenesis of parathyroid cancer.

The correlation pattern of acquired copy number changes in 164 ETV6/RUNX1-positive childhood acute lymphoblastic leukemias.

The ETV6/RUNX1 fusion gene, present in 25% of B-lineage childhood acute lymphoblastic leukemia (ALL), is thought to represent an initiating event, which requires additional genetic changes for leukemia development. To identify additional genetic alterations, 24 ETV6/RUNX1-positive ALLs were analyzed using 500K single nucleotide polymorphism arrays. The results were combined with previously published data sets, allowing us to ascertain genomic copy number aberrations (CNAs) in 164 cases. In total, 45 recurrent CNAs were identified with an average number of 3.5 recurrent changes per case (range 0-13). Twenty-six percent of cases displayed a set of recurrent CNAs identical to that of other cases in the data set. The majority (74%), however, displayed a unique pattern of recurrent CNAs, indicating a large heterogeneity within this ALL subtype. As previously demonstrated, alterations targeting genes involved in B-cell development were common (present in 28% of cases). However, the combined analysis also identified alterations affecting nuclear hormone response (24%) to be a characteristic feature of ETV6/RUNX1-positive ALL. Studying the correlation pattern of the CNAs allowed us to highlight significant positive and negative correlations between specific aberrations. Furthermore, oncogenetic tree models identified ETV6, CDKN2A/B, PAX5, del(6q) and +16 as possible early events in the leukemogenic process.

Genomic profiling of adult acute lymphoblastic leukemia by single nucleotide polymorphism oligonucleotide microarray and comparison to pediatric acute lymphoblastic leukemia.

BACKGROUND: Differences in survival have been reported between pediatric and adult acute lymphoblastic leukemia. The inferior prognosis in adult acute lymphoblastic leukemia is not fully understood but could be attributed, in part, to differences in genomic alterations found in adult as compared to in pediatric acute lymphoblastic leukemia. DESIGN AND METHODS: We compared two different sets of high-density single nucleotide polymorphism array genotyping data from 75 new diagnostic adult and 399 previously published diagnostic pediatric acute lymphoblastic leukemia samples. The patients' samples were randomly acquired from among Caucasian and Asian populations and hybridized to either Affymetrix 50K or 250K single nucleotide polymorphism arrays. The array data were investigated with Copy Number Analysis for GeneChips (CNAG) software for allele-specific copy number analysis. RESULTS: The high density single nucleotide polymorphism array analysis of 75 samples of adult acute lymphoblastic leukemia led to the identification of numerous cryptic and submicroscopic genomic lesions with a mean of 7.6 genomic alterations per sample. The patterns and frequencies of lesions detected in the adult samples largely reproduced known genomic hallmarks detected in previous single nucleotide polymorphism-array studies of pediatric acute lymphoblastic leukemia, such as common deletions of 3p14.2 (FHIT), 5q33.3 (EBF), 6q, 9p21.3 (CDKN2A/B), 9p13.2 (PAX5), 13q14.2 (RB1) and 17q11.2 (NF1). Some differences between adult and pediatric acute lymphoblastic leukemia were identified when the pediatric data set was partitioned into hyperdiploid and non-hyperdiploid cases and then compared to the nearly exclusively non-hyperdiploid adult samples. In this analysis, adult samples had a higher rate of deletions of chromosome 17p (TP53) and duplication of 17q. CONCLUSIONS: Our analysis of adult acute lymphoblastic leukemia cases led to the identification of new potential target lesions relevant for the pathogenesis of acute lymphoblastic leukemia. However, no unequivocal pattern of submicroscopic genomic alterations was found to separate adult acute lymphoblastic leukemia from pediatric acute lymphoblastic leukemia. Therefore, apart from different therapy regimen, differences of prognosis between adult and pediatric acute lymphoblastic leukemia are probably based on genetic subgroups according to cytogenetically detectable lesions but not focal genomic copy number microlesions.

Variation in CDKN2A at 9p21.3 influences childhood acute lymphoblastic leukemia risk.

Using data from a genome-wide association study of 907 individuals with childhood acute lymphoblastic leukemia (cases) and 2,398 controls and with validation in samples totaling 2,386 cases and 2,419 controls, we have shown that common variation at 9p21.3 (rs3731217, intron 1 of CDKN2A) influences acute lymphoblastic leukemia risk (odds ratio = 0.71, P = 3.01 x 10(-11)), irrespective of cell lineage.

Prevalence and prognostic impact of allelic imbalances associated with leukemic transformation of Philadelphia chromosome-negative myeloproliferative neoplasms.

Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) including polycythemia vera, essential thrombocythemia, and primary myelofibrosis show an inherent tendency for transformation into leukemia (MPN-blast phase), which is hypothesized to be accompanied by acquisition of additional genomic lesions. We, therefore, examined chromosomal abnormalities by high-resolution single nucleotide polymorphism (SNP) array in 88 MPN patients, as well as 71 cases with MPN-blast phase, and correlated these findings with their clinical parameters. Frequent genomic alterations were found in MPN after leukemic transformation with up to 3-fold more genomic changes per sample compared with samples in chronic phase (P < .001). We identified commonly altered regions involved in disease progression including not only established targets (ETV6, TP53, and RUNX1) but also new candidate genes on 7q, 16q, 19p, and 21q. Moreover, trisomy 8 or amplification of 8q24 (MYC) was almost exclusively detected in JAK2V617F(-) cases with MPN-blast phase. Remarkably, copy number-neutral loss of heterozygosity (CNN-LOH) on either 7q or 9p including homozygous JAK2V617F was related to decreased survival after leukemic transformation (P = .01 and P = .016, respectively). Our high-density SNP-array analysis of MPN genomes in the chronic compared with leukemic stage identified novel target genes and provided prognostic insights associated with the evolution to leukemia.

SNP array analysis of tyrosine kinase inhibitor-resistant chronic myeloid leukemia identifies heterogeneous secondary genomic alterations.

To elucidate whether tyrosine kinase inhibitor (TKI) resistance in chronic myeloid leukemia is associated with characteristic genomic alterations, we analyzed DNA samples from 45 TKI-resistant chronic myeloid leukemia patients with 250K single nucleotide polymorphism arrays. From 20 patients, matched serial samples of pretreatment and TKI resistance time points were available. Eleven of the 45 TKI-resistant patients had mutations of BCR-ABL1, including 2 T315I mutations. Besides known TKI resistance-associated genomic lesions, such as duplication of the BCR-ABL1 gene (n = 8) and trisomy 8 (n = 3), recurrent submicroscopic alterations, including acquired uniparental disomy, were detectable on chromosomes 1, 8, 9, 17, 19, and 22. On chromosome 22, newly acquired and recurrent deletions of the IGLC1 locus were detected in 3 patients, who had previously presented with lymphoid or myeloid blast crisis. This may support a hypothesis of TKI-induced selection of subclones differentiating into immature B-cell progenitors as a mechanism of disease progression and evasion of TKI sensitivity.

Single nucleotide polymorphism genomic arrays analysis of t(8;21) acute myeloid leukemia cells.

Translocation of chromosomes 8 and 21, t(8;21), resulting in the AML1-ETO fusion gene, is associated with acute myeloid leukemia. We searched for additional genomic abnormalities in this acute myeloid leukemia subtype by performing single nucleotide polymorphism genomic arrays (SNP-chip) analysis on 48 newly diagnosed cases. Thirty-two patients (67%) had a normal genome by SNP-chip analysis (Group A), and 16 patients (33%) had one or more genomic abnormalities including copy number changes or copy number neutral loss of heterozygosity (Group B). Two samples had copy number neutral loss of heterozygosity on chromosome 6p including the PIM1 gene; and one of these cases had E135K mutation of Pim1. Interestingly, 38% of Group B and only 13% of Group A samples had a KIT-D816 mutation, suggesting that genomic alterations are often associated with a KIT-D816 mutation. Importantly, prognostic analysis revealed that overall survival and event-free survival of individuals in Group B were significantly worse than those in Group A.

Chromosomal abnormalities and novel disease-related regions in progression from Barrett's esophagus to esophageal adenocarcinoma.

Barrett's esophagus (BE) is a metaplastic condition caused by chronic gastroesophageal reflux which represents an early step in the development of esophageal adenocarcinoma (EAC). Single-nucleotide polymorphism microarray (SNP-chip) analysis is a novel, precise, high-throughput approach to examine genomic alterations in neoplasia. Using 250K SNP-chips, we examined the neoplastic progression of BE to EAC, studying 11 matched sample sets: 6 sets of normal esophagus (NE), BE and EAC, 4 of NE and BE and 1 of NE and EAC. Six (60%) of 10 total BE samples and 4 (57%) of 7 total EAC samples exhibited 1 or more genomic abnormalities comprising deletions, duplications, amplifications and copy-number-neutral loss of heterozygosity (CNN-LOH). Several shared abnormalities were identified, including chromosome 9p CNN-LOH [2 BE samples (20%)], deletion of CDKN2A [4 BE samples (40%)] and amplification of 17q12-21.2 involving the ERBB2, RARA and TOP2A genes [3.1 Mb, 2 EAC (29%)]. Interestingly, 1 BE sample contained a homozygous deletion spanning 9p22.3-p22.2 (1.2 Mb): this region harbors only 1 known gene, basonuclin 2 (BNC2). Real-time PCR analysis confirmed the deletion of this gene and decreased the expression of BNC2 mRNA in the BE sample. Furthermore, transfection and stable expression of BNC2 caused growth arrest of OE33 EAC cells, suggesting that BNC2 functions as a tumor suppressor gene in the esophagus and that deletion of this gene occurs during the development of EAC. Thus, this SNP-chip analysis has identified several early cytogenetic events and novel candidate cancer-related genes that are potentially involved in the evolution of BE to EAC.