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Supplementary Fig. 5. Kaplan-Meier analysis of the survival of patients with different mutations.

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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.

Supplementary Fig. 4. p53 pathway is dysregulated in ALL.

Supplementary Fig. 2. Mutations found in the ASXL family of genes.

Supplementary Fig. 1. Mutational spectrum of pediatric ALL.

Supplementary Table 1. Samples used for whole exome and targeted sequencing. Supplementary Table 2. Clinical information of patients. Supplementary Table 3. 560 Genes screened in the targeted sequencing. Supplementary Table 4. Sequences of shRNA or CRISPR-Cas9 sgRNA used in this study. Supplementary Table 5. Sequences of real-time PCR primers used in this study. Supplementary Table 6. Sequences of primers used for Sanger validation of CRISPR-Cas9 indel. Supplementary Table 7. Mutations of KRAS identified in this study have been found in a variety of cancers. Supplementary Table 8. Mutations of PTPN11 in other cancers occurring in the same location as in our ALL cohort. Supplementary Table 9. Recurrent mutation sites of FLT3 in different cancers. Supplementary Table 10. Cell lines or cancer samples harboring either E1099K or T1150A hotspot mutation of WHSC1. Supplementary Table 11. R1446 mutations of CREBBP recurrently occurred in a variety of cancers. Supplementary Table 12. Mutational hotspot (D1399) of EP300 occurring in other cancers occurring in the same location as in our ALL cohort.Supplementary Table 13. Rare mutations occurring in our ALL cohort (mutated in one individual each), but these mutations have been recurrently documented in the COSMIC cancer mutations database. Supplementary Table 14. Mutations of genes involved in DNA repair pathway in relapse samples.

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

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.

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.

Chronic myeloid leukemia (CML) is a hematological malignancy caused by an abnormal tyrosine kinase, BCR-ABL1. Leukemic stem cells (LSCs) in this disease proliferate indefinitely. Also, the LSCs normally differentiate into mature neutrophils. Although tyrosine kinase inhibitors (TKIs) that target the kinase activity of BCR-ABL1 improved prognosis of CML patients, many patients still have residual diseases and need life-long treatment with the TKIs. This is due to a small number of CML LSCs that cannot be completely eradicated by the TKIs. It has been hypothesized that not only chemotherapy alone, but also immune surveillance systems of patients are needed to completely eradicate cancerous cells from their body. We hypothesized that residual CML cells might escape from the immune surveillance. Surfaces molecules associated with immune surveillance are examined on CML cells. At first, we confirmed expression levels of CD80 and PD-L1 by flowcytometry using CML cell lines. Our data showed that CD80 was expressed on surfaces of CML derived cell, K562, not PD-L1. Moreover, an engineered K562 subline is generated with the constitutive active JAK2V617F gene and became dependent on JAK-STAT signaling, but not BCR-ABL. This subline did not show CD80 on the surface. The TKIs inhibited expression of CD80 on the parental K562. Meanwhile, an ERK inhibitor decreased CD80 expression in K562. These results suggested that CD80 was downstream of BCR-ABL-ERK signaling pathway, not JAK-STAT pathway. When K562 cells were differentiated into mature neutrophils by ATRA, levels of CD80 decreased. Cytotoxicity assays using K562 and NK cells from normal individuals showed that the parental K562 cells and K562 cells with decreased levels of CD80 were similarly killed. These data suggested that CD80 on K562 played a minimum role in NK cell immune surveillance. We also found that only a small fraction of CD11b positive monocytes in normal peripheral blood showed expression of CD80. Also, blastic cells of CML blastic phase showed CD80 expression. These results suggested that only blastic cells of CML and a fraction of monocytes expressed CD80. These cells were distinguished by expression of CD11b as a marker. Although it is currently impossible to distinguish normal stem cells from CML LSCs, our study suggested that CD80/CD11b might be helpful to distinguish the CML LSCs in patients' bone marrow. It may be possible to collect a very small number of CML LSCs in CML patients under the TKI treatment using these antibodies and characterize the residual LSCs. Citation Format: Huixin Li, Shunichiro Yasuda, Satoru Aoyama, Chenyang Zhang, Yohei Kawano, Miyuki Azuma, Norihiko Kawamata. CD80 can be the marker of chronic myeloid leukemia stem cells and regulated by BCR-ABL signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3117.

【Introduction】 Although Ruxolitinib (RUX), a JAK1/2-inhibitor, is an effective treatment option for primary myelofibrosis, tumor cells become resistant to this drug in many MPN patients, causing poor prognosis of MPN patients. Until now, various studies have elucidated the mechanisms of RUX-resistance in JAK2V617F-mutant MPN cells, including 1) increased JAK2 heterodimerization leading to sustained JAK2 activation, 2) JAK2 kinase domain mutations, and 3) JAK1/JAK3 activations. However, mechanisms of RUX-resistance in MPN cells with CALR mutations have not been fully characterized to date. In this study, we have clarified a mechanism of RUX-resistance in MPN tumor cells with CALR mutations. 【Materials and Methods】 At first, we have created several human cell lines with exogenous MPL expressions (exMPL) and CALR +1 frameshift mutations (CALR-fs) by introductions of V5-tagged MPL and/or FLAG-tagged CALR Del52/Ins5 expressing vectors or the CRISPR/Cas9 technology. We have confirmed that these cell lines had increased JAK/STAT signaling and respond to RUX-treatment. To establish RUX-resistant cell lines, we have cultured these cell lines with low-dose RUX (0.2μM), and gradually increased the concentrations of RUX by 0.1μM every week. We have successfully established RUX-resistant cells that proliferated in the presence of RUX at 0.8μM. Then, we characterized the RUX-resistant cells with CALR-fs/exMPL. To examine whether RUX-resistant cells shows the resistant-phenotype in vivo, we have subcutaneously implanted RUX-resistant cells as well as RUX-sensitive cells into immunocompromised mice. Three weeks after injections of the tumor cells, the mice were euthanized, and the subcutaneous tumors pathologically examined. MPL knockdown experiments showed that high levels of MPL were indispensable in the resistant cell lines. Co-immunoprecipitation assaies showed the interactions of mutant CALR and MPL proteins in RUX-resistant cells. To examine reversibility of RUX-resistance, RUX-resistant cells were cultured without RUX for three months. Finally, we examined pathological features of bone marrow samples of MPN patients with CALR mutations by immunohistochemical staining. 【Results】We have found that RUX-resistant cells had high MPL transcripts, overexpression of both immature and mature MPL, and JAK2. We also found that RUX-resistant cells had increased phosphorylations of JAK1, JAK2, JAK3 STAT5, MEK and ERK. In vivo assay using immunocompromised mice showed the immunohistochemical staining of MPL in the tumors from RUX-resistant cells showed high expression of MPL in the tumor cells. We also found that mature MPL proteins were more stable since proteasome-dependent degradation of mature MPL proteins was impaired in RUX-resistant cells. Knockdown of MPL of RUX-resistant cells by shRNAs decreased intensity of phosphorylations of JAK1, JAK2, STAT5, MEK and ERK, suggesting that the high expression of MPL leads to more potent signaling. Notably, when mutant-CALR proteins were immunoprecipitated, both immature and mature MPL proteins were co-immunoprecipitated: more MPL proteins were pulled down in RUX-resistant cells. In a reciprocal experiment, when MPL proteins were immunoprecipitated, mutant-CALR proteins were co-immunoprecipitated: more mutant-CALR proteins were pulled down in RUX-resistant cells. Reduction of mutated CALR decreased proliferation of the resistant cells, suggesting that a high level of mutant-CALR/MPL complex contributed to RUX-resistance in these cells. When the resistant cells were cultured in the absence of RUX, RUX-resistance was reversed with reduction of MPL transcripts, mature MPL and JAK2 proteins and mutant-CALR/MPL complex. Immunohistochemical staining showed that MPL staining intensity of megakaryocytes of MF patients with CALR mutations were higher than those of patients with JAK2V617F or normal individuals. MPL expressions were higher after RUX-treatment in MF patients with both JAK2V617F mutations and CALR mutations. 【Discussion】 Overexpression of MPL is a common mechanism after RUX-treatment in MPN cells with both JAK1V617F mutations and CALR mutations. In RUX-resistant cells with CALR mutations, MPL overexpression leads to the high level of mutant-CALR/MPL complex, causing resistance to RUX. This novel mechanism could be a new therapeutic target to overcome RUX-resistance in MPN cells with CALR mutations. Disclosures Komatsu: Otsuka Pharmaceutical Co., Ltd., Shire Japan KK, Novartis Pharma KK, PharmaEssentia Japan KK, Fuso Pharmaceutical Industries, Ltd., Fujifilm Wako Pure Chemical Corporation, Chugai Pharmaceutical Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Takeda Pharmaceutica: Research Funding; Otsuka Pharmaceutical Co., Ltd., PharmaEssentia Japan KK, AbbVie GK, Celgene KK, Novartis Pharma KK, Shire Japan KK, Japan Tobacco Inc: Consultancy; Takeda Pharmaceutical Co., Ltd, Novartis Pharma KK, Shire Japan KK: Speakers Bureau; AbbVie: Other: member of safety assessment committee in M13-834 clinical trial.; PPMX: Consultancy, Research Funding; Meiji Seika Pharma Co., Ltd.: Patents & Royalties: PCT/JP2020/008434, Research Funding.

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 (CNN‑LOH) 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.

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.

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.

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.

【 Introduction 】 CAR-T cell therapy has shown excellent therapeutic effects against some malignancies including refractory B cell lymphoma or leukemia. This adoptive T cell transfer therapy is an attractive methodology; however, there are several disadvantages to be overcome. Current CAR-T cell therapy targets single cell-surface molecule, which leads to damage of normal cells expressing the target protein. Such "On-target / off-tumor" effect is one of the major adverse effects. Improvement of target-cell-specificity is needed to avoid this serious adverse effect and to expand target diseases of this therapy. Here, we show the protease-mediated "Double-Arm" CAR-T cell system, which improved the specificity of CAR-T cell therapy by recognizing two distinct cell-surface proteins. We designed two types of CARs: "Effector CAR" and "Scissors CAR". The "Effector CAR" is constituted of a single chain Fv fragment (scFv) targeting a cell-surface protein (protein X) on tumor cells, Human Immunodeficiency Virus protease (HIVPR) recognition polypeptide sequence, and a functional domain of CD3-zeta. The "scissors CAR" is constituted of a recognition portion targeting another protein (protein Y) and HIVPR. The HIVPR induces cleavage of the recognition polypeptide sequence in the effector CAR leading to inactivation of the effector CAR when the CAR-T cells contact with cells expressing both proteins X and Y. 【 Material and Methods 】 For proof of principle, we first constructed "anti-CD19 mCherry CAR" harboring mCherry fluorescence protein in the cytoplasmic region under the HIVPR recognition polypeptide sequence. Also, we constructed "anti-CD19 scissors CAR", low affinity "anti-HER2 (4D5-3) scissors CAR" and high affinity "anti-HER2 (4D5-8) scissors CAR". To analyze the target-cell-dependent cleavage of mCherry CAR, 293T cells expressing these CARs were co-cultured with target cells, including K562 (CD19-, HER2-), Raji (CD19+, HER2+), or SK-BR-3 (CD19-, HER2+). To obtain target cells expressing both CD19 and HER2, Raji and SK-BR-3 cells were molecularly manipulated. (1) To evaluate efficiency of this system, after co-cultivation of CAR-transduced 293T and the target cells (K562, Raji, SK-BR-3), the localization of mCherry was examined under the microscopy and Western blotting. (2) To assess the T cell activation, we constructed "anti-CD19 effector CAR" and established Jurkat cells expressing both the "effector CAR" and the "anti-HER2 scissors CAR". These cells were co-cultured with wild type or engineered Raji or SK-BR-3 cells. T cell activation was analyzed with flowcytometric analysis. (3) To assess the CAR activity of this system, we transduced these CARs to primary T cells from healthy donors. After co-cultivation with the target cells, we measured target-specific cytotoxic activity. 【 Results 】 (1) Transduced "anti-CD19 mCherry CAR" was detected as a membrane-bound protein in 293T cells. Co-cultivation of 293T cells expressing both the "anti-CD19 mCherry CAR" and "anti-HER2 scissors CAR" with engineered Raji cells expressing both CD19 and HER2 induced cleavage of the recognition site and translocation of the mCherry from the membrane to the cytoplasm. These results suggested that this system would regulate activities of CAR-T cell through HIVPR-mediated cleavage of the "effector CAR" in vitro. (2) Jurkat cells expressing "anti-CD19 effector CAR" were activated through a target-cell-dependent manner. Furthermore, "anti-HER2 scissors CAR" attenuated T cell activation driven by "anti-CD19 effector CAR" when Jurkat cells expressing both the "anti-CD19 effector CAR" and the "anti-HER2 scissors CAR" contacted with target-cells expressing both CD19 and HER2. In addition, high affinity 4D5-8 scissors CAR showed more potent attenuation than the low affinity 4D5-3 scissors CAR. (3) Primary human T cells expressing "anti-CD19 effector CAR" showed CD19-expressing target-cell-specific cytotoxic activity. We also demonstrated that "Double Arm" primary human CAR-T cells showed tumor-cell-specificity as seen in cell-line models described above. 【Discussion】 Our "Double-Arm" CAR-T cell system has improved tumor-cell-specificity even in primary human T-cells as we expected. This system would attenuate the adverse effects of clinical CAR-T cell therapies. Disclosures No relevant conflicts of interest to declare.

[Introduction] CAR-T cell therapy is an attractive methodology in the field of cancer immunotherapy. The anti-CD19 CAR-T cell therapy shows excellent therapeutic effects against refractory B-cell malignancies. However, the CAR-T cells attack not only target-tumor-cells, but also normal tissues expressing the target molecule, for example, normal B-cells with CD19. For the CAR-T cell therapy, such “on-target / off-tumor” effect is one of major obstacles to be overcome. Here, we show the protease-mediated regulatory CAR-T cell system, which improved the target-specificity by recognizing two distinct antigens. We designed two types of CAR: "effector CAR" and "scissors CAR". The “effector CAR” is constituted of a single chain Fv fragment (scFv) targeting an antigen (protein X) on tumor cells, Human Immunodeficiency Virus protease (HIVPR) recognition site, and a functional domain of CD3-ζ. The “scissors CAR” is constituted of a scFv targeting another antigen (protein Y) and HIVPR. The “scissors CAR” induces cleavage of the “effector CAR” leading to inactivation when the CAR-T cells contact with cells expressing both proteins X and Y. [Material and Methods] For proof of principle, we first constructed the anti-CD19 “mCherry CAR” harboring mCherry fluorescence protein in the cytoplasmic region under the HIVPR recognition site. Also, we constructed the anti-HER2 “scissors CAR”. To analyze the target-cell dependent cleavage of “mCherry CAR”, 293T cells expressing the CARs were co-cultured with target-cells, including Raji (CD19+, HER2-) and engineered SK-BR-3 (CD19+, HER2+). Locations of mCherry were analyzed under the microscopy and by Western blotting. Next, to assess T-cell activation, we established Jurkat cells expressing both the anti-CD19 “effector CAR” and the anti-HER2 “scissors CAR”. These cells were co-cultured with the target-cells described above. T-cell activation was analyzed by both flowcytometric analysis and measurement of IL-2 mRNA expression. [Results] Transduced anti-CD19 “mCherry CAR” was detected as a membranous protein in 293T cell. Co-cultivation of 293T cell expressing both the anti-CD19 “mCherry CAR” and the anti-HER2 “scissors CAR” with Raji (CD19+, HER2-) did not change the localization of mCherry. However, engineered SK-BR-3 (CD19+, HER2+) induced cleavage of the recognition site and translocation of the mCherry from the membrane to cytoplasm. Furthermore, the anti-HER2 “scissors CAR” attenuated T-cell activation driven by the anti-CD19 “effector CAR” when Jurkat cells expressing both the CARs contacted with the target-cells expressing both CD19 and HER2. [Discussion] This novel protease-mediated CAR-T system recognized expression pattern of target molecules and regulated T-cell activity. Our CAR-T system would attenuate the adverse effects and contribute to expansion in application of CAR-T cell therapy. Citation Format: Satoru Aoyama, Shunichiro Yasuda, Daisuke Watanabe, Hiroki Akiyama, Keigo Okada, Yoshihiro Umezawa, Ayako Nogami, Osamu Miura, Norihiko Kawamata. A protease-mediated regulatory chimeric antigen receptor (CAR): “Scissors” CAR improves target-specificity through regulation of T-cell activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2167.

【Introduction】 Although Ruxolitinib (Rux), a JAK1/2-inhibitor, is an effective treatment option for primary myelofibrosis, tumor cells become resistant to this drug in many MPN patients. Currently, Rux-resistance is a major problem and contributes to poor prognosis of MPN patients. Mechanisms of Rux-resistance in MPN cells with JAK2V617F mutations have already been reported. On the other hand, MPN cells with CALR mutations resistant to Rux-treatment have not been fully characterized yet. In this study, we have clarified a mechanism of Rux-resistance in MPN tumor cells with CALR mutations in both cell lines and clinical samples. 【Materials and Methods】To establish human cell lines with CALR mutations, we have introduced deletion of 46-nucleotides generating the CALR frame-shift mutation (del46), one of the type1-like mutations, to UT-7/EPO by the CRISPR/Cas9 system. Then, we introduced exogenous MPL to UT-7/EPO with the CALR mutations by transfection. We confirmed that UT-7/EPO with CALR frame-shift mutations (CALR-fs) and exogenous MPL (exMPL) activate STAT5 and showed cytokine-independent cell growth. To establish Rux-resistant cell lines, we have cultured these cell lines with low-dose Rux (0.2μM), and gradually increased the concentrations of Rux by 0.1μM every week. Cells were considered as resistant when the half-maximal inhibitory concentrations (IC50) of the cells was double the IC50 of the parental cells. We have successfully established Rux-resistant cells that proliferated in the presence of Rux at 0.8μM. Then, we characterized the Rux-resistant cells with CALR-fs/exMPL. Next, reversibility of Rux-resistance, the Rux-resistant cells was examined. To examine whether Rux-resistant cells shows the resistant-phenotype in vivo, we have subcutaneously implanted Rux-resistant cells as well as Rux-sensitive cells into immunocompromised mice and treated the animals with Rux. Three weeks after injections of the tumor cells, the mice were euthanized, and the subcutaneous tumors were excised and weighted. Finally, we examined pathological features of bone marrow samples of MPN patients with CALR mutations by immunohistochemical staining. 【Results】We found that Rux-resistant cells had overexpressions of both MPL and JAK2, and increased phosphorylations of both JAK2 and STAT5. We found that high levels of MPL transcripts in the resistant cells. Cycloheximide treatment assay showed that mature MPL proteins were more stable in Rux-resistant cells. Then, we examined mechanisms of stability of mature MPL proteins in Rux-resistant cells. Our data suggested that the proteasome/system degrading mature MPL was attenuated in Rux-resistant cells. When Rux-resistant cells were cultured in the absence of Rux, Rux-resistance was reversed with reduction of mature MPL and JAK2. In vivo assay using immunocompromised mice showed the Rux resistant cells were not sufficiently suppressed by Rux-treatment. Since our data using the cell lines suggested that high MPL expression causes resistance to Rux in MPN cells with CALR mutations, we examined levels of MPL proteins in bone marrow samples of MPN patients with CALR mutations by immunohistochemical staining. Immunohistochemical staining of MPL showed that the megakaryocytes of MPN patients with high-risk and/or resistant to ruxolitinib treatment expressed higher MPL. 【Discussion】 MPL overexpression is one of mechanisms of MPN cells with CALR mutations to gain resistance to Rux. This mechanism may be a key element to overcome Rux-resistance in this fatal disease. Disclosures Komatsu: Novartis K.K: Speakers Bureau; Wako Pure Chemical Industries, Ltd.: Research Funding; Fuso Pharmaceutical Industries, Ltd.: Research Funding; Takeda Pharmaceutical Company Limited: Research Funding, Speakers Bureau; Pharma Essentia: Research Funding, Speakers Bureau.

【 Introduction 】 CAR-T cell therapy is an attractive methodology in the field of cancer immunotherapy. Anti-CD19 CAR is used for treating refractory B cell malignancies and shows excellent therapeutic effects. However, there are several disadvantages to be overcome in this therapy. "On-target / off-tumor" effect is one of such adverse effects. Current CAR-T cell therapy targets single cell-surface molecule, which leads to damage of normal cells expressing the target protein. Improvement of target-cell-specificity is one of important issues in CAR-T cell therapy to avoid this serious adverse effect. Here, we show the protease-mediated "Double-Arm" CAR-T cell system (Figure), which improved the specificity of CAR-T cell therapy by recognizing two distinct cell-surface proteins. We designed two types of CAR: "Effector CAR" and "Scissors CAR" (Figure, left panel). The "effector CAR" is constituted of a single chain Fv fragment (scFv) targeting a cell-surface protein (protein X) on tumor cells, Human Immunodeficiency Virus protease (HIVPR) recognition polypeptide sequence, and a functional domain of CD3-zeta. The "scissors CAR" is constituted of a recognition portion targeting another protein (protein Y) and HIVPR. The HIVPR induces cleavage of the recognition polypeptide sequence in the effector CAR leading to inactivation of the effector CAR when the CAR-T cells contact with cells expressing both protein X and Y (Figure, right panel). 【 Material and Methods 】 For proof of principle, we first constructed "anti-CD19 mCherry CAR" harboring mCherry fluorescence protein in the cytoplasmic region under the HIVPR recognition polypeptide sequence. Also, we constructed "anti-CD19 scissors CAR" and "anti-HER2 scissors CAR". To analyze the target-cell-dependent cleavage of mCherry CAR, 293T cells expressing these CARs were co-cultured with target cells, including K562 (CD19-, HER2-), Raji (CD19+, HER2+), or SK-BR-3 (CD19-, HER2+). To obtain target cells expressing both CD19 and HER2, Raji and SK-BR-3 cells were molecularly manipulated. (1) To evaluate efficiency of this system, after co-cultivation of CAR-transduced 293T cell and target cells (K562, Raji, SK-BR-3), the localization of mCherry was examined under the microscopy and Western blotting. (2) To assess the T cell activation, we constructed "anti-CD19 effector CAR" and established Jurkat cells expressing both the "effector CAR" and the "anti-HER2 scissors CAR". These cells were co-cultured with wild type or engineered Raji or SK-BR-3 cells. T cell activation was analyzed with flowcytometric analysis and IL-2 mRNA expression measured with qRT-PCR. 【 Results 】 (1) Transduced "anti-CD19 mCherry CAR" was detected as a membrane-bound protein in 293T cell. Co-cultivation of 293T cell expressing both the "anti-CD19 mCherry CAR" and "anti-HER2 scissors CAR" with engineered Raji cells expressing both CD19 and HER2 induced cleavage of the recognition site and translocation of the mCherry from the membrane to cytoplasm. In addition, the cleavage was inhibited by a HIVPR inhibitor, Saquinavir. These results suggested that this novel system would regulate CAR-T cell activities through HIVPR-mediated cleavage of the "effector CAR" in vitro. (2) Jurkat cells expressing "anti-CD19 effector CAR" were activated through the target-cell-dependent manner. In addition, "anti-HER2 scissors CAR" attenuated T cell activation driven by "anti-CD19 effector CAR" when Jurkat cells expressing both the "anti-CD19 effector CAR" and the "anti-HER2 scissors CAR" contacted with the target cells expressing both CD19 and HER2. 【Discussion】 This is a novel protease-mediated controllable CAR system. The "scissors CAR" regulated activity of CAR-T cells depending on expression pattern of target molecules on the target cells. Our "Double-Arm" CAR-T cell system (Figure) would improve target specificity. It would attenuate the adverse effects and contribute to expansion in application of CAR-T cell therapy other than B cell malignancies. Figure Disclosures No relevant conflicts of interest to declare.

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.

【Introduction 】 Myeloproliferative neoplasm (MPN) is a heterogeneous group of disorders, including Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF). It is known that mutations of JAK2, CALR, or MPL gene are causative alternations of MPNs. However, 10-15% of MPNs have no mutations in any of these three genes, so-called "triple negative" (TN) MPN. It remains unclear that what genetic alternations cause TN-MPN. Cell lines derived from clonal hematopoietic disorders are useful tools to characterize diseases as well as to identify effective reagents for such diseases. To our knowledge, there is no cell line derived from TN-MPN although there are several lines with JAK2 V617F or CALR mutation. We have encountered an aggressive ET case with TN phenotype. Three years after initial diagnosis, blastic transformation occurred in this patient. Both conventional chemotherapy and allogeneic transplantation failed to cure the patient. After relapse, various therapies including ruxolitinib (JAK inhibitor) failed to control the disease. We have successfully established a cell line from the leukemic cells of this patient. We have also characterized the cell line by whole genome sequencing (WGS), RNA sequencing (RNA-Seq), as well as in vitro assays. 【Material and Methods 】 We have established a novel cell line, TNET-2, from the leukemic cells. WGS and RNA-Seq were performed at Beijing Genome Institute (BGI) Japan. Proliferation assays were performed in the presence or absence of ruxolitinib, AC220 (FLT3 inhibitor), giltertinib (FLT3/AXL inhibitor), etoposide, cytarabine or hydroxycarbamide. 【Results 】 The patient's leukemic cells have been cultured in RPMI1640 culture media with 10% fetal bovine serum and antibiotics. The cells were exponentially growing more than one year and the cell line, TNET-2, has been established. WGS has found 116 missense mutations in 110 genes encoding proteins. Also, five nonsense mutations causing early termination codons were found. InDel mutations causing frame-shifts were found in 28 genes. In-frame deletions were found in seven genes and in-frame insertions were found in nine genes. No mutations were found in JAK2, MPL, or CALR gene. A nonsense mutation was found in ASXL1 gene. Multiple genes encoding proteins associated with myeloid proliferation/differentiation, including CBL, A-RAF, HOXB9 and BCL11B, were mutated. Genes involved in DNA repair, for example ERCC6, were also mutated. Genes involved in apoptosis including CASP7, TRAK1 and SOCS3 were mutated. RNA-Seq revealed high expression of IRAK1 mRNA as well as low expression of TET2. Cell proliferation assays showed TNET-2 cells were resistant to ruxolitinib and conventional chemotherapeutic reagents including etoposide, cytarabine and hydroxycarbamide. However, both AC220 and giltertinib induced cell death. 【Discussion 】 It has been reported that alternations of ASXL1 and/or TET2 genes are common in TN-MPNs. Consistent with the reports, a nonsense mutation of ASXL1 gene and low expression of TET2 were detected in TNET-2 cells. Besides these well-known genetic changes, we have found more than 150 mutated genes. Some of them seem "actionable" / "targetable" mutations. There are no optimal treatments targeting an altered signaling pathway for TN-MPNs. Our novel cell line is a useful tool to characterize this subtype of MPN and find novel treatment options for this rare type of disease. In summary, we have, for the first time, established a cell line derived from TN-MPN and found novel altered signaling pathways by WGS, RNA-Seq and in vitro assays. Disclosures No relevant conflicts of interest to declare.

Chromosome 1p36.23 is frequently deleted in glioblastoma multiforme (GBM). miR-34a localizes in this region. Our experiments found that miR-34a was often deleted and epidermal growth factor receptor (EGFR) was frequently amplified in genomic DNA of 55 GBMs using single-nucleotide polymorphism DNA microarray. Notably, we found that the mean survival time was significantly shortened for patients whose GBMs had both EGFR amplification and miR-34a deletion. Expression of miR-34a was significantly lower in GBM samples compared with normal brain tissue. Forced expression of miR-34a in GBM cells decreased their ability to migrate and profoundly decreased their levels of cyclin-A1, -B1, -D1, and -D3, as well as cyclin-dependent kinase and increased expression of cyclin kinase inhibitor proteins (p21, p27). Also, human GBM cells (U251) stable overexpressing mir-34a formed smaller tumors when growing as xenografts in immunodeficient mice compared with wild-type U251 GBM cells. Furthermore, the protein expression of EGFR decreased in the cells with forced overexpression of miR-34a. Additional studies showed that mir-34a targeted Yin Yang-1 (YY1) and YY1 is a transcription factor that can stimulate the expression of EGFR. Thus, our data suggest that miR-34a acts as a tumor suppressor by inhibiting growth of GBM cells in vitro and in vivo associated with moderating the expression of cell-cycle proteins and EGFR. Moreover, we discovered for the first time that both deletion of miR-34a and amplification of EGFR were associated with significantly decreased overall survival of GBM patients.

[Introduction]Mutations of the calreticulin gene (CALR) are frequently found in patients with myeloproliferative neoplasms (MPN). Five-nucleotide insertion (Ins5) and 52-nucleotide deletion (Del52) in the last exon of the CALR gene are common mutations, generating an altered CALR protein with a mutated peptide sequence at the carboxy end due to the frame-shifts. Cell-lines with such CALR mutations have not been reported yet. CRISPR/Cas9 system is the novel technique commonly used in genetic engineering. CRISPR/Cas9 technology allows us to “edit” genome in mammalian cells. We introduced 46-nucleotide deletion (46Del) in the last exon of the CALR gene in a human erythroleukemia cell line, HEL. This 46Del abnormality lead to generation of an altered CALR protein with the abnormal peptide sequence at the carboxy end as seen in MPN cells with 52Del or Ins5. We compared the CALR-engineered HEL cells with the parental cells in the presence/absence of a Jak2 kinase inhibitor, ruxolitinib (RUX). [Methods and Results]To introduce 46Del abnormality, we have designed two guide RNAs. We co-transfected vectors encoding the Cas9 with a fluorescence protein as a marker as well as the two guide RNAs into HEL cells using nucleoporation. Fluorescence protein positive cells were isolated and the single cells were cultured in 96-well plates for 3 weeks. Genomic DNA of each the clone was extracted, and genomic status of CALR was examined by PCR/sequencing. We have isolated one clone with 46Del (HEL-46DEL), of which expression of abnormal transcripts was confirmed by RT-PCR/sequencing. We also confirmed expressions of abnormal CALR protein by western blot analysis. Since the JAK2-STAT5 pathway is known to be activated in MPN cells, phosphorylation status of STAT5 in HEL and HEL-DEL46 was examined, showing comparable levels in each the line. Proliferation of HEL and HEL-DEL46 was examined with XTT assay, showing that the two lines proliferated similarly. RUX inhibited proliferation of HEL cells as well as HEL-DEL46. HEL cells have a JAK2 mutation which causes constitutive activation of the JAK2-STAT5 pathway. Introduction of the Del46 mutation into HEL cells might not enhance activation of the pathway because the JAK2 mutation already activates the pathway at the maximum level. Citation Format: Shunichiro Yasuda, Satoru Aoyama, Daisuke Watanabe, Norihiko Kawamata. Introduction of CALR mutations to a human erythroleukemia cell line, HEL, by CRISPR/Cas9 systems [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 4338.

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.

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.

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.

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.

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 examining 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 one 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 (3.1Mb, 2 EAC [29%]). Interestingly, one BE sample contained a homozygous deletion spanning 9p22.3-p22.2 (1.2 Mb): this region harbors only one known gene, basonuclin 2 (BNC2). Real-time PCR analysis confirmed deletion of this gene and decreased 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.

金沢大学医薬保健研究域医学系, 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. ©2009 Ferrata Storti Foundation.

Glioblastoma multiforme (GBM) is an extremely malignant brain tumor. To identify new genomic alterations in GBM, genomic DNA of tumor tissue/explants from 55 individuals and 6 GBM cell lines were examined using single nucleotide polymorphism DNA microarray (SNP-Chip). Further gene expression analysis relied on an additional 56 GBM samples. SNP-Chip results were validated using several techniques, including quantitative PCR (Q-PCR), nucleotide sequencing, and a combination of Q-PCR and detection of microsatellite markers for loss of heterozygosity with normal copy number [acquired uniparental disomy (AUPD)]. Whole genomic DNA copy number in each GBM sample was profiled by SNP-Chip. Several signaling pathways were frequently abnormal. Either the p16(INK4A)/p15(INK4B)-CDK4/6-pRb or p14(ARF)-MDM2/4-p53 pathways were abnormal in 89% (49 of 55) of cases. Simultaneous abnormalities of both pathways occurred in 84% (46 of 55) samples. The phosphoinositide 3-kinase pathway was altered in 71% (39 of 55) GBMs either by deletion of PTEN or amplification of epidermal growth factor receptor and/or vascular endothelial growth factor receptor/platelet-derived growth factor receptor α. Deletion of chromosome 6q26-27 often occurred (16 of 55 samples). The minimum common deleted region included PARK2, PACRG, QKI, and PDE10A genes. Further reverse transcription Q-PCR studies showed that PARK2 expression was decreased in another collection of GBMs at a frequency of 61% (34 of 56) of samples. The 1p36.23 region was deleted in 35% (19 of 55) of samples. Notably, three samples had homozygous deletion encompassing this site. Also, a novel internal deletion of a putative tumor suppressor gene, LRP1B, was discovered causing an aberrant protein. AUPDs occurred in 58% (32 of 55) of the GBM samples and five of six GBM cell lines. A common AUPD was found at chromosome 17p13.3-12 (included p53 gene) in 13 of 61 samples and cell lines. Single-strand conformational polymorphism and nucleotide sequencing showed that 9 of 13 of these samples had homozygous p53 mutations, suggesting that mitotic recombination duplicated the abnormal p53 gene, probably providing a growth advantage to these cells. A significantly shortened survival time was found in patients with 13q14 (RB) deletion or 17p13.1 (p53) deletion/AUPD. Taken together, these results suggest that this technique is a rapid, robust, and inexpensive method to profile genome-wide abnormalities in GBM.(Mol Cancer Res 2009;7(5):665–77)

Background T-cell prolymphocytic leukemia is a rare aggressive lymphoproliferative disease with a mature T-cell phenotype and characteristic genomic lesions such as inv(14)(q11q34), t(14;14)(q11;q32) or t(X;14)(q28;q11), mutation of the ATM gene on chromosome 11 and secondary alterations such as deletions of chromosome 8p and duplications of 8q.Design and Methods We analyzed malignant cells from 18 patients with T-cell prolymphocytic leukemia using high density 250K single nucleotide polymorphism arrays and molecular allelokaryotyping to refine understanding of known alterations and identify new target genes.Results Our analyses revealed that characteristic disruptions of chromosome 14 are frequently unbalanced. In the commonly deleted region on chromosome 11, we found recurrent microdeletions targeting the microRNA 34b/c and the transcription factors ETS1 and FLI1. On chromosome 8, we identified genes such as PLEKHA2, NBS1, NOV and MYST3 to be involved in breakpoints. New recurrent alterations were identified on chromosomes 5p, 12p, 13q, 17 and 22 with a common region of acquired uniparental disomy in four samples on chromosome 17q. Single nucleotide polymorphism array results were confirmed by direct sequencing and quantitative real-time polymerase chain reaction.Conclusions The first high density single nucleotide polymorphism array allelokaryotyping of T-cell prolymphocytic leukemia genomes added substantial new details about established alterations in this disease and moreover identified numerous new potential target genes in common breakpoints, deletions and regions of acquired uniparental disomy.

金沢大学医薬保健研究域医学系, Acute promyelocytic leukemia (APL) is a hematopoietic malignant disease characterized by the chromosomal transloca-tion t(15;17), resulting in the formation of the PML-RARA gene. Here, 47 t(15;17) APL samples were analyzed with high-density single-nucleotide polymorphism microarray (50-K and 250-K SNP-chips) using the new algorithm AsCNAR (allele-specific copy-number analysis using anonymous references). Copy-number-neutral loss of heterozygosity (CNN-LOH) was identified at chromosomes 10q (3 cases), 11p (3 cases), and 19q (1 case). Twenty-eight samples (60%) did not have an obvious alteration (normal-copy-number [NC] group). Nineteen samples (40%) showed either one or more genomic abnormalities: 8 samples (17%) had trisomy 8 either with or without an additional duplication, deletion, or CNN-LOH (+8 group); and 11 samples (23%) had genomic abnormalities without trisomy 8 (other abnormalities group). These chromosomal abnormalities were acquired somatic mutations. Interestingly, FLT3-ITD mutations (11/47 cases) occurred only in the group with no genomic alteration (NC group). Taken together, these results suggest that the pathway of development of APL differs in each group: FLT3-ITD, tri-somy 8, and other genomic changes. Here, we showed for the first time hidden abnormalities and novel disease-related genomic changes in t(15;17) APL. © 2009 by The American Society of Hematology.

Molecular-targeted therapy is a hopeful approach for pancreatic cancer. Silencing of tumor suppressor genes can occur by histone deacetylation and/or DNA methylation in the promoter. Here, we identified epigenetically silenced genes in pancreatic cancer cells. Pancreatic cancer cell line, PANC-1 cells were treated either with or without 5Aza-dC (a DNA methyltransferase inhibitor) and suberoylanilide hydroxamic acid (SAHA, a histone deacetylase inhibitor), and mRNA was isolated from these cells. Oligonucleotide microarray analysis revealed that 30 genes including UCHL1, C/EBPalpha, TIMP2 and IRF7 were up-regulated after treatment with 5Aza-dC and SAHA in PANC-1. The induction of these 4 genes was validated by real-time PCR in several pancreatic cancer cell lines. Interestingly, expression of C/EBPalpha was significantly restored in 6 of 6 pancreatic cancer cell lines. Chromatin immunoprecipitation assay revealed that histone H3 of the promoter region of C/EBPalpha was acetylated in PANC-1 treated with SAHA; and bisulfate sequencing showed methylation of its promoter region in several pancreatic cancer cell lines. Forced expression of C/EBPalpha markedly suppressed clonal proliferation of PANC-1 cells. Co-immunoprecipitation assay showed the interaction of C/EBPalpha and E2F1; and the interaction caused the inhibition of E2F1 transcriptional activity. Immunohistochemical analysis revealed that C/EBPalpha localized in the cytoplasm in pancreatic adenocarcinoma cells, whereas it localized predominantly in the nucleus in normal pancreatic cells. Our data demonstrated that aberrant silencing, as well as, inappropriate cytoplasmic localization of C/EBPalpha causes dysregulation of its function, suggesting that C/EBPalpha is a novel candidate tumor suppressor gene in pancreatic cancer cells.

Background Acute myeloid leukemia is a clonal hematopoietic malignant disease; about 45–50% of cases do not have detectable chromosomal abnormalities. Here, we identified hidden genomic alterations and novel disease-related regions in normal karyotype acute myeloid leukemia/myelodysplastic syndrome samples.Design and Methods Thirty-eight normal karyotype acute myeloid leukemia/myelodysplastic syndrome samples were analyzed with high-density single-nucleotide polymorphism microarray using a new algorithm: allele-specific copy-number analysis using anonymous references (AsCNAR). Expression of mRNA in these samples was determined by mRNA microarray analysis.Results Eighteen samples (49%) showed either one or more genomic abnormalities including duplication, deletion and copy-number neutral loss of heterozygosity. Importantly, 12 patients (32%) had copy-number neutral loss of heterozygosity, causing duplication of either mutant FLT3 (2 cases), JAK2 (1 case) or AML1/RUNX1 (1 case); and each had loss of the normal allele. Nine patients (24%) had small copy-number changes (< 10 Mb) including deletions of NF1, ETV6/TEL, CDKN2A and CDKN2B. Interestingly, mRNA microarray analysis showed a relationship between chromosomal changes and mRNA expression levels: loss or gain of chromosomes led, respectively, to either a decrease or increase of mRNA expression of genes in the region.Conclusions This study suggests that at least one half of cases of normal karyotype acute myeloid leukemia/myelodysplastic syndrome have readily identifiable genomic abnormalities, as found by our analysis; the high frequency of copy-number neutral loss of heterozygosity is especially notable.

Asparagine synthetase (ASNS) is an enzyme expressed ubiquitously in mammalian cells. Here, we discovered two 14-bp tandem repeat (2R, wild-type) sequences in the first intron of the gene. The 14-bp sequence is similar to the three GC-boxes (GC-I, -II, and -III) found in the promoter region of the ASNS gene, as well as, the binding site of transcription factor Sp-1. Approximately 75% of acute lymphoblastic leukemia (ALL) samples had the 2R sequence in both allele; however, 20% and 3% ALL samples had three (3R) and four (4R) 14-bp tandem repeats in one allele, respectively; the other allele had 2R. The tandem repeat sequence was not specific to the leukemia cells but represents a novel germline polymorphism. Interestingly, the 14-bp sequence functioned as a transcriptional enhancer element as shown by reporter analysis and formed a protein-DNA complex in vitro. Our data for the first time show that the ASNS gene has tandem repeated sequences as a polymorphism, and it can function as a transcriptional element; increased number of tandem repeat producing increased activity. Clinical significance in ALL requires further studies.

Purpose: The importance of the BRCA gene products in maintaining genomic stability led us to hypothesize that BRCA-associated and sporadic ovarian cancers would have distinctive genetic profiles despite similarities in histologic appearance.Experimental Design: A whole-genome copy number analysis of fresh, frozen, papillary serous ovarian cancer DNA was done using the Affymetrix 50K Xba Mapping Array using each patient's normal genomic DNA as the matched control. Loss of heterozygosity and copy number abnormalities were summarized to define regions of amplification, deletion, or uniparental disomy (UPD), defined as loss of one allele and duplication of the remaining allele. Genomic abnormalities were compared between BRCA-associated and sporadic tumors.Results: We compared 6 BRCA-associated with 14 sporadic papillary serous ovarian carcinomas. Genetic instability, measured by percentage of genome altered, was more pronounced in BRCA-associated tumors (median, 86.6%; range, 54-100%) than sporadic tumors (median, 43.6%; range, 2-83%; P = 0.009). We used frequency plots to show the proportion of cases affected by each type abnormality at each genomic region. BRCA-associated tumors showed genome-wide loss of heterozygosity primarily due to the occurrence of UPD rather than deletion. UPD was found in 100% of the BRCA-associated and 50% of the sporadic tumors profiled.Conclusions: This study reports on a previously underappreciated genetic phenomenon of UPD, which occurs frequently in ovarian cancer DNA. We observed distinct genetic patterns between BRCA-associated and sporadic ovarian cancers, suggesting that these papillary serous tumors arise from different molecular pathways.

Purpose To identify a biomarker of ovarian cancer response to chemotherapy. Patients and Methods Study participants had epithelial ovarian cancer treated with surgery followed by platinum-based chemotherapy. DNA and RNA were isolated from frozen tumors and normal DNA was isolated from matched peripheral blood. A whole-genome loss of heterozygosity (LOH) analysis was performed using a high-density oligonucleotide array. Candidate genomic areas that predicted enhanced response to chemotherapy were identified with Cox proportional hazards methods. Gene expression analyses were performed through microarray experiments. Candidate genes were tested for independent effects on survival using Cox proportional hazards models, Kaplan-Meier survival curves, and the log-rank test. Results Using a whole-genome approach to study the molecular determinants of ovarian cancer response to platinum-based chemotherapy, we identified LOH of a 13q region to predict prolonged progression-free survival (PFS; hazard ratio, 0.23; P = .006). ERCC5 was identified as a candidate gene in this region because of its known function in the nucleotide excision repair pathway, the unique DNA repair pathway that removes platinum-DNA adducts. We found LOH of the ERCC5 gene locus and downregulation of ERCC5 gene expression to predict prolonged PFS. Integration of genomic and gene expression data shows a correlation between 13q LOH and ERCC5 gene downregulation. Conclusion ERCC5 is a novel biomarker of ovarian cancer prognosis and a potential therapeutic target of ovarian cancer response to platinum chemotherapy.

Mantle cell lymphoma (MCL) has a chromosomal translocation resulting in the expression of the cyclin D1 gene driven by the powerful enhancer of the immunoglobulin heavy chain gene, leading to uncontrolled, overexpressed cyclin D1 protein. We showed that suberoylanilide hydroxamic acid (SAHA; vorinostat), one of the histone deacetylase inhibitors derived from hydroxamic acid, caused a dramatic decrease (90%) in protein levels of cyclin D1 after 8-hour exposure to SAHA (5 μM) in MCL lines (SP49, SP53, Jeko1). mRNA levels and protein stability of cyclin D1 were minimally affected by SAHA over 8 hours. In contrast, metabolic labeling assays showed that SAHA decreased incorporation of [35S]methionine into cyclin D1 protein. The drug also decreased levels of phosphorylated Akt, mammalian target of Rapamycin (mTOR), and eukaryotic translation initiation factor 4E binding protein (eIF4E-BP) and lowered the cap site binding activity of eIF4E in the MCL cells. In vitro phosphatidyl inositol (PI) kinase assay demonstrated that SAHA directly inhibited kinase activity of PI 3′ kinase. Taken together, SAHA caused a rapid decrease of cyclin D1 in MCL by blocking the translation of cyclin D1 by inhibiting the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR/eIF4E-BP pathway, probably by PI3K inhibition.

Purpose: Histone acetylation is one of the main mechanisms involved in regulation of gene expression. During carcinogenesis, tumor-suppressor genes can be silenced by aberrant histone deacetylation. This epigenetic modification has become an important target for tumor therapy. The histone deacetylation inhibitor, suberoylanilide hydroxamic acid (SAHA), can induce growth arrest in transformed cells. The aim of this study is to examine the effects of SAHA on gene expression and growth of glioblastoma multiforme (GBM) cells in vitro and in vivo. Experimental Design: The effect of SAHA on growth of GBM cell lines and explants was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Changes of the cell cycle and relative gene expression were detected by fluorescence-activated cell sorting, real-time reverse transcription-PCR, and Western blotting. After glioma cells were implanted in the brains of mice, the ability of SAHA to decrease tumor growth was studied. Results: Proliferation of GBM cell lines and explants were inhibited in vitro by SAHA (ED50, 2 × 10−6 to 2 × 10−5 mol/L, 5 days). SAHA exposure of human U87 and T98G glioma cell lines, DA66 and JM94 GBM explants, as well as a murine GL26 GBM cell line resulted in an increased accumulation of cells in G2-M of the cell cycle. Many proapoptotic, antiproliferative genes increased in their expression (DR5, TNFα, p21WAF1, p27KIP1), and many antiapoptotic, progrowth genes decreased in their levels (CDK2, CDK4, cyclin D1, cyclin D2) as measured by real-time reverse transcription-PCR and/or Western blot after these GBM cells were cultured with SAHA (2.5 × 10−6 mol/L, 1 day). Chromatin immunoprecipitation assay found that acetylation of histone 3 on the p21WAF1 promoter was markedly increased by SAHA. In vivo murine experiments suggested that SAHA (10 mg/kg, i.v., or 100 mg/kg, i.p.) could cross the blood-brain barrier as shown by prominent increased levels of acetyl-H3 and acetyl-H4 in the brain tissue. Furthermore, the drug significantly (P < 0.05) inhibited the proliferation of the GL26 glioma cells growing in the brains of mice and increased their survival. Conclusions: Taken together, SAHA can slow the growth of GBM in vitro and intracranially in vivo. SAHA may be a welcome addition for the treatment of this devastating disease.

BACKGROUND Human epidermal growth factor receptor 2 (HER2) is overexpressed in 25–30% of ovarian carcinoma cases and a correlation between increased HER2 expression and decreased survival has been demonstrated. HER2 is a ligand-less member of the HER family that functions as the preferred coreceptor for epidermal growth factor receptor (EGFR), HER3, and HER4. METHODS An approach was developed to target HER2's role as a coreceptor using a monoclonal antibody, 2C4, which sterically hinders HER2's recruitment into a functional HER complex. RESULTS HER2 was robustly expressed in all eight ovarian carcinoma cell lines; expression of EGFR and HER3 was variable. Even though four of the eight cell lines responded to EGF, 2C4 antibody moderately inhibited in vitro proliferation of only two cell lines (OVCA433 and SK-OV-3). Furthermore, ligand-stimulated p-MAPK expression was inhibited by 2C4 only in these two cell lines after exposure to EGF. Immunoprecipitation and eTag analysis revealed that OVCA433 expressed heterodimers of EGFR/HER2, and these heterodimers were disrupted after treatment with 2C4, whereas OVCA432 cells did not have these heterodimers. In murine xenograft experiments, the in vivo growth of OVCA433, but not of OVCA432, ovarian carcinoma cells was significantly inhibited by 2C4 treatment of the mice. CONCLUSION 2C4 is able to disrupt the HER signaling pathway and inhibit the in vitro and in vivo growth of ovarian carcinoma cell lines. The response appears limited to lines in which HER2 heterodimers were able to transduce proliferative signals. Our findings suggest a strong rationale to conduct clinical trials of 2C4 in a subset of patients with ovarian tumors. Cancer 2005. © 2005 American Cancer Society.

Udgivelsesdato: Aug Dlk1 (Pref-1) is a transmembrane and secreted protein, which is a member of the epidermal growth factor-like family, homologous to Notch/Delta/Serrate. We have found by real-time RT-PCR that Dlk1 mRNA levels were high in CD34(+) cells in 10 of 12 MDS samples compared with CD34(+) cells from 11 normals. Also, Dlk1 mRNA was elevated in mononuclear, low density bone marrow cells from 11/38 MDS patients, 5/11 AML M6 and 2/4 AML M7 samples. Furthermore, 5/6 erythroleukemia and 2/2 megakaryocytic leukemia cell lines highly expressed Dlk1 mRNA. Levels of Dlk1 mRNA markedly increased during megakaryocytic differentiation of both CMK megakaryoblasts as well as normal CD34(+) hematopoietic stem cells. High serum levels of Dlk1 occurred in RA (4/10) and essential thrombocythemia (2/10) patients. Functional studies showed that forced expression of Dlk1 enhanced proliferation of K562 cells growing in 1% fetal bovine serum. Analysis of hematopoiesis of Dlk1 knockout mice suggested that Dlk1 contributed to granulocyte, megakaryocyte and B-cell clonogenic growth and was needed for generation of splenic B-cells. In summary, Dlk1 is overexpressed in selected samples of MDS (especially RA and RAEB) and AML (particularly M6, M7), and it appears to be associated with normal development of megakaryocytes and B cells.

Natural killer (NK) cell disorders are rare diseases. Genetic abnormalities of the several tumor suppressor genes, including p15INK4B, p16INK4A/p14ARF, p53, p73, and Rb genes have been reported. Deletions and point mutations of these genes are frequently detected in these diseases. It has been reported that tumor suppressor genes are inactivated by DNA methylation of the promoter region and/or first exon of the genes in a variety of human cancers. In this study we analyze the methylation status of the genes associated with cell cycle regulation, including p16INK4A, p15INK4B, p21/Waf1/Cip1, p27/Kip1, p73, and p14ARF, by methylation specific (MS) PCR and/or bisulfite sequencing. We examined 29 cases of NK cell disorders (five aggressive NK cell leukemia/lymphoma, three blastic NK cell lymphoma/leukemia, five nasal NK cell lymphoma, three myeloid/NK cell precursor acute leukemia, 13 chronic NK lymphocytosis). We found methylation of the first exon of the p16INK4A gene in two cases (one aggressive, one blastic), and methylation of the p14ARF gene in one aggressive NK cell leukemia. Bisulfite sequencing revealed that methylation of the p15 and p27 genes was rare in these disorders. MS-PCR suggested that the p73 and p21 genes were methylated in seven cases, respectively (p73: one blastic, one nasal, five chronic; p21: one myeloid/NK, one aggressive, one nasal, and four chronic); bisulfite sequencing confirmed that methylated alleles of these genes were dominant in the samples except three cases (one myeloid/NK, one aggressive, and one chronic) in which methylated alleles of the p21 genes were less than 34% of all alleles. These results suggested that inactivation of the cell cycle regulatory genes by DNA methylation could be associated with tumorigenesis in NK cell disorders, not only aggressive subtypes but also chronic subtype.