Huan-Chau Lin

Upfront maintenance therapy with arsenic trioxide in acute promyelocytic leukemia provides no benefit for non-t(15;17) subtype.

Aims:  The optimal maintenance therapy for patients with acute promyelocytic leukemia (APL) who achieved complete remission (CR) and complete consolidation chemotherapy is still controversial. Whether the use of arsenic trioxide (ATO) alone or along with all‐trans retinoic acid (ATRA) improves overall survival (OS) or disease‐free survival (DFS) is still debated.

Germline variations at JAK2 , TERT , HBS1L - MYB and MECOM and the risk of myeloproliferative neoplasms in Taiwanese population

Germline variations at JAK2, TERT, HBS1L-MYB and MECOM have been found to associate with myeloproliferative neoplasms (MPNs) in European populations. Whether these germline variations are associated with MPNs in Taiwanese population is obscure. Here we aimed to evaluate the association of five germline variations (JAK2 46/1 haplotype tagged by rs12343867, JAK2 intron 8 rs12339666, TERT rs2736100, HBS1L-MYB rs9376092 and MECOM rs2201862) and the risk of MPNs in Taiwanese population. A total of 178 MPN patients (109 essential thrombocythemia, 54 polycythemia vera and 15 primary myelofibrosis) were enrolled into this study. The information of 17033 control subjects was obtained from Taiwan Biobank database. The JAK2 46/1 haplotype, JAK2 rs12339666 and TERT rs2736100 were significantly associated with Taiwanese MPNs (P = 3.6×10-19, 1.9×10-19 and 3.1×10-6, respectively), and JAK2V617F-positive MPNs (n=121) (P = 5.6×10-21, 4.4×10-21 and 8.6×10-7, respectively). In JAK2V617F-negative cases (n=55), only the JAK2 46/1 haplotype and JAK2 rs12339666 remained statistically significant (P= 0.009 and 0.007, respectively). When stratified by disease subtypes, the JAK2 46/1 haplotype and JAK2 rs12339666 were significantly associated with all three MPN subtypes, but TERT rs2736100 was only associated with essential thrombocythemia and polycythemia vera. We did not find any association of these five SNPs with CALR mutations in our cohort. Furthermore, the risk alleles of MECOM rs2201862 and HBS1L-MYB rs9376092 were demonstrated to be negatively associated with the risk of developing polycythemia vera. In conclusion, germline variations at JAK2 (both the 46/1 haplotype and rs12339666) and TERT rs2736100 were associated with MPNs in Taiwanese population.Germline variations at JAK2, TERT, HBS1L-MYB and MECOM have been found to associate with myeloproliferative neoplasms (MPNs) in European populations. Whether these germline variations are associated with MPNs in Taiwanese population is obscure. Here we aimed to evaluate the association of five germline variations (JAK2 46/1 haplotype tagged by rs12343867, JAK2 intron 8 rs12339666, TERT rs2736100, HBS1L-MYB rs9376092 and MECOM rs2201862) and the risk of MPNs in Taiwanese population. A total of 178 MPN patients (109 essential thrombocythemia, 54 polycythemia vera and 15 primary myelofibrosis) were enrolled into this study. The information of 17033 control subjects was obtained from Taiwan Biobank database. The JAK2 46/1 haplotype, JAK2 rs12339666 and TERT rs2736100 were significantly associated with Taiwanese MPNs (P = 3.6x10-19, 1.9x10-19 and 3.1x10-6, respectively), and JAK2V617F-positive MPNs (n=121) (P = 5.6x10-21, 4.4x10-21 and 8.6x10-7, respectively). In JAK2V617F-negative cases (n=55), only the JAK2 46/1 haplotype and JAK2 rs12339666 remained statistically significant (P= 0.009 and 0.007, respectively). When stratified by disease subtypes, the JAK2 46/1 haplotype and JAK2 rs12339666 were significantly associated with all three MPN subtypes, but TERT rs2736100 was only associated with essential thrombocythemia and polycythemia vera. We did not find any association of these five SNPs with CALR mutations in our cohort. Furthermore, the risk alleles of MECOM rs2201862 and HBS1L-MYB rs9376092 were demonstrated to be negatively associated with the risk of developing polycythemia vera. In conclusion, germline variations at JAK2 (both the 46/1 haplotype and rs12339666) and TERT rs2736100 were associated with MPNs in Taiwanese population.

Targeted next-generation sequencing identified novel mutations in triple-negative myeloproliferative neoplasms

Mutations in JAK2, MPL and CALR genes have been identified in the majority of myeloproliferative neoplasm (MPN) patients, and patients negative for these three mutations are the so-called triple-negative (TN) MPN. In this study, we examined the mutational profiles of 16 triple-negative MPN patients including 7 essential thrombocythemia (ET), 1 primary myelofibrosis and 8 polycythemia vera (PV). Targeted next-generation sequencing was performed using the ACTOnco Comprehensive Cancer Panel (Ion AmpliSeq Comprehensive Cancer Panel, Life Technologies) to target all coding exons of 409 cancer-related genes. Overall, 30 nonsynonymous somatic mutations were detected in 12 (75%) patients with a range of 1–5 mutations per sample. Notably, one ET patient was found to have JAK2V617F and KITP551L mutations at very low allele frequency. One MPLP70L and 1 MPLM602T mutations were identified each in 1 ET and 1 PV, respectively. Other recurrent mutations were also identified including KMT2C, KMT2D, IRS2, SYNE1, PDE4DIP, SETD2, ATM, TNFAIP3 and CCND2. In addition, germline mutations were also found in some cancer-related genes. Copy number changes were rare in this cohort of TN MPNs. In conclusion, both somatic and germline mutations can be detected in TN MPN patients.

Epidermal growth factor induces STAT1 expression to exacerbate the IFNr-mediated PD-L1 axis in epidermal growth factor receptor-positive cancers

The epidermal growth factor (EGF) receptor (EGFR) overexpressed in many cancers, including lung and head and neck cancers, and is involved in cancer cell progression and survival. PD‐L1, increases in tumor cells to evade and inhibit CD8+ T cells, is a clinical immunotherapeutic target. This study investigated the molecular mechanism of EGF on regulating PD‐L1 in EGFR‐positive cancers and determined potential agents to reduce PD‐L1 expression. RNA sequencing (RNAseq) and bioinformatics analysis were performed to determine potential driver genes that regulate PD‐L1 in tumor cells‐derived tumorspheres which mimicking cancer stem cells. Then, the specific inhibitors targeting EGFR were applied to reduce the expression of PD‐L1 in vitro and in vivo. We validated that EGF could induce PD‐L1 expression in the selected EGFR‐positive cancers. RNAseq results revealed that STAT1 increased as a driver gene in KOSC‐3‐derived tumorspheres; these data were analyzed using PANTHER followed by NetworkAnalyst. The blockade of EGFR by afatinib resulted in decreased STAT1 and IRF‐1 levels, both are transcriptional factors of PD‐L1, and disabled the IFNr‐STAT1‐mediated PD‐L1 axis in vitro and in vivo. Moreover, STAT1 knockdown significantly reduced EGF‐mediated PD‐L1 expression, and ruxolitinib, a JAK1/JAK2 inhibitor, significantly inhibited STAT1 phosphorylation to reduce the IFNr‐mediated PD‐L1 axis. These results indicate that EGF exacerbates PD‐L1 by increasing the protein levels of STAT1 to enforce the IFNr‐JAK1/2‐mediated signaling axis in selected EGFR‐positive cancers. The inhibition of EGFR by afatinib significantly reduced PD‐L1 and may be a potential strategy for enhancing immunotherapeutic efficacy.

Increased B cell activation is present in JAK2 V617F-mutated, CALR -mutated and triple-negative essential thrombocythemia

Essential thrombocythemia (ET) is a BCL-ABL1-negative myeloproliferative neoplasm. We have reported that increased activated B cells can facilitate platelet production mediated by cytokines regardless JAK2 mutational status in ET. Recently, calreticulin (CALR) mutations were discovered in ~30% JAK2/MPL-unmutated ET and primary myelofibrosis. Here we sought to screen for CALR mutations and to evaluate B cell immune profiles in a cohort of adult Taiwanese ET patients. B cell populations, granulocytes/monocytes membrane-bound B cell-activating factor (mBAFF) levels, B cells toll-like receptor 4 (TLR4) expression and intracellular levels of interleukin (IL)-1β/IL-6 and the expression of CD69, CD80, and CD86 were quantified by flow cytometry. Serum BAFF concentration was measured by ELISA. 48 healthy adults were used for comparison. 19 (35.2%) of 54 ET patients harbored 8 types of CALR exon 9 mutations including 4 (7.4%) patients with concomitant JAK2V617F mutations. Compared to JAK2V617F mutation, CALR mutations correlated with younger age at diagnosis (p=0.04), higher platelet count (p=0.004), lower hemoglobin level (p=0.013) and lower leukocyte count (p=0.013). Multivariate analysis adjusted for age, sex, follow-up period and hematological parameters confirmed that increased activated B cells were universally present in JAK2-mutated, CALR-mutated and triple-negative ET patients when compared to healthy adults. JAK2- and CALR-mutated ET have significantly higher fraction of B cells with TLR4 expression when compared to triple-negative ET (p=0.019 and 0.02, respectively). CALR-mutated ET had significantly higher number of CD69-positive activated B cells when compared to triple-negative ET (p=0.035). In conclusion, increased B cell activation is present in ET patients across different mutational subgroups.

STAT3 exacerbates survival of cancer stem-like tumorspheres in EGFR-positive colorectal cancers: RNAseq analysis and therapeutic screening

BackgroundCancer stem cells are capable of undergoing cell division after surviving cancer therapies, leading to tumor progression and recurrence. Inhibitory agents against cancer stem cells may be therapeutically used for efficiently eradicating tumors. Therefore, the aim of this study was to identify the relevant driver genes that maintain cancer stemness in epidermal growth factor receptor (EGFR)-positive colorectal cancer (CRC) cells and to discover effective therapeutic agents against these genes.MethodsIn this study, EGFR-positive cancer stem-like cells (CSLCs) derived from HCT116 and HT29 cells were used as study models for in vitro inductions. To identify the differential genes that maintain CSLCs, RNAseq analysis was conducted followed by bioinformatics analysis. Moreover, a panel containing 172 therapeutic agents targeting the various pathways of stem cells was used to identify effective therapeutics against CSLCs.ResultsRNAseq analysis revealed that 654 and 840 genes were significantly upregulated and downregulated, respectively, in the HCT116 CSLCs. Among these genes, notably, platelet-derived growth factor A (PDGFA) and signal transducer and activator of transcription 3 (STAT3) were relevant according to the cancer pathway analyzed using NetworkAnalyst. Furthermore, therapeutic screening revealed that the agents targeting STAT3 and Wnt signaling pathways were efficient in reducing the cell viabilities of both HCT116 and HT29 cells. Consequently, we discovered that STAT3 inhibition using homoharringtonine and STAT3 knockdown significantly reduced the formation and survival of HT29-derived tumorspheres. We also observed that STAT3 phosphorylation was regulated by epidermal growth factor (EGF) to induce PDGFA and Wnt signaling cascades.ConclusionsWe identified the potential genes involved in tumorsphere formation and survival in selective EGFR-positive CRCs. The results reveal that the EGF-STAT3 signaling pathway promotes and maintains CRC stemness. In addition, a crosstalk between STAT3 and Wnt activates the Wnt/β-catenin signaling pathway, which is also responsible for cancer stemness. Thus, STAT3 is a putative therapeutic target for CRC treatment.

Abstract 934: EGFR induces ILF3 and G9a expressions to maintain Oct4-mediated stemness property in lung cancer

Cancer stem cells survive and lead to tumor recurrence in tumor therapeutic treatments. Lung cancer cells highly express epidermal growth factor receptor (EGFR), which is critical for tumor progression. We hypothesized that EGFR may play a pivotal role in the induction of cancer stemness cells (CSCs). The aim of this study attempted to investigate the molecular mechanism of EGFR-mediated cancer stemness, and to discover the potent therapeutic agents. A high expression level of cancer stemness markers, ALDH1, CD133, Oct4, and Nanog was observed in tumorspheres derived from HCC827. External EGF further elevated the expression of Oct4 in HCC827 cells. Moreover, chemical inhibition and genetic knockdown of EGFR reduced the Oct4 expression and also tumorsphere formation. We found that inhibitors targeting to EGFR (afatinib), ILF3 (YM155), and G9a (UNC0642) are potential to reduce the formations of tumorspheres. The results revealed that EGFR triggered the formation of tumorspheres through elevating the activations of G9a-mediated stemness genes. In addition, YM155 effectively inhibited tumor growth and formation of tumorspheres as an anti-lung cancer agent blocking the EGFR-G9a cell initiating program pathway. This study demonstrated that EGFR participated in maintain of cancer stemness property through expressing ILF3 and G9a expressions. YM155 inhibited the EGFR-G9a stemness pathway, and reduced the formation of tumorspheres as a potent anti-lung cancer agent. Citation Format: Chun-Chia Cheng, Jungshan Chang, Huan-Chau Lin, Ai-Sheng Ho, Ken-Hong Lim, Chun-Chao Chang, Yi-Fang Chang, Cheng-Wen Wu. EGFR induces ILF3 and G9a expressions to maintain Oct4-mediated stemness property in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 934. doi:10.1158/1538-7445.AM2017-934

Targeted re-sequencing of TET2 in Taiwanese patients with myelodysplastic/myeloproliferative neoplasms.

The myelodysplastic/myeloproliferative neoplasms (MDS/ MPN) are clonal myeloid neoplasms that overlap the myelodysplastic syndrome (MDS) and MPN categories. At the time of initial diagnosis, MDS/MPN exhibit some clinical, laboratory, or morphologic findings supporting the diagnosis of MDS and other proliferative features consistent with MPN. These disorders include chronic myelomonocytic leukemia (CMML), atypical chronic myeloid leukemia (BCR-ABL1 negative), juvenile myelomonocytic leukemia, and MDS/MPN-unclassifiable (MDS/MPN-U). Many cytogenetic and molecular abnormalities have been identified in MDS/MPN and implicate a wide range of molecular mechanisms in the pathogenesis of this disease [1]. The teneleven translocation 2 gene (TET2) is located at chromosome 4q24. TET2 methylcytosine dioxygenase enzymatically catalyzes the conversion of methylcytosine to 5-hydroxymethylcytosine. Recent studies have shown that TET2 mutations are important in the pathogenesis and treatment of MDS and MDS/MPN. To screen for TET2 mutations, we carried out next-generation sequencing using the Illumina MiSeq platform in ten Taiwanese patients with MDS/MPN (five CMML, three CMML in blastic transformation and three MDS/MPN-U). All coding regions of the TET2 gene were sequenced. The screening of mutation was approved by the Institutional Review Board of the MacKay Memorial Hospital, and all patients provided written informed consent. Genomic DNA derived from bone marrow, bone marrow granulocytes, peripheral whole blood, or peripheral blood granulocytes was used for the screening. We processed sequencing data using BWA alignment and GATK variant calling on Illumina’s BaseSpace platform. Variants were considered if consistent with the following criteria: absent from dbSNP database, NGS coverage[ 100 9 and variant frequency[ 10 %. We identified three single nucleotide variants, one insertion and one deletion in four patients (40 %) (Table 1). Three variants were predicted to cause TET2 truncation. Our results confirmed that TET2 is frequently mutated in Taiwanese patients with CMML. However, TET2 mutations did not impact either the overall survival or leukemia-free survival in CMML [2]. In contrast, ASXL1 mutations (nonsense and frameshift) have been shown to negatively impact the overall survival in CMML and have been incorporated into prognostic models [2, 3]. Our study was limited by small case number, and larger study is warranted to determine the prognostic role of TET2 mutations in Taiwanese patients with CMML.

The Successful Closure of Jejunostomy in Sigmoid Colon Cancer with Advanced Intra-Abdominal Carcinomatosis Rescued by Cytoreductive Surgery and Systemic Chemotherapy

Intra-abdominal carcinomatosis with intestinal obstruction is usually a terminal and refractory event. A 52-year-old woman underwent cytoreductive surgery and jejunostomy following a diagnosis of ileus obstruction due to recurrent sigmoid colon adenocarcinoma with advanced intra-abdominal carcinomatosis. She was further treated with biweekly chemotherapy with Oxaliplatin plus 5-FU (FOLFOX regimen). After 4 cycles of treatment of FOLFOX post-operatively, the patient started oral intake with tapered TPN. Despite good oral intake, the patient was dependent on large amount of intravenous fluid at home due to massive fluid loss from jejunostomy. After two more cycles of FOLFOX, and complete abdominal workup indicating good bowel movement, the patient had successful closure of the jejunostomy and became independent of intravenous fluid supplement. Our experience suggested aggressive multi-modality approach may be helpful in this difficult clinical situation. Meanwhile, early closure of enterostomy to assure quality of life in cancer patients is also feasible.