AIM: To evaluate occurrence, variety, structural peculiarities and prognostic meaning of cytogenetic abnormalities in adult patients with Ph-negative acute lymphoblastic leukemia (ALL) receiving therapy according to ALL-2009 protocol.
MATERIALS AND METHODS: The study included 115 adult patients with firstly diagnosed Ph-negative ALL: 58 male and 57 female aged from 15 to 61 years (mean age 26.5 years), who underwent treatment from September 2009 to September 2015 in National Medical Research Center for Hematology MH RF (n=101) and in hematology departments of regional hospitals (n=14). All patients received therapy of ALL-2009 protocol (ClinicalTrials.gov, NCT01193933). The median follow-up was 24.5 months (0.2-94.4 months). As a part of the study results of a standard cytogenetic assay (SCA) were analyzed and fluorescence hybridization in situ (FISH) with the use of DNA-probes was performed on archived biological material for structural changes in gene locuses MLL/t(11q23), с-MYC/t(8q24), TP53/ deletion 17p13, CDKN2A/ deletion 9p21, translocation t(1;19)/E2A-PBX1 и t(12;21)/ETV6-RUNX1; iAMP21 identification.
RESULTS: Karyotype was defined using SCA in 86% of patients. Normal karyotype was found in 48.5% of them, chromosome aberrations in 51.5% (structural changes were found in 19.2%, hyperploidy in 27.2%, and hypoploidy in 5.1%). In 17.2% of patients complex karyotype abnormalities were found. With the use of FISH technique aberrations were found in 67% of patients: 9p21/CDKN2A deletion in 24.3%, MLL/t(11q23) gene abnormalities in 7.8%, 17p13/TP53 deletion in 5.2%, abnormalities of c-MYC/t(8q24) in 1.7%, t(1;19)/E2A-PBX1 in 0.8%, and iAMP21 in 0.8%, other abnormalities (additional signals/absence of signals from gene locuses) in 26.4%, t(12;21)/ETV6-RUNX1 was not found. FISH technique use in addition to SCA allows to increase aberrant karyotype location from 51.5 to 67%. A statistically significant correlation of 9p21/CDKN2A deletion with high serum lactate dehydrogenase activity (p=0.02); MLL/t(11q23) gene abnormalities - with leucocytosis and high blast cells level in blood (p=0.0016), hyperploidy - with normal leukocyte count (p=0.02) was shown. In groups with different cytogenetic abnormalities no statistically significant differences of treatment with ALL-2009 protocol were found (in terms of complete remission, early mortality and treatment resistance). When connection of cytogenetic abnormalities and their combinations with long-term results were analyzed according to ALL-2009 protocol, only two characteristics - MLL/t(11q23) and c MYC/t(8q24) gene abnormalities had a statistically significant influence on disease-free survival (HR - 176.9; p<0.0001) and chance of recurrence (HR - 6.4; p=0.02).
CONCLUSION: Adverse prognostic factors in terms of therapeutic management provided in ALL-2009 protocol were MLL/t(11q23) and с-MYC/t(8q24) genes abnormalities. CDKN2A/9p21 and TP53/17p13 genes deletions, quantative and complex karyotype abnormalities were not prognostic factors in adult patients with Ph-negative ALL in ALL-2009 protocol use.

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) with TCF3-PBX1 fusion gene expression has constitutively elevated levels of Wnt16b and ROR1 (receptor tyrosine kinase-like orphan receptor), a ligand and a receptor from the Wnt signaling pathway, respectively. Although survival rate is usually high after the initial chemotherapy, many TCF3-PBX1 BCP-ALL patients relapse and subsequently develop treatment resistance, resulting in poor prognosis. Here, we aimed to investigate the molecular signaling associated with Wnt16b and ROR1 overexpression in TCF3-PBX1 cell lines and primary samples, and to identify effective treatment options via ROR1 targeting. We detected higher ROR1 expression on TCF3-PBX1 leukemic cells even at a later stage of patient relapse, providing a strong rationale for the use of ROR1-targeted therapy. We found that Wnt5a-ROR1 signaling enhances proliferation of TCF3-PBX1 cells via RhoA/Rac1 GTPases activation and STAT3 upregulation. Wnt16b also activated the RhoA/Rac1 signaling cascade suggesting the activation of a non-canonical Wnt pathway in TCF3-PBX1 cells. Wnt16 could interact with ROR1 but not in TCF3-PBX1 cells, suggesting that Wnt5a is the ligand signaling via ROR1 in TCF3-PBX1 cells. By high throughput drug-sensitivity testing of TCF3-PBX1 cells before and after ROR1 knockdown we found that targeting ROR1 significantly improves the therapeutic efficacy of Bcl-2 family inhibitors venetoclax and navitoclax, and this synergism was confirmed ex vivo using a drug-resistant primary sample from a relapsed TCF3-PBX1 patient. Our work underlines a new type of targeted combination therapy that could be clinically advantageous for patients with TCF3-PBX1 BCP-ALL.

BACKGROUND: Chromosome translocations are a hallmark of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Additional genomic aberrations are also crucial in both BCP-ALL leukemogenesis and treatment management. Herein, we report the phenotypic and molecular cytogenetic characterization of an extremely rare case of BCP-ALL harboring two concomitant leukemia-associated chromosome translocations: t(1;19)(q23;q13.3) and t(9;17)(p13;q11.2). Of note, we described a new rearrangement between exon 6 of PAX5 and a 17q11.2 region, where intron 3 of SPECC1 is located. This rearrangement seems to disrupt PAX5 similarly to a PAX5 deletion. Furthermore, a distinct karyotype between diagnosis and relapse samples was observed, disclosing a complex clonal evolution during leukemia progression.
CASE PRESENTATION: A 16-year-old boy was admitted febrile with abdominal and joint pain. At clinical investigation, he presented with anemia, splenomegaly, low white blood cell count and 92% lymphoblast. He was diagnosed with pre-B ALL and treated according to high risk GBTLI-ALL2009. Twelve months after complete remission, he developed a relapse in consequence of a high central nervous system and bone marrow infiltration, and unfortunately died.
CONCLUSIONS: To our knowledge, this is the first report of a rearrangement between PAX5 and SPECC1. The presence of TCF3-PBX1 and PAX5-rearrangement at diagnosis and relapse indicates that both might have participated in the malignant transformation disease maintenance and dismal outcome.

Most B cell precursor acute lymphoblastic leukemia (BCP ALL) can be classified into known major genetic subtypes, while a substantial proportion of BCP ALL remains poorly characterized in relation to its underlying genomic abnormalities. We therefore initiated a large-scale international study to reanalyze and delineate the transcriptome landscape of 1,223 BCP ALL cases using RNA sequencing. Fourteen BCP ALL gene expression subgroups (G1 to G14) were identified. Apart from extending eight previously described subgroups (G1 to G8 associated with

Fusion genes are major molecular biological abnormalities in hematological malignancies. To depict the common recurrent gene-fusion landscape in acute lymphoblastic leukemia (ALL), 36 recurrent fusion genes in hematologic malignancies were assessed using multiplex-nested RT-PCR in 2479 patients with de novo ALL. 17 kinds of distinct fusion genes were detected in 712 (28.72%) cases. Co-occurrence of different fusion genes was observed in 6 (0.24%) patients. Incidence of fusion genes in B-ALL was significantly higher than in T-ALL (31.40% vs. 14.50%, P < 0.001). Pediatric ALL had higher prevalence of ETV6-RUNX1, TCF3-PBX1, and STIL-TAL1, while BCR-ABL1 and SET-NUP214 were more common in adult ALL. BCR-ABL1, TCF3-PBX1, KMT2A-AFF1 and ETV6-RUNX1 were more frequent in B-ALL. On the contrary, KMT2A-MLLT4, SET-NUP214 and STIL-TAL1 were of higher incidence in T-ALL. In comparison with Western cohorts, the incidence of BCR-ABL1 (5.94%) was much higher in our series, while the occurrence of ETV6-RUNX1 (13.19%) was significantly lower in pediatric B-ALL patients in our study than in Western reports. This study provides a genetic landscape of common fusion genes in ALL patients and may serve as a foundation for further improvement of a fusion gene screening panel for clinical applications.

OBJECTIVE: The aim of this study was to review clinical outcomes and prognosis of paediatric patients with acute lymphoblastic leukaemia (ALL) with TCF3-PBX1 rearrangement.
PATIENTS: All children in Hong Kong diagnosed with ALL with TCF3-PBX1 rearrangement over the past two decades were included.
METHODS: Six hundred and twenty-four newly diagnosed patients with ALL from four consecutive studies were enrolled from 1997 to 2016. Patients carrying TCF3-PBX1 rearrangement and patients at intermediate risk without the gene expression were compared for clinical characteristics, overall survival and event-free survival (EFS).
RESULTS: The TCF3-PBX1 rearrangement was detected in 30 of 624 patients (4.8%). Results were consistent across the consecutive clinical trials employed in the past two decades. Compared with 239 intermediate risk patients without TCF3-PBX1 rearrangement, the 5-year overall survival and EFS for patients with TCF3-PBX1 rearrangement was superior, with both at 100% (P = 0.12 and P = 0.029).
CONCLUSION: This population-based study over the past 20 years demonstrated that patients with TCF3-PBX1 rearrangement had favourable EFS compared with other intermediate risk patients treated with a similar chemotherapy backbone.

BACKGROUND: High HOX gene expression has been described in many cancers, including oral squamous cell carcinoma and the functional roles of these genes are gradually being understood. The pattern of overexpression suggests that inhibition may be useful therapeutically. Inhibition of HOX protein binding to PBX cofactors by the use of synthetic peptides, such as HXR9, results in apoptosis in multiple cancers.
METHODS: Activity of the HOX-PBX inhibiting peptide HXR9 was tested in immortalised normal oral (NOK), potentially-malignant (PMOL) and squamous cell carcinoma (OSCC) cells, compared to the inactive peptide CXR9. Cytotoxicity was assessed by LDH assay. Expression of PBX1/2 and c-Fos was assessed by qPCR and western blotting. Apoptosis was assessed by Annexin-V assay.
RESULTS: PMOL and OSCC cells expressed PBX1/2. HOX-PBX inhibition by HXR9 caused death of PMOL and OSCC cells, but not NOKs. HXR9 treatment resulted in apoptosis and increased expression of c-Fos in some cells, whereas CXR9 did not. A correlation was observed between HOX expression and resistance to HXR9.
CONCLUSION: Inhibition of HOX-PBX interactions causes selective apoptosis of OSCC/PMOL, indicating selective toxicity that may be useful clinically.

The E2A-PBX1 rearrangement is common in B cell acute lymphoblastic leukemia (B-ALL). However, whether this fusion gene can be used as a reliable marker for minimal residual disease (MRD) following allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains unknown. In this study, clinical data were collected from 28 consecutive B-ALL patients who received allo-HSCT. Their MRD was evaluated by E2A-PBX1 and leukemia-associated immunophenotype (LAIP). The median follow-up was 374 days (55-2342 days). Of the enrolled patients, seven (25%) patients died of leukemia relapse. A total of nine (32.1%) patients experienced relapse at a median of 164 days (75-559 days) after transplantation. The median expression level in the first positive sample was 0.14% (0.0071-902.4%). The duration from E2A-PBX1-positive results to hematological relapse was 74 days (30-469 days). E2A-PBX1 expression generally became positive prior to flow cytometry. Patients with positive E2A-PBX1 gene expression pre-transplantation were more likely to have positive E2A-PBX1 expression after transplantation. Taken all together, E2A-PBX1 expression determined by real-time quantitative reverse transcriptase polymerase chain reaction (RQ-PCR) could be used to evaluate MRD status after allo-HSCT. Patients with positive E2A-PBX1 expression after transplant will have a poor prognosis.

PBX1 was abnormally overexpressed and its intracellular localization was found to be frequently amplified in many types of cancer, including renal clear cell carcinoma. PBX1 displays oncogenic activity in several different types of cells, but little is known about how signaling pathways are altered, and the function of PBX1 in renal clear cell carcinoma has not been well investigated. In this study, we demonstrate that the expression of PBX1 was significantly upregulated in 30 pairs of human tissues compared to adjacent normal tissues and the overall survival rate of PBX1-high group was significantly worse than that of PBX1-low group. Furthermore, JAK2 expression is significantly correlated to PBX1 expression in human clinical specimen and PBX1 knockdown inhibits STAT3 phosphorylation and reduced transcription of STAT3 target genes Cyclin D1. More interestingly, PBX1 knockdown inhibits ccRCC cell viability, proliferation and cell cycle progression in vivo and in vitro. Thus, our results demonstrate that PBX1 plays an oncogenic role in ccRCC via JAK2/STAT3 pathway and indicate its potential application for the treatment of human ccRCC in future.

The survival rate of children with acute lymphoblastic leukemia (ALL) has increased to approximately 90% after substantial progress in risk-oriented treatment strategies. Between 2005 and 2013, the Tokyo Children's Cancer Study Group (TCCSG) conducted a risk-oriented, non-randomized study, L04-16. The principal aim of this study was to assemble background characteristics and treatment outcomes, and gather genetic information on leukemic cells under central diagnosis. This report outlines the background characteristics and treatment outcomes of 1033 children with ALL treated according to a TCCSG platform. The 5-year event-free and overall survival (OS) rates for all children were 78.1 ± 1.3 and 89.6 ± 1.0%, respectively. The OS rate was significantly higher in children with B-cell precursor (BCP)-ALL (91.9 ± 1.0%, n = 916) than in those with T-ALL (71.9 ± 4.3%, n = 117, p < 0.001). In univariate analysis for BCP-ALL, children aged 1-6 years (5y-OS: 94.2 ± 1.0%), with an initial white blood cell count of < 20,000/μL (94.0 ± 1.0%), high hyperdiploidy (95.4 ± 1.6%), ETV6-RUNX1 (97.4 ± 1.2%) or TCF3-PBX1 (96.9 ± 2.1%), and "Day8NoBlasts" (96.4 ± 1.1%) had the best outcomes. Genetic investigation revealed two novel fusion genes within this cohort: ETV6-ZNF385A and ZNF362-TCF4. Our study highlighted the clinical aspects of genomic features of ALL in Japanese children. We provide fundamental information for the further molecular investigation of this disease.

AIM: To determine whether methylation differences between mostly fatal TCF3-HLF and curable TCF3-PBX1 pediatric acute lymphoblastic leukemia subtypes can be associated with differential gene expression and remission.
MATERIALS & METHODS: Five (extremely rare) TCF3-HLF versus five (very similar) TCF3-PBX1 patients were sampled before and after remission and analyzed using reduced representation bisulfite sequencing and RNA-sequencing.
RESULTS: We identified 7000 differentially methylated CpG sites between subtypes, of which 78% had lower methylation levels in TCF3-HLF. Gene expression was negatively correlated with CpG sites in 23 genes. KBTBD11 clearly differed in methylation and expression between subtypes and before and after remission in TCF3-HLF samples.
CONCLUSION: KBTBD11 hypomethylation may be a promising potential target for further experimental validation especially for the TCF3-HLF subtype.

OBJECTIVE: Breast cancer is a severe risk to public health and has adequately convoluted pathogenesis. Therefore, the description of key molecular markers and pathways is of much importance for clarifying the molecular mechanism of breast cancer-associated fibroblasts initiation and progression. Breast cancer-associated fibroblasts gene expression dataset was downloaded from Gene Expression Omnibus database.
METHODS: A total of nine samples, including three normal fibroblasts, three granulin-stimulated fibroblasts and three cancer-associated fibroblasts samples, were used to identify differentially expressed genes (DEGs) between normal fibroblasts, granulin-stimulated fibroblasts and cancer-associated fibroblasts samples. The gene ontology (GO) and pathway enrichment analysis was performed, and protein-protein interaction (PPI) network of the DEGs was constructed by NetworkAnalyst software.
RESULTS: Totally, 190 DEGs were identified, including 66 up-regulated and 124 down-regulated genes. GO analysis results showed that up-regulated DEGs were significantly enriched in biological processes (BP), including cell-cell signalling and negative regulation of cell proliferation; molecular function (MF), including insulin-like growth factor II binding and insulin-like growth factor I binding; cellular component (CC), including insulin-like growth factor binding protein complex and integral component of plasma membrane; the down-regulated DEGs were significantly enriched in BP, including cell adhesion and extracellular matrix organization; MF, including N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase activity and calcium ion binding; CC, including extracellular space and extracellular matrix. WIKIPATHWAYS analysis showed the up-regulated DEGs were enriched in myometrial relaxation and contraction pathways. WIKIPATHWAYS, REACTOME, PID_NCI and KEGG pathway analysis showed the down-regulated DEGs were enriched endochondral ossification, TGF beta signalling pathway, integrin cell surface interactions, beta1 integrin cell surface interactions, malaria and glycosaminoglycan biosynthesis-chondroitin sulfate/dermatan sulphate. The top 5 up-regulated hub genes, CDKN2A, MME, PBX1, IGFBP3, and TFAP2C and top 5 down-regulated hub genes VCAM1, KRT18, TGM2, ACTA2, and STAMBP were identified from the PPI network, and subnetworks revealed these genes were involved in significant pathways, including myometrial relaxation and contraction pathways, integrin cell surface interactions, beta1 integrin cell surface interaction. Besides, the target hsa-mirs for DEGs were identified. hsa-mir-759, hsa-mir-4446-5p, hsa-mir-219a-1-3p and hsa-mir-26a-5p were important miRNAs in this study.
CONCLUSIONS: We pinpoint important key genes and pathways closely related with breast cancer-associated fibroblasts initiation and progression by a series of bioinformatics analysis on DEGs. These screened genes and pathways provided for a more detailed molecular mechanism underlying breast cancer-associated fibroblasts occurrence and progression, holding promise for acting as molecular markers and probable therapeutic targets.

RAS pathway mutations have been linked to relapse and chemotherapy resistance in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, comprehensive data on the frequency and prognostic value of subclonal mutations in well-defined subgroups using highly sensitive and quantitative methods are lacking. Targeted deep sequencing of 13 RAS pathway genes was performed in 461 pediatric BCP-ALL cases at initial diagnosis and in 19 diagnosis-relapse pairs. Mutations were present in 44.2% of patients, with 24.1% carrying a clonal mutation. Mutation frequencies were highest in high hyperdiploid, infant t(4;11)-rearranged, BCR-ABL1-like and B-other cases (50-70%), whereas mutations were less frequent in ETV6-RUNX1-rearranged, and rare in TCF3-PBX1- and BCR-ABL1-rearranged cases (27-4%). RAS pathway-mutated cells were more resistant to prednisolone and vincristine ex vivo. Clonal, but not subclonal, mutations were linked to unfavorable outcome in standard- and high-risk-treated patients. At relapse, most RAS pathway mutations were clonal (9 of 10). RAS mutant cells were sensitive to the MEK inhibitor trametinib ex vivo, and trametinib sensitized resistant cells to prednisolone. We conclude that RAS pathway mutations are frequent, and that clonal, but not subclonal, mutations are associated with unfavorable risk parameters in newly diagnosed pediatric BCP-ALL. These mutations may designate patients eligible for MEK inhibitor treatment.

BACKGROUND: We aimed to investigate the frequencies and the association with genetic/cytogenetic abnormalities as well as prognostic relevance of RAS pathway mutations in Taiwanese children with B-precursor acute lymphoblastic leukemia (ALL), the largest cohort in Asians.
PROCEDURE: Between 1995 and 2012, marrow samples at diagnosis from 535 children were studied for NRAS, KRAS, and PTPN11 mutations. The mutational status of each gene was correlated with the clinico-hematological features, recurrent genetic abnormalities, and outcomes for those treated with TPOG-ALL-2002 protocol (n = 346).
RESULTS: The frequencies of NRAS, KRAS, and PTPN11 mutations were 10.8% (57/530), 10.2% (54/530), and 3.0% (16/526), respectively. NRAS mutations were associated with a higher frequency of hyperdiploidy (P = 0.01) and lower frequency of ETV6-RUNX1 (P < 0.01), whereas KRAS mutations were associated with younger age (P < 0.01), a higher frequency of KMT2A rearranged (P < 0.01) but no significant difference if infants with ALL were excluded, and inferior event-free survival (66.6% vs. 80.5%, P = 0.04). None of patients with TCF3-PBX1 had KRAS mutation (P = 0.02).
CONCLUSIONS: Our study showed that the frequency of KRAS mutations in Taiwan was significantly higher than that reported in Caucasians. The occurrence of RAS pathway mutations was associated with recurrent genetic/cytogenetic abnormalities in pediatric B-precursor ALL.

AIM: To clarify the mechanisms of connexin 32 (Cx32) downregulation by potential transcriptional factors (TFs) in
METHODS: Approximately 25 specimens at each developmental stage of gastric carcinogenesis [non-atrophic gastritis, chronic atrophic gastritis, intestinal metaplasia, dysplasia and gastric carcinoma (GC)] with
RESULTS: Incremental PBX1 was first detected by TF microarray in
CONCLUSION: PBX1 is one of the determinants in the Cx32 promoter targeting site, preventing further damage of gap junction protein in

B cell leukaemia is one of the most frequent malignancies in the paediatric population, but also affects a significant proportion of adults in developed countries. The majority of infant and paediatric cases initiate the process of leukaemogenesis during foetal development (in utero) through the formation of a chromosomal translocation or the acquisition/deletion of genetic material (hyperdiploidy or hypodiploidy, respectively). This first genetic insult is the major determinant for the prognosis and therapeutic outcome of patients. B cell leukaemia in adults displays similar molecular features as its paediatric counterpart. However, since this disease is highly represented in the infant and paediatric population, this review will focus on this demographic group and summarise the biological, clinical and epidemiological knowledge on B cell acute lymphoblastic leukaemia of four well characterised subtypes: t(4;11) MLL-AF4, t(12;21) ETV6-RUNX1, t(1;19) E2A-PBX1 and t(9;22) BCR-ABL1.

Acute lymphoblastic leukaemia (ALL) occurs in approximately 1:1500 children and is less frequently found in adults. The most common immunophenotype of ALL is the B cell lineage and within B cell precursor ALL, specific genetic aberrations define subtypes with distinct biological and clinical characteristics. With more advanced genetic analysis methods such as whole genome and transcriptome sequencing, novel genetic subtypes have recently been discovered. One novel class of genetic aberrations comprises tyrosine kinase-activating lesions, including translocations and rearrangements of tyrosine kinase and cytokine receptor genes. These newly discovered genetic aberrations are harder to detect by standard diagnostic methods such as karyotyping, fluorescent in situ hybridisation (FISH) or polymerase chain reaction (PCR) because they are diverse and often cryptic. These lesions involve one of several tyrosine kinase genes (among others, v-abl Abelson murine leukaemia viral oncogene homologue 1 (ABL1), v-abl Abelson murine leukaemia viral oncogene homologue 2 (ABL2), platelet-derived growth factor receptor beta polypeptide (PDGFRB)), each of which can be fused to up to 15 partner genes. Together, they compose 2-3% of B cell precursor ALL (BCP-ALL), which is similar in size to the well-known fusion gene BCR-ABL1 subtype. These so-called BCR-ABL1-like fusions are mutually exclusive with the sentinel translocations in BCP-ALL (BCR-ABL1, ETV6-RUNX1, TCF3-PBX1, and KMT2A (MLL) rearrangements) and have the promising prospect to be sensitive to tyrosine kinase inhibitors similar to BCR-ABL1. In this review, we discuss the types of tyrosine kinase-activating lesions discovered, and the preclinical and clinical evidence for the use of tyrosine kinase inhibitors in the treatment of this novel subtype of ALL.

BACKGROUND: Endoplasmic reticulum (ER) calcium storage and release play important roles in B lymphocyte maturation, survival, antigen-dependent cell activation and immunoglobulin synthesis. Calcium is accumulated in the endoplasmic reticulum (ER) by Sarco/Endoplasmic Reticulum Calcium ATPases (SERCA enzymes). Because lymphocyte function is critically dependent on SERCA activity, it is important to understand qualitative and quantitative changes of SERCA protein expression that occur during B lymphoid differentiation and leukemogenesis.
METHODS: In this work we investigated the modulation of SERCA expression during the pharmacologically induced differentiation of leukemic precursor B lymphoblast cell lines that carry the E2A-PBX1 fusion oncoprotein. Changes of SERCA levels during differentiation were determined and compared to those of established early B lymphoid differentiation markers. SERCA expression of the cells was compared to that of mature B cell lines as well, and the effect of the direct inhibition of SERCA-dependent calcium transport on the differentiation process was investigated.
RESULTS: We show that E2A-PBX1
CONCLUSION: These data show that the calcium pump composition of the ER is concurrent with increased SERCA3 expression during the differentiation of precursor B acute lymphoblastic leukemia cells, that a cross-talk exists between SERCA function and the control of differentiation, and that SERCA3 may constitute an interesting new marker for the study of early B cell phenotype.

Gene fusions resulting from structural rearrangements are an established mechanism of tumorigenesis in pediatric cancer. In this clinical cohort, 1,350 single nucleotide polymorphism (SNP)-based chromosomal microarrays from 1,211 pediatric cancer patients were evaluated for copy number alterations (CNAs) associated with gene fusions. Karyotype or fluorescence in situ hybridization studies were performed in 42% of the patients. Ten percent of the bone marrow or solid tumor specimens had SNP array-associated CNAs suggestive of a gene fusion. Alterations involving ETV6, ABL1-NUP214, EBF1-PDGFRB, KMT2A(MLL), LMO2-RAG, MYH11-CBFB, NSD1-NUP98, PBX1, STIL-TAL1, ZNF384-TCF3, P2RY8-CRLF2, and RUNX1T1-RUNX1 fusions were detected in the bone marrow samples. The most common alteration among the low-grade gliomas was a 7q34 tandem duplication resulting in a KIAA1549-BRAF fusion. Additional fusions identified in the pediatric brain tumors included FAM131B-BRAF and RAF1-QKI. COL1A1-PDGFB, CRTC1-MAML2, EWSR1, HEY1, PAX3- and PAX7-FOXO1, and PLAG1 fusions were determined in a variety of solid tumors and a novel potential gene fusion, FGFR1-USP6, was detected in an aneurysmal bone cyst. The identification of these gene fusions was instrumental in tumor diagnosis. In contrast to hematologic and solid tumors in adults that are predominantly driven by mutations, the majority of hematologic and solid tumors in children are characterized by CNAs and gene fusions. Chromosomal microarray analysis is therefore a robust platform to identify diagnostic and prognostic markers in the clinical setting.

Pre-B-cell leukemia homeobox 1 (PBX1) was originally identified as a proto-oncogene in human leukemia. Although this protein has been shown to contribute to cellular development and tumorigenesis, the role of PBX1 in gastric carcinoma (GC) remains unclear. In this study, we observed increased expression of PBX1 in GC tissues compared with adjacent normal tissues. This increase in PBX1 expression levels negatively correlated with HOXB9 mRNA expression and was also associated with malignancy and metastasis. PBX1 promoted proliferation and metastasis of GC cells both in vitro and in vivo.These phenomena were also accompanied by epithelial-to-mesenchymal transition (EMT). Additionally, we observed that PBX1 promotes the expression of tumor growth and angiogenic factors. A structural model of the PBX1-HOX complex revealed that hydrophobic binding between PBX1 and the hexapeptide motif might be required for EMT induction. This analysis also demonstrated that the Phe252 residue in the first helix of the TALE homeodomain is involved in the latter hydrophobic binding reaction. In vitro data from PBX1 mutants suggest that PBX1 cannot promote tumorigenesis of GC cells via EMT induction when Phe252 residues lose hydrophobicity. It is likely that the presence of this residue is essential in facilitating hydrophobic binding with the hexapeptide motif. These findings suggest that PBX1 may be a potential target for GC treatment and this study provides a platform to elucidate the molecular mechanisms that underpin the role of PBX1 in GC tumorigenesis.

B-cell precursor acute lymphocytic leukemia (B-ALL) represents a worldwide public health issue. Particularly, Mexico is one of the countries with the highest incidence of ALL in children. Between the multiple factors involved in ALL etiology, genetic alterations are clearly one of the most relevant features. In this work, a group of 24 B-ALL patients, all negative for the four most frequent gene fusions (ETV6-RUNX1, BCR-ABL1, TCF3-PBX1 and MLL-AF4), were included in a high-resolution microarray analysis in order to evaluate genomic copy-number alterations (CNAs). The results of this preliminary report showed a broad genomic heterogeneity among the studied samples; 58% of the patients were hyperdiploid and 33% displayed a chromosome 9p deletion of variable length affecting genes CDKN2A/B, two patients displayed genomic instability with a high number of focal CNAs, three patients presented unique duplications affecting 2q, 12p and 1q, respectively, and one patient displayed no copy number imbalances. The copy-number profile of 44 genes previously related to B-ALL was heterogeneous as well. Overall results highlight the need for a detailed description of the genetic alterations in ALL cancer cells in order to understand the molecular pathogenesis of the disease and to identify any prognostic markers with clinical significance.

BACKGROUND: The translocation t(1;19)(q23;p13), which results in the TCF3-PBX1 chimeric gene, is one of the most frequent rearrangements observed in B cell acute lymphoblastic leukemia. It appears in both adult and pediatric patients with B cell acute lymphoblastic leukemia at an overall frequency of 3 to 5%. Most cases of pre-B cell acute lymphoblastic leukemia carrying the translocation t(1;19) have a typical immunophenotype with homogeneous expression of CD19, CD10, CD9, complete absence of CD34, and at least diminished CD20. Moreover, the translocation t(1;19) correlates with known clinical high risk factors, such as elevated white blood cell count, high serum lactate dehydrogenase levels, and central nervous system involvement; early reports indicated that patients with translocation t(1;19) had a poor outcome under standard treatment.
CASE PRESENTATION: We report the case of a 15-year-old Syrian boy with pre-B cell acute lymphoblastic leukemia with abnormal karyotype with a der(19)t(1;19)(q21.1;p13.3) and two yet unreported chromosomal aberrations: an interstitial deletion 6q12 to 6q26 and a der(13)t(1;13)(q21.1;p13). According to the literature, cases who are translocation t(1;19)-positive have a significantly higher incidence of central nervous system relapse than patients with acute lymphoblastic leukemia without the translocation. Of interest, central nervous system involvement was also seen in our patient.
CONCLUSIONS: To the best of our knowledge, this is the first case of childhood pre-B cell acute lymphoblastic leukemia with an unbalanced translocation t(1;19) with two additional chromosomal aberrations, del(6)(q12q26) and t(1;13)(q21.3;p13), which seem to be recurrent and could influence clinical outcome. Also the present case confirms the impact of the translocation t(1;19) on central nervous system relapse, which should be studied for underlying mechanisms in future.

Activating mutations in

Risk biomarkers for estrogen receptor (ER)-negative breast cancer have clear value for breast cancer prevention. We previously reported a set of lipid metabolism (LiMe) genes with high expression in the contralateral unaffected breasts (CUBs) of ER-negative cancer cases. We now further examine LiMe gene expression in both tumor and CUB, and investigate the role of Pre-B-cell leukemia homeobox-1 (PBX1) as a candidate common transcription factor for LiMe gene expression. mRNA was extracted from laser-capture microdissected epithelium from tumor and CUB of 84 subjects (28 ER-positive cases, 28 ER-negative cases, 28 healthy controls). Gene expression was quantitated by qRT-PCR. Logistic regression models were generated to predict ER status of the contralateral cancer. Protein expression of HMGCS2 and PBX1 was measured using immunohistochemistry. The effect of PBX1 on LiMe gene expression was examined by overexpressing PBX1 in MCF10A cells with or without ER, and by suppressing PBX1 in MDA-MB-453 cells. The expression of DHRS2, HMGCS2, UGT2B7, UGT2B11, ALOX15B, HPGD, UGT2B28 and GLYATL1 was significantly higher in ER-negative versus ER-positive CUBs, and predicted ER status of the tumor in test and validation sets. In contrast, LiMe gene expression was significantly lower in ER-negative than ER-positive tumors. PBX1 overexpression in MCF10A cells up-regulated most LiMe genes, but not in MCF10A cells overexpressing ER. Suppressing PBX1 in MDA-MB-453 cells resulted in decrease of LiMe gene expression. Four binding sites of PBX1 and cofactor were identified in three lipid metabolism genes using ChIP-qPCR. These data suggest a novel role for PBX1 in the regulation of lipid metabolism genes in benign breast, which may contribute to ER-negative tumorigenesis.

BACKGROUND: Acute lymphocytic leukemia (ALL) is the most common hematologic malignancy in early childhood. In children with acute lymphoblastic leukemia (ALL), the activity of glycogen synthase kinase (GSK-3β) has been associated with changes in the transcriptional activity and expression of nuclear factor kappa beta (NFKB) in the mononuclear cells of bone marrow.
OBJECTIVES: The aim of the study was to determine the possible role of glycogen synthase kinase 3beta (GSK-3β) and nuclear factor kappa beta (NFKB) as prognostic variables in pediatric patients with ALL.
MATERIAL AND METHODS: This was a descriptive, transversal, and observational study. Bone marrow and blood samples were obtained from 30 children with newly-diagnosed ALL, who were seen at the Hematology-Oncology Service, Hospital para el Niño (HPN), Toluca, Mexico, from 2014‒2015. Anthropometric variables, clinical lab results, immunophenotype and cytogenetic abnormalities were registered. GSK-3β was evaluated through immunohistochemistry, and NFKB messenger RNA (mRNA) with real-time polymerase chain reaction (qPCR). The cases of ALL were classified into two groups of risk: high and habitual.
RESULTS: Thirty patients were included in this study, with a mean age of 7.1 years (range 2‒13 years). Twenty-one were male and 9 female. Employing the morphological classification, 26 patients had type L1 ALL and the remaining 4 patients had type L2 ALL. Abnormal genes were found in 7 (23.33%) patients, ETV-RUNX1 in 3, followed by TCF3-PBX1 (two), STL1-TAL1 (one), and BCR-ABL1 (one). NFKB relative expression levels, in comparison to the GSK-3β immunohistochemistry results of the bone marrow samples, showed significant differences between positive and negative cases (p = 0.001) and between weak-positive and negative cases (p = 0.002).
CONCLUSIONS: These results suggest that GSK-3β may be a prognostic biomarker in childhood ALL.

BACKGROUND: Recent studies have shown that the high mortality of patients with colorectal cancer (CRC) is related to its ability to spread the surrounding tissues, thus there is a need for designing and developing new drugs.
OBJECTIVE: Here, we proposed a combinational therapy strategy, an inhibitory peptide in combination with miRNA targeting, for modulating CRC metastasis. In this study, some of the recent patents were also reviewed.
METHODS: After data analysis with GEO2R and gene annotation using DAVID server, regulatory interactions of differentially expressed genes (DEGs) were obtained from STRING, GeneMANIA, KEGG and TRED databases. In parallel, the corresponding validated microRNAs (miRNAs) were obtained from mirDIP web server and a miRNA-DEG regulatory network was also reconstructed. Clustering and topological analyses of the regulatory networks were performed using Cytoscape plug-ins.
RESULTS: We found the HOXB family as the most important functional complex in DEG-derived regulatory network. Accordingly, an anti-HOXB7 peptide was designed based on the binding interface of its coactivator, PBX1. Topological analysis of miRNA-DEG network indicated that hsa-miR-222 is one of the most important oncomirs involved in regulation of DEGs activities. Thus, this miRNA, along with HOXB7, was also considered as the potential target for inhibiting CRC metastasis. Molecular docking studies exhibited that the designed peptide can bind to desired binding pocket of HOXB7 in a highaffinity manner. Further confirmations were also observed in Molecular dynamics (MD) simulations carried out by GROMACS v5.0.2 simulation package.
CONCLUSION: In conclusion, our findings suggest that simultaneous targeting of key regulatory genes and miRNAs may be a useful strategy for prevention of CRC metastasis.

The evolution of chemoresistance is a fundamental characteristic of cancer that ultimately hampers its clinical management. However, it may be possible to improve patient outcomes significantly by a better understanding of resistance mechanisms, which cancers rely upon during the evolution to an untreatable state. Here we report an essential role of the stem cell reprogramming factor, PBX1, in mediating chemoresistance in ovarian carcinomas. In the clinical setting, high levels of PBX1 expression correlated with shorter survival in post-chemotherapy ovarian cancer patients. In tumor cells with low endogenous levels of PBX1, its enforced expression promoted cancer stem cell-like phenotypes, including most notably an increase in resistance to platinum-based therapy used most commonly for treating this disease. Conversely, silencing PBX1 in platinum-resistant cells that overexpressed PBX1 sensitized them to platinum treatment and reduced their stem-like properties. An analysis of published genome-wide chromatin immunoprecipitation data indicated that PBX1 binds directly to promoters of genes involved in stem cell maintenance and the response to tissue injury. We confirmed direct regulation of one of these genes, STAT3, demonstrating that the PBX1 binding motif at its promoter acted to positively regulate STAT3 transcription. We further demonstrated that a STAT3/JAK2 inhibitor could potently sensitize platinum-resistant cells to carboplatin and suppress their growth in vivo Our findings offer a mechanistic rationale to target the PBX1/STAT3 axis to antagonize a key mechanism of chemoresistance in ovarian cancers and possibly other human cancers. Cancer Res; 76(21); 6351-61. ©2016 AACR.

There is limited understanding of how signaling pathways are altered by oncogenic fusion transcription factors that drive leukemogenesis. To address this, we interrogated activated signaling pathways in a comparative analysis of mouse and human leukemias expressing the fusion protein E2A-PBX1, which is present in 5%-7% of pediatric and 50% of pre-B-cell receptor (preBCR

Fusion genes involving ZNF384 have recently been identified in B-cell precursor acute lymphoblastic leukemia, and 7 fusion partners have been reported. We further characterized this type of fusion gene by whole transcriptome sequencing and/or polymerase chain reaction. In addition to previously reported genes, we identified BMP2K as a novel fusion partner for ZNF384 Including the EP300-ZNF384 that we reported recently, the total frequency of ZNF384-related fusion genes was 4.1% in 291 B-cell precursor acute lymphoblastic leukemia patients enrolled in a single clinical trial, and TCF3-ZNF384 was the most recurrent, with a frequency of 2.4%. The characteristic immunophenotype of weak CD10 and aberrant CD13 and/or CD33 expression was revealed to be a common feature of the leukemic cells harboring ZNF384-related fusion genes. The signature gene expression profile in TCF3-ZNF384-positive patients was enriched in hematopoietic stem cell features and related to that of EP300-ZNF384-positive patients, but was significantly distinct from that of TCF3-PBX1-positive and ZNF384-fusion-negative patients. However, clinical features of TCF3-ZNF384-positive patients are markedly different from those of EP300-ZNF384-positive patients, exhibiting higher cell counts and a younger age at presentation. TCF3-ZNF384-positive patients revealed a significantly poorer steroid response and a higher frequency of relapse, and the additional activating mutations in RAS signaling pathway genes were detected by whole exome analysis in some of the cases. Our observations indicate that ZNF384-related fusion genes consist of a distinct subgroup of B-cell precursor acute lymphoblastic leukemia with a characteristic immunophenotype, while the clinical features depend on the functional properties of individual fusion partners.

BACKGROUND: The World Health Organization (WHO) classification system defines recurrent chromosomal translocations as the sole diagnostic and prognostic criteria for acute leukemia (AL). These fusion transcripts are pivotal in the pathogenesis of AL. Clinical laboratories universally employ conventional karyotype/FISH to detect these chromosomal translocations, which is complex, labour intensive and lacks multiplexing capacity. Hence, it is imperative to explore and evaluate some newer automated, cost-efficient multiplexed technologies to accommodate the expanding genetic landscape in AL.
METHODS: "nCounter® Leukemia fusion gene expression assay" by NanoString was employed to detect various fusion transcripts in a large set samples (n = 94) utilizing RNA from formalin fixed paraffin embedded (FFPE) diagnostic bone marrow biopsy specimens. This series included AL patients with various recurrent translocations (n = 49), normal karyotype (n = 19), or complex karyotype (n = 21), as well as normal bone marrow samples (n = 5). Fusion gene expression data were compared with results obtained by conventional karyotype and FISH technology to determine sensitivity/specificity, as well as positive /negative predictive values.
RESULTS: Junction probes for PML/RARA; RUNX1-RUNX1T1; BCR/ABL1 showed 100 % sensitivity/specificity. A high degree of correlation was noted for MLL/AF4 (85 sensitivity/100 specificity) and TCF3-PBX1 (75 % sensitivity/100 % specificity) probes. CBFB-MYH11 fusion probes showed moderate sensitivity (57 %) but high specificity (100 %). ETV6/RUNX1 displayed discordance between fusion transcript assay and FISH results as well as rare non-specific binding in AL samples with normal or complex cytogenetics.
CONCLUSIONS: Our study presents preliminary data with high correlation between fusion transcript detection by a throughput automated multiplexed platform, compared to conventional karyotype/FISH technique for detection of chromosomal translocations in AL patients. Our preliminary observations, mandates further vast validation studies to explore automated molecular platforms in diagnostic pathology.