Somatic Mutations In Exon Research Articles

Abstract 3187: Genomic abnormalities in air pollution-related lung cancer

Abstract The lung cancer incidence in Xuanwei (XW) City and Fuyuan (FY) County in Yunnan Province of China is among the highest in China, owing to high levels of air pollution produced by combustion of smoky coal. This epidemiology profile provides a unique opportunity for identifying the carcinogenic mechanism underlying air pollution. Here, we analyze the genome sequences of 14 XW/FY non-small-cell lung cancers and their paired normal tissues, and report a mean of 289 somatic exonic mutations and 71 genomic rearrangements/tumor. Among the recurring mutated genes, calcium signaling and ion channel genes are the most frequently altered gene category. Higher mutation rate in calcium signaling genes is confirmed by genomic resequencing in 65 XW/FY patients and 85 patients from control regions. Small interfering RNA mediated silencing shows that inactivation of calcium channel leads to inhibition of lung cancer cells, suggesting a role for calcium signaling molecules in air pollution-related carcinogenesis leading to lung cancer. Citation Format: Guang-Biao Zhou, Xian-Jun Yu, Li-Chuan Wu, Yun-Chao Huang, Gao-Feng Li, Zhe-Sheng Wen, Sai-Juan Chen, Yi Cao, Sheng-Yue Wang, Min-Jun Yang, Zhu Chen. Genomic abnormalities in air pollution-related lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3187. doi:10.1158/1538-7445.AM2014-3187

Abstract 1526: Integrative and comparative genomic analysis of East-Asian lung squamous cell carcinomas

Abstract Purpose Lung squamous cell carcinoma (SqCC) is the second most prevalent type of lung cancer. Currently, no targeted-therapeutics are approved for treatment of this cancer, largely due to a lack of systematic understanding of the molecular pathogenesis of the disease. To identify therapeutic targets and perform comparative analyses of lung SqCC, we probed somatic genome alterations of lung SqCC cases from Korean patients. Patients and Methods We performed whole-exome sequencing of DNA from 104 lung SqCC samples from Korean patients and matched normal DNA. In addition, copy number analysis and transcriptome analysis were conducted for a subset of these samples. Clinical association with cancer-specific somatic alterations was investigated. Results This cancer cohort is characterized by a very high mutational burden with an average of 261 somatic exonic mutations per tumor and a mutational spectrum showing a signature of cigarette-smoke exposure. Seven genes demonstrated statistical enrichment for mutation (TP53, RB1, PTEN, NFE2L2, KEAP1, MLL2 and PIK3CA). Comparative analysis between Korean and North American lung SqCC demonstrated similar spectrum of alterations in these two populations, in contrast to the differences seen in lung adenocarcinoma. We also uncovered recurrent occurrence of therapeutically actionable FGFR3-TACC3 fusion in lung SqCC. Conclusion These findings provide new steps towards the identification of genomic target candidates for precision medicine in lung SqCC, a disease with a significant unmet medical need. Note: This abstract was not presented at the meeting. Citation Format: Youngwook Kim, Peter S. Hammerman, Jaegil Kim, Gad Getz, Matthew Meyerson, Keunchil Park. Integrative and comparative genomic analysis of East-Asian lung squamous cell carcinomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1526. doi:10.1158/1538-7445.AM2014-1526

Constitutive AKT activation in follicular lymphoma

BackgroundThe phosphoinositide 3- kinase (PI3K) pathway is involved in the growth of various human cancers, including lymphoid malignancies. However its role in the pathogenesis of follicular lymphoma (FL) has not been yet described.MethodsTo clarify this point, biopsy tissue samples from 38 human FL cases were investigated for PIK3CA somatic mutations in exon 9 and 20 using direct sequencing. The same samples were analyzed using western blotting and immunohistochemistry to detect expression of AKT, phosphorylated AKT (pAKT), and PTEN proteins. Two cases of benign lymphadenitis were used as controls.ResultsAKT expression was present in all FL and lymphadenitis cases. 14/38 (37%) FL and 2/2 lymphadenitis cases expressed pAKT. 9/38 (24%) FL samples showed high level of pAKT, whereas 5/38 (13%) FL cases and 2/2 benign lymphadenitis samples expressed low level of pAKT. PTEN expression was observed in 30/38 (79%) FL and 2/2 benign lymphadenitis cases, whereas 8/38 (21%) FL cases showed loss of PTEN expression. 3 cases with positive pAKT did not express PTEN. PIK3CA mutations were not detected in any sample.ConclusionsThese data suggest that the PI3K/AKT signaling pathway could be activated in a subset of FL cases, due to either AKT phosphorylation or PTEN downregulation, in the absence of PIK3CA mutations.

Uterine Leiomyoma-Linked MED12 Mutations Disrupt Mediator-Associated CDK Activity

Somatic mutations in exon 2 of the RNA polymerase II transcriptional Mediator subunit MED12 occur at very high frequency (∼70%) in uterine leiomyomas. However, the influence of these mutations on Mediator function and the molecular basis for their tumorigenic potential remain unknown. To clarify the impact of these mutations, we used affinity-purification mass spectrometry to establish the global protein-protein interaction profiles for both wild-type and mutant MED12. We found that uterine leiomyoma-linked mutations in MED12 led to a highly specific decrease in its association with Cyclin C-CDK8/CDK19 and loss of Mediator-associated CDK activity. Mechanistically, this occurs through disruption of a MED12-Cyclin C binding interface that we also show is required for MED12-mediated stimulation of Cyclin C-dependent CDK8 kinase activity. These findings indicate that uterine leiomyoma-linked mutations in MED12 uncouple Cyclin C-CDK8/19 from core Mediator and further identify the MED12/Cyclin C interface as a prospective therapeutic target in CDK8-driven cancers.

Integrative and Comparative Genomic Analysis of Lung Squamous Cell Carcinomas in East Asian Patients

Lung squamous cell carcinoma (SCC) is the second most prevalent type of lung cancer. Currently, no targeted therapeutics are approved for treatment of this cancer, largely because of a lack of systematic understanding of the molecular pathogenesis of the disease. To identify therapeutic targets and perform comparative analyses of lung SCC, we probed somatic genome alterations of lung SCC by using samples from Korean patients. We performed whole-exome sequencing of DNA from 104 lung SCC samples from Korean patients and matched normal DNA. In addition, copy-number analysis and transcriptome analysis were conducted for a subset of these samples. Clinical association with cancer-specific somatic alterations was investigated. This cancer cohort is characterized by a high mutational burden with an average of 261 somatic exonic mutations per tumor and a mutational spectrum showing a signature of exposure to cigarette smoke. Seven genes demonstrated statistical enrichment for mutation: TP53, RB1, PTEN, NFE2L2, KEAP1, MLL2, and PIK3CA). Comparative analysis between Korean and North American lung SCC samples demonstrated a similar spectrum of alterations in these two populations in contrast to the differences seen in lung adenocarcinoma. We also uncovered recurrent occurrence of therapeutically actionable FGFR3-TACC3 fusion in lung SCC. These findings provide new steps toward the identification of genomic target candidates for precision medicine in lung SCC, a disease with significant unmet medical needs.

Expression of PIK3CA, PTEN mRNA and PIK3CA mutations in primary breast cancer: association with lymph node metastases

PurposeHigh activity of the intracellular phosphatidylinositol-3 kinase (PI3K) pathway is common in breast cancer. Here, we explore differences in expression of important PI3K pathway regulators: the activator, phosphatidylinositol-3-kinase catalytic subunit alpha (PIK3CA), and the tumour suppressor, phosphatase and tensin homolog (PTEN), in breast carcinoma tissue and normal breast tissue. Furthermore, we examine whether expression of PIK3CA and PTEN mRNA and occurrence of PIK3CA mutations are associated with lymph node metastases in patients with primary breast cancer.MethodsPaired tissue samples of breast carcinoma and normal breast tissue were obtained from 175 breast cancer patients at the time of primary surgery, of these 105 patients were lymph node positive. Expression of PIK3CA and PTEN mRNA was quantified with Quantitative Real Time PCR. Somatic mutations in exon 9 and exon 20 of the PIK3CA gene were identified by genotyping.ResultsBoth PIK3CA and PTEN mRNA expression was significantly increased in breast carcinoma tissue compared to normal breast tissue (p = 2 × 10-11) and (p < 0.001), respectively. PIK3CA mutations were present in 68 out of 175 patients (39%), but were not associated with PIK3CA expression (p = 0.59). Expression of PIK3CA and PTEN mRNA, and PIK3CA mutations in breast carcinomas were not associated with presence of lymph node metastases.ConclusionsThe expression of PTEN and PIK3CA mRNA is increased in breast carcinoma tissue compared to normal breast tissue, and PIK3CA mutations are frequent in primary breast carcinoma, however these factors were not associated with lymph node metastases.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-2-464) contains supplementary material, which is available to authorized users.

Overexpression of GATA1 Confers Resistance to Chemotherapy in Acute Megakaryocytic Leukemia

It has been previously shown that acute myeloid leukemia (AML) patients with higher levels of GATA1 expression have poorer outcomes. Furthermore, pediatric Down syndrome (DS) patients with acute megakaryocytic leukemia (AMKL), whose blast cells almost universally harbor somatic mutations in exon 2 of the transcription factor gene GATA1, demonstrate increased overall survival relative to non-DS pediatric patients, suggesting a potential role for GATA1 in chemotherapy response. In this study, we confirmed that amongst non-DS patients, GATA1 transcripts were significantly higher in AMKL blasts compared to blasts from other AML subgroups. Further, GATA1 transcript levels significantly correlated with transcript levels for the anti-apoptotic protein Bcl-xL in our patient cohort. ShRNA knockdown of GATA1 in the megakaryocytic cell line Meg-01 resulted in significantly increased cytarabine (ara-C) and daunorubicin anti-proliferative sensitivities and decreased Bcl-xL transcript and protein levels. Chromatin immunoprecipitation (ChIP) and reporter gene assays demonstrated that the Bcl-x gene (which transcribes the Bcl-xL transcripts) is a bona fide GATA1 target gene in AMKL cells. Treatment of the Meg-01 cells with the histone deacetylase inhibitor valproic acid resulted in down-regulation of both GATA1 and Bcl-xL and significantly enhanced ara-C sensitivity. Furthermore, additional GATA1 target genes were identified by oligonucleotide microarray and ChIP-on-Chip analyses. Our findings demonstrate a role for GATA1 in chemotherapy resistance in non-DS AMKL cells, and identified additional GATA1 target genes for future studies.

Comparison of COBAS 4800 KRAS, TaqMan PCR and High Resolution Melting PCR assays for the detection of KRAS somatic mutations in formalin-fixed paraffin embedded colorectal carcinomas

Many studies documented the influence of KRAS mutation status on the response of patients with metastatic colorectal cancer (mCRC) to anti-EGFR monoclonal antibodies. The COBAS 4800 KRAS is an assay using real time PCR and TaqMelt technology, CE-IVD validated, for the detection of 19 KRAS somatic mutations in exons 2 and 3. We compared COBAS with previously validated PCR TaqMan and High Resolution Melting (HRM) assays on 156 formalin-fixed paraffin embedded (FFPE) specimens of colorectal carcinoma. DNA extraction procedures, using the Qiagen QiAMP kit and the Roche COBAS DNA kit, were also compared. Of the 156 samples, 132 were interpretable using COBAS and TaqMan and 92 using COBAS and HRM. No statistically significant difference was found between COBAS/TaqMan and COBAS/HRM (k = 0.937; p < 0.001 - four discordant cases were found, mostly concerning codon 61 mutations and k = 0.891; p < 0.001 - five discordant cases were found, three regarding codon 61 and two on codon 12/13, respectively). No difference was found between the two DNA extraction methods (t = 1.7185; dol = 39; α = 5 %). The three assays were found suitable to detect accurately KRAS mutations in colon FFPE specimens. COBAS and TaqMan were found to be more robust than HRM, as they yielded fewer non-interpretable results. DNA extraction kits were found to provide equivalent results. The present study shows that pre-screening using COBAS with further TaqMan mutation characterization constitutes an easy and reliable approach for routine diagnostic purposes.

38. Intralesional molecular heterogeneity in a BRAF-mutant BRAF inhibitor resistant melanoma: a case illustrating the challenges for personalised medicine

Background Mutations of the serine/threonine-protein kinase (BRAF) gene occur in approximately half of all patients with met-astatic melanoma. The BRAF inhibitors have proven highly active in treating BRAF mutant metastatic melanoma patients. Eventually, almost all patients develop resistance to BRAF inhibition and relapse. Aims We sought to determine whether the patient’s initial vemurafe nib resistance developed through the reactivation of the MAPK pathway due to additional mutations to the NRAS or BRAF genes. Methods Sanger sequencing was performed on the patient’s ve-murafenib progressing metastasis to detect somatic mutations in exon 1 and exon 2 of the NRAS gene and in exon 11 and exon 15 of the BRAF gene. Immunohistochemical staining, macrodissection and RT-PCR was performed to identify any mutational heterogeneity within the tumour. Results Sequencing revealed a G13R NRAS mutation in addition to the original BRAF V600E mutation within the same vemu-rafenib progressing metastasis. Immunohistochemical staining for p-ERK1/2 expression clearly identified two areas within the tumour with differential staining. Macrodissection of these areas revealed a NRAS wt and BRAF V600E mutant subpopulation, while the other subclone was NRAS G13R mutant and BRAF V600E mutant. Conclusion This study demonstrates the heterogeneous nature of melanoma, even at the intra-lesional level, highlighting that personalising or adjusting therapies based on genotyping of a portion of a single metastasis, might not accurately depict the drivers of mutagenesis across the entire patient’s metastatic melanoma tumour cell population.

Identification of Two Novel NPM1 Mutations in Patients with Acute Myeloid Leukemia

BackgroundGenetic abnormalities in adult AML are caused most frequently by somatic mutations in exon 12 of the NPM1 gene, which is observed in approximately 35% of AML patients and up to 60% of patients with cytogenetically normal AML (CN-AML).MethodsWe performed mutational analysis, including fragment analysis and direct sequencing of exon 12 of the NPM1 gene, on 83 AML patients to characterize the NPM1 mutations completely.ResultsIn this study, NPM1 mutations were identified in 19 (22.9%) of the 83 AML patients and in 12 (42.9%) of the 28 CN-AML patients. Among the 19 patients with NPM1 mutations, type A NPM1 mutations were identified in 16 (84.2%) patients, whereas non-A type NPM1 mutations were observed in 3 (15.8%) patients. Two of the 3 non-A type NPM1 mutations were novel: c.867_868insAAAC and c.869_873indelCTTTAGCCC. These 2 novel mutant proteins display a nuclear export signal motif (L-xxx-L-xx-V-x-L) less frequently and exhibit a mutation at tryptophan 290 that disrupts the nucleolar localization signal.ConclusionsThis study suggests that novel NPM1 mutations may be non-rare and that supplementary sequence analysis is needed along with conventional targeted mutational analysis to detect non-A types of NPM1 mutations.

COLD-PCR and Innovative Microarray Substrates for Detecting and Genotyping MPL Exon 10 W515 Substitutions

Myeloproliferative neoplasms (MPNs) include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Somatic mutations in exon 10 of the MPL (myeloproliferative leukemia virus oncogene) gene, mainly substitutions encoding W515 variants, have recently been described in a minority of patients with ET or PMF. We optimized analytically sensitive methods for detecting and genotyping MPL variants. We used DNA previously isolated from circulating granulocytes of 60 patients with MPN that had previously been analyzed by high-resolution melting (HRM), direct sequencing, and the TaqMan allelic-discrimination assay. We developed conditions for enriching tumor mutant alleles with COLD-PCR (coamplification at lower denaturation temperature PCR) and coupled it with direct sequencing. Assays were designed for identifying MPL W515 substitutions with full COLD-PCR protocols. In parallel, we used innovative microarray substrates to develop assays for evaluating the mutant burden in granulocyte cells. Mutations that were present at very low levels in patients who had previously been scored as having an MPL variant by HRM and as wild type by direct sequencing were successfully identified in granulocyte DNA. Notably, the microarray approach displayed analytical sensitivities of 0.1% to 5% mutant allele, depending on the particular mutation. This analytical sensitivity is similar to that obtained with COLD-PCR. The assay requires no enrichment strategy and allows both the characterization of each variant allele and the evaluation of its proportion in every patient. These procedures, which are transferable to clinical diagnostic laboratories, can be used for detecting very low proportions of minority mutant alleles that cannot be identified by other, conventional methods.

WT1, WTX and CTNNB1 mutation analysis in 43 patients with sporadic Wilms’ tumor

Wilms' tumor (WT) is a heterogeneous neoplasia characterized by a number of genetic abnormalities, involving tumor suppressor genes, oncogenes and genes related to the Wnt signaling pathway. Somatic biallelic inactivation of WT1 is observed in 5-10% of sporadic WT. Somatic mutations in exon 3 of CTNNB1, which encodes β-catenin, were initially observed in 15% of WT. WTX encodes a protein that negatively regulates the Wnt/β-catenin signaling pathway and mediates the binding of WT1. In this study, we screened germline and somatic mutations in selected regions of WT1, WTX and CTNNB1 in 43 WT patients. Mutation analysis of WT1 identified two single-nucleotide polymorphisms, one recurrent nonsense mutation (p.R458X) in a patient with proteinuria but without genitourinary findings of Denys-Drash syndrome (DDS) and one novel missense mutation, p.C428Y, in a patient with Denys-Drash syndrome phenotype. WT1 SNP rs16754A>G (R369R) was observed in 17/43 patients, and was not associated with significant difference in age at diagnosis distribution, or with 60-month overall survival rate. WTX mutation analysis identified five sequence variations, two synonymous substitutions (p.Q1019Q and p.D379D), a non-synonymous mutation (p.F159L), one frameshift mutation (p.157X) and a novel missense mutation, p.R560W. Two sequence variations in CTNNB1 were identified, p.T41A and p.S45C. Overall survival of bilateral cases was significantly lower (p=0.005). No difference was observed when survival was analyzed among patients with WT1 or with WTX mutations. On the other hand, the survival of two patients with the CTNNB1 p.T41A mutation was significantly lower (p=0.000517) than the average.

MIS5-2 - Low Frequencies of BRAF and b-Catenin (CTNNB1) Mutations in Advanced Hepatocellular Carcinoma

ABSTRACT Background The association of specific mutations in mitogen activated protein kinase and Wnt/β-catenin pathways and tumorigenesis of hepatocellular carcinoma (HCC) have been demonstrated. However, the frequency and significance of BRAF and CTNNB1 (the coding gene for β-catenin) mutations in patients with advanced HCC remain unclear. Methods Patients with advanced HCC who were screened for enrollment in clinical trials of first-line molecular targeted therapy between March 2006 and August 2009 at National Taiwan University Hospital were reviewed. We identified those with adequate pathologic tissue obtained at the time of developing advanced disease. Somatic mutations in exon 15 of BRAF and exon 3 of CTNNB1 were analyzed by direct sequencing. Clinical variables were recorded and their associations with the mutations were examined. Results A total of 81 patients were included in the study, with the median age of 56.3 years. Among them, 55 (67.9%) patients were seropositive for hepatitis B virus (HBV) surface antigen; 19 (23.5%) patients were seropositive for antibody against hepatitis C virus (HCV); 77 (95.1%) patients had BCLC stage C; 42 (51.9%) patients had a CLIP score ≥3. BRAF mutations were identified in four (4.9%) patients, including three missense and one silent mutations. Missense mutations of CTNNB1 were found in 10 (12.3%) patients. The two gene mutations were mutually exclusive. The median overall survival of patients with and without BRAF mutation were 2.8 and 6.6 months, respectively (P = 0.350). The median overall survival of patients with and without CTNNB1 mutation were 7.5 and 6.5 months, respectively (P = 0.430). There were no statistically significant associations between BRAF or CTNNB1 mutation and the gender, the ECOG performance status, HBV status, HCV status, alpha-fetoprotein levels, prior local treatment, macrovascular invasion, extrahepatic metastasis, BCLC stages, Okuda stages or CLIP scores. Patients with CTNNB1 mutations were more likely to be >70 years old than patients without CTNNB1 mutations (40% versus 11.3%, P = 0.017). Conclusions BRAF and CTNNB1 mutations were uncommon and were not associated with prognosis in advanced HCC.

Molecular therapeutic advances in personalized therapy of melanoma and non-small cell lung cancer.

The incorporation of individualized molecular therapeutics into routine clinical practice for both non-small cell lung cancer (NSCLC) and melanoma are amongst the most significant advances of the last decades in medical oncology. In NSCLC activating somatic mutations in exons encoding the tyrosine kinase domain of the Epidermal Growth Factor Receptor (EGFR) gene have been found to be predictive of a response to treatment with tyrosine kinase inhibitors (TKI), erlotinib or gefitinib. More recently the EML4-ALK fusion gene which occurs in 3–5% of NSCLC has been found to predict sensitivity to crizotinib an inhibitor of the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase. Similarly in melanoma, 50% of cases have BRAF mutations in exon 15 mostly V600E and these cases are sensitive to the BRAF inhibitors vemurafenib or dabrafenib. In a Phase III study of advanced melanoma cases with this mutation vemurafenib improved survival from 64% to 84% at 6 months, when compared with dacarbazine. In both NSCLC and melanoma clinical benefit is not obtained in patients without these genomic changes, and moreover in the case of vemurafenib the therapy may theoretically induce proliferation of cases of melanoma without BRAF mutations. An emerging clinical challenge is that of acquired resistance after initial responses to targeted therapeutics. Resistance to the TKI’s in NSCLC is most frequently due to acquisition of secondary mutations within the tyrosine kinase of the EGFR or alternatively activation of alternative tyrosine kinases such as C-MET. Mechanisms of drug resistance in melanoma to vemurafenib do not involve mutations in BRAF itself but are associated with a variety of molecular changes including RAF1 or COT gene over expression, activating mutations in RAS or increased activation of the receptor tyrosine kinase PDGFRβ. Importantly these data support introducing re-biopsy of tumors at progression to continue to personalize the choice of therapy throughout the patient’s disease course.

KRAS mutation in relation to HER2 overexpression/amplification in colorectal cancer

The development of targeted therapies for KRAS, EGFR and HER2 may increase the range of response in patients with colorectal cancer. Mutation at codons 12 and 13 of the KRAS gene has been shown to be predictive of cetuximab response in colorectal cancer. However, due to the combined effects of multiple oncogenes involved in disease progression of patients with colorectal cancer, it seems to be important to identify the molecular factors that characterize therapy-resistance phenotype of tumors. Forty six paraffin-embedded colorectal cancer specimens were analyzed for KRAS mutation and HER2 overexpression/amplification. A high–resolution melting (HRM) assay and single-nucleotide polymorphisms (SNPs) were used to detect somatic mutation in exon 2 notably condons 12 and 13 of the KRAS gene. HER2 overexpression was detected using monoclonal antibody and confirmed by fluorescence in situ hybridization (FISH) analysis. KRAS mutations for codons 12 and 13 were identified in 16/46 (34.7%) of patients by SNP. The alterations in KRAS gene were observed in similar percentage of both codons. Colorectal cancer showed mainly heterozygous 35G>A and 38G>A KRAS gene mutations. HRM analysis showed presence of KRAS exon 2 mutation in 13/46 (28.2%) colorectal cancers. Despite positive SPN results in three cases , HRM technique did not reveal KRAS gene alteration. The concordance rate between the two methodologies was high at 87.5%. KRAS mutation was more frequently observed in poorly differentiated tumors and adenocarcinomas than in other histological types. HER2 overexpression was found in 37/46 (80.3%) of all colorectal cancers and in 62.1%, 61.5% of KRAS mutation-positive cases detected by SNP and HRM techniques respectively. HER2 overexpression was accompanied by amplification of HER2 gene. The subgroup of colorectal cancers with KRAS mutation and HER2 overexpression/amplification was poorly differentiated. In summary, the presence of HER2 overexpression/amplification in KRAS mutation–positive colorectal cancers suggests a possible role for the use of specific tyrosine kinase inhibitors in the treatment of disease.

The Irf4 Gene, a Susceptibility Locus for Chronic Lymphocytic Leukemia (CLL), Controls Establishment of Follicular and Marginal Zone B Cell Compartments in Mice

Abstract 285Chronic lymphocytic leukemia (CLL) originates from the malignant transformation of mature B cells. Recently, single nucleotide polymorphisms (SNPs) in the 3'UTR region of the Irf4 gene have been associated with an increased risk of developing CLL in independent patient cohorts. IRF4 is a member of the interferon regulatory factor (IRF) family of transcription factors, and in the B lineage is essential for plasma cell differentiation in T-dependent immune responses. Irf4 has been demonstrated to act as an oncogene in multiple myeloma. Conversely, evidence suggests that the corresponding SNPs in the Irf4 gene lead to a reduction of IRF4 mRNA expression, and it was reported that tumor cells in half of CLL cases show reduced IRF4 protein expression compared to normal B cells. Together, these observations suggest that aberrant downregulation of IRF4 expression in mature B cells may contribute to CLL development. However, the normal function of IRF4 in mature B cells is incompletely understood. In order to investigate how IRF4 deficiency affects the biology of mature B cells, we investigated the consequences of deleting Irf4 specifically in B cells in vivo using a conditional Irf4 knockout mouse line.We and others had previously observed that Irf4−/− mice develop an expansion of B cells with a marginal zone (MZ) phenotype (IgMhiIgDloCD23–CD21+). By demarcating the MZ with the MOMA-1 marker, we here show that B cells in Irf4−/− mice localized preferentially in the MZ area, causing MZ hyperplasia. In contrast, the area where follicular B cells normally home contained few B cells. B cell autonomy of the observed phenotype was ascertained by crossing a “floxed” Irf4 allele with CD19-Cre mice to achieve B cell conditional deletion. We then crossed the floxed Irf4 allele with a transgenic mouse that allows inducible deletion of Irf4 specifically in B cells. Whereas flow-cytometric analysis revealed an unchanged ratio between cells with a follicular (IgMloIgDhiCD23+CD21int) vs. a MZ B cell surface phenotype upon Irf4 deletion, immunohistochemical (IHC) stainings of spleen sections for a marker protein (eGFP) that signals gene deletion demonstrated that the Irf4-deleted cells localized preferentially in the MZ, leading to MZ hyperplasia. Together, these results suggest that deletion of Irf4 in B cells alters chemokine responsiveness and migratory capacity. In agreement, global gene expression profile analysis of B cells purified from Irf4−/− and Irf4+/+ mice identified a set of differentially expressed genes with known functions in cell migration and homing. Notably, PLXND1 and the chemokine receptor CXCR7 showed 4.5 and 6.5-fold upregulation, respectively, while the G protein coupled receptor RGS13 and the adhesion molecule ALCAM (CD166) showed 3.5 and 5-fold downregulation in Irf4−/− vs. Irf4+/+ B cells.Unexpectedly, we observed in the profiling analysis that expression of the known NOTCH target genes Deltex1 and Hes5 was significantly upregulated (3 and 5-fold) in Irf4−/− vs. Irf4+/+ B cells. Despite unchanged NOTCH1 and NOTCH2 mRNA levels, Western blot and immunofluorescence analysis showed that Notch2, a gene known to be indispensible for MZ B cell development, was strongly upregulated in Irf4−/− B cells, suggesting that IRF4 is indirectly involved in NOTCH2 repression at a post-transcriptional level. Together with the altered migratory properties of Irf4−/− vs. Irf4+/+ B cells, these findings indicate that a balanced expression of IRF4 and NOTCH2 in B cells is required for establishing the follicular and MZ B cell compartments in mice, and suggest that IRF4 maintains the cellular identity of follicular B cells.The results may have implications for understanding CLL pathogenesis, as both NOTCH1 and NOTCH2 transmembrane receptors were reported to be expressed and activated in CLL B cells, and since NOTCH1 was recently found to be aberrantly activated in a fraction of CLL cases due to genetic mutations. Alterations in the balance of the transcriptional network established by NOTCH and IRF4 either through mutations, polymorphisms, or microenvironmental factors may disrupt normal B cell physiology and thereby contribute to tumorigenesis by an as yet unknown mechanism. Recently, a small fraction of CLL patients were identified that have a recurrent heterozygous somatic mutation in exon 2 of Irf4, providing additional rationale for determining how alterations in IRF4 function may promote CLL pathogenesis. Disclosures:No relevant conflicts of interest to declare.

JAK2 exon 14 Skipping in Patients with Primary Myelofibrosis (PMF),

Abstract 3844In recent years the diagnosis and treatment of patients with myeloproliferative disorders has focused on the JAK2 gene. Recently, an alternative splicing of JAK2 transcript lacking the entire exon 14 (88bp), has been described (Ma et al. (2010) PLoS ONE, 5, e12165). This mRNA has a stop codon in exon 15 and is translated into a truncated protein that has a deletion within the JH2 pseudokinase domain and complete deletion of the kinase domain (JH1). The alternative transcript was detected in plasma of patients with myeloproliferative neoplasms (MPNs) (58% of patients positive for the V617F mutation and 33% of negative) in proportions ranging from 2% to 26% compared to the normal transcript, but never in healthy subjects. However, quantification of the transcript was performed by quantitative fragment length analysis, which is not the most appropriate method for such determination especially if, as in this case, the PCR is not limited to the exponential phase (Cirigliano, V. et al. (2001) Mol. Hum. Reprod., 7, 1001–1006).We investigate the mRNA levels of JAK2 and its splicing variant lacking exon 14, in granulocytes isolated from peripheral blood of 9 healthy individuals and 41 patients diagnosed with primary myelofibrosis (19 negative and 22 positive for the JAK2 V617F mutation). To overcome the limitations of quantitative fragment length analysis, the levels of the splice variant were assessed by qPCR, using a boundary-spanning primer designed on the junction between exons 13 and 15 (Vandenbroucke II, et al. (2001) Nucleic Acids Res., 29:E68). The JAK2 V617F allele burden was measured as previously described (Lippert, E. et al. (2006) Blood, 108, 1865–1867).Low levels of the alternative-spliced product was found in all RNA samples from patients and from all healthy individuals, in proportions ranging from 0.3 % to 3.5% compared to the normal transcript. The wild-type patients (95% C.I.: 0.3–0.8%) did not differ from controls (95% C.I.: 0.4–0.7%) while, in patients with the JAK2 V617F mutation, the levels correlates with allele burden (R^2= 0.513; p= 0.0002) (Figure 1). [Display omitted] To explain this observation we performed a bioinformatic analysis of the exon 14 sequence, using the software Human Splice Finder 2.4.1 (Desmet, F.O. et al. (2009) Nucleic Acids Res37 (e67) and ESE finder 3.0 (Cartegni, L. et al. (2003) Nucleic Acids Res31 (13)). The G/T transversion causing the aminoacidic substitution V617F also breaks an high-score, putative, Exon Splicing Enhancer (ESE) sequence that potentially binds the splicing regulator protein SRP55 (TGTGTC; positions 70 to 75 of exon 14). The higher level of the splice variant found in patients with the mutation could be due to an alteration of the binding of molecular factors that determine the inclusion of exon 14 in mature transcripts. The low levels of alternative transcripts observed, could be due to the presence of a stop codon in exon 15 as a result of the frameshift caused by the exclusion of exon 14. We know that a stop codon at more than 55 bases upstream of the last exon junction (in this case between exons 24 and 25) determines the activation of the Nonsense Mediated mRNA Decay (NMD) system and the consequent destruction of the messenger. In order to knowhow a small percentage of mRNA, subjected to degradation by NMD system, can produce the predicted 70 KDa protein that has been detected after immunoprecipitation and western blot analysis using JAK2 N-terminal antibodies, we are characterizing the 3' end of the alternative transcript.We show that the somatic mutation in exon 14 of JAK2 gene, in addition to the effects caused by the V617F amino acid change, has a moderate influence on splicing, increasing the levels of a splicing variant that is produced in small amounts also in healthy subjects. Our study demonstrates the efficacy of qPCR technique and the predictive power of bioinformatics tools used here. Further studies are needed to understand how this isoform is processed at the post-transcriptional level and whether there are significant effects on clinical phenotype. Disclosures:Barosi:Novartis: Membership on an entity's Board of Directors or advisory committees.

2.15 The Irf4 Gene, a Susceptibility Locus for Chronic Lymphocytic Leukemia, Controls Establishment of Follicular and Marginal Zone B-Cell Compartments in Mice

Abstract 285 Chronic lymphocytic leukemia (CLL) originates from the malignant transformation of mature B cells. Recently, single nucleotide polymorphisms (SNPs) in the 39UTR region of the Irf4 gene have been associated with an increased risk of developing CLL in independent patient cohorts. IRF4 is a member of the interferon regulatory factor (IRF) family of transcription factors, and in the B lineage is essential for plasma cell differentiation in T-dependent immune responses. Irf4 has been demonstrated to act as an oncogene in multiple myeloma. Conversely, evidence suggests that the corresponding SNPs in the Irf4 gene lead to a reduction of IRF4 mRNA expression, and it was reported that tumor cells in half of CLL cases show reduced IRF4 protein expression compared to normal B cells. Together, these observations suggest that aberrant downregulation of IRF4 expression in mature B cells may contribute to CLL development. However, the normal function of IRF4 in mature B cells is incompletely understood. In order to investigate how IRF4 deficiency affects the biology of mature B cells, we investigated the consequences of deleting Irf4 specifically in B cells in vivo using a conditional Irf4 knockout mouse line. We and others had previously observed that Irf4 −/− mice develop an expansion of B cells with a marginal zone (MZ) phenotype (IgM hi IgD lo CD23 – CD21 + ). By demarcating the MZ with the MOMA-1 marker, we here show that B cells in Irf4 −/− mice localized preferentially in the MZ area, causing MZ hyperplasia. In contrast, the area where follicular B cells normally home contained few B cells. B cell autonomy of the observed phenotype was ascertained by crossing a “floxed” Irf4 allele with CD19-Cre mice to achieve B cell conditional deletion. We then crossed the floxed Irf4 allele with a transgenic mouse that allows inducible deletion of Irf4 specifically in B cells. Whereas flow-cytometric analysis revealed an unchanged ratio between cells with a follicular (IgM lo IgD hi CD23 + CD21 int ) vs. a MZ B cell surface phenotype upon Irf4 deletion, immunohistochemical (IHC) stainings of spleen sections for a marker protein (eGFP) that signals gene deletion demonstrated that the Irf4 -deleted cells localized preferentially in the MZ, leading to MZ hyperplasia. Together, these results suggest that deletion of Irf4 in B cells alters chemokine responsiveness and migratory capacity. In agreement, global gene expression profile analysis of B cells purified from Irf4 −/− and Irf4 +/+ mice identified a set of differentially expressed genes with known functions in cell migration and homing. Notably, PLXND1 and the chemokine receptor CXCR7 showed 4.5 and 6.5-fold upregulation, respectively, while the G protein coupled receptor RGS13 and the adhesion molecule ALCAM (CD166) showed 3.5 and 5-fold downregulation in Irf4 −/− vs. Irf4 +/+ B cells. Unexpectedly, we observed in the profiling analysis that expression of the known NOTCH target genes Deltex1 and Hes5 was significantly upregulated (3 and 5-fold) in Irf4 −/− vs. Irf4 +/+ B cells. Despite unchanged NOTCH1 and NOTCH2 mRNA levels, Western blot and immunofluorescence analysis showed that Notch2 , a gene known to be indispensible for MZ B cell development, was strongly upregulated in Irf4 −/− B cells, suggesting that IRF4 is indirectly involved in NOTCH2 repression at a post-transcriptional level. Together with the altered migratory properties of Irf4 −/− vs. Irf4 +/+ B cells, these findings indicate that a balanced expression of IRF4 and NOTCH2 in B cells is required for establishing the follicular and MZ B cell compartments in mice, and suggest that IRF4 maintains the cellular identity of follicular B cells. The results may have implications for understanding CLL pathogenesis, as both NOTCH1 and NOTCH2 transmembrane receptors were reported to be expressed and activated in CLL B cells, and since NOTCH1 was recently found to be aberrantly activated in a fraction of CLL cases due to genetic mutations. Alterations in the balance of the transcriptional network established by NOTCH and IRF4 either through mutations, polymorphisms, or microenvironmental factors may disrupt normal B cell physiology and thereby contribute to tumorigenesis by an as yet unknown mechanism. Recently, a small fraction of CLL patients were identified that have a recurrent heterozygous somatic mutation in exon 2 of Irf4 , providing additional rationale for determining how alterations in IRF4 function may promote CLL pathogenesis. Disclosures: No relevant conflicts of interest to declare.

Somatic mutations in exon 17 of the TEK gene in vascular tumors and vascular malformations

As a common disease, the molecular etiology of noninherited vascular anomalies is still poorly understood. Recently, somatic mutations in exon 17 of the endothelial cell tyrosine kinase receptor Tie-2 (encoded by TEK) were identified in 49.1% of patients with common sporadic venous malformation, a subtype of vascular anomalies. We assessed whether such a mutational region also had a role in the Chinese population or in other subtypes of noninherited vascular anomalies (vascular tumors and vascular malformations). Direct sequencing of polymerase chain reaction (PCR)-amplified DNA, extracted from 139 lesions in 129 individuals with noninherited vascular anomalies (vascular tumors or vascular malformations) and 60 control samples, was used for detecting the mutations in exon 17 of the TEK gene. Mutations were confirmed by allele-specific PCR. Clone sequences were then used for the mutations identified for the first time. We also explored the associations between these mutations and clinical characteristics (gender, onset age, number of lesions, severity, category, and recurrence of the disease) in both vascular tumors and vascular malformations. Two somatic TEK mutations (Y897C, R915C) were identified in vascular tumors, and seven somatic TEK mutations (Y897H, Y897C, L914F, R915C, S917I, R918C, R918H) were identified in vascular malformations. Among these mutations, R918C (2,752 C > T) and R918H (2,753 G > A) were first identified in noninherited vascular anomalies. Somatic TEK mutations were detected in lesions from 4 of 23 (17.4%) vascular tumors and 35 of 106 (33.0%) vascular malformations, where most mutations were single substitutions in vascular tumors (100%) and vascular malformations (88.6%), while the remainders were double substitutions. In addition to the reported venous malformation, such mutations were identified in some other subtypes of vascular anomalies, including vascular tumors (infantile hemangioma, pyogenic granuloma, and epithelioid hemangioma) and vascular malformations (capillary malformation, arteriovenous malformation, capillary lymphatic malformation, and capillary arteriovenous malformation). By contrast, these mutations were absent from the control tissues or blood. However, mutations showed no association (P > .05) with clinical characteristics in vascular anomalies or either of its two types (vascular tumors or vascular malformations). Our study revealed that somatic mutations in exon 17 of the TEK gene were more common in noninherited vascular anomalies than previously reported. Furthermore, such substitutions may shed new light on the molecular etiology, diagnosis, and potential therapeutic targets of vascular anomalies.

A case of mast cell leukaemia with exon 9 KIT mutation and good response to imatinib

Mastocytosis is a myeloproliferative neoplasm characterized by the excessive proliferation of mast cells. Mast cell leukaemia (MCL), the aggressive form of this disease, requires cytoreductive therapy, such as cladribine, interferon-alpha-2b and, most recently, tyrosine kinase inhibitors - dasatinib or imatinib. We present a case of a 56-yr-old female patient with aleukaemic MCL in whom the typical KIT-D816V mutation was not detected. Sequencing of the entire coding sequence of KIT gene revealed a somatic mutation in exon 9 (p.A502_Y503dup). This mutation was previously reported in patients with gastrointestinal stromal tumours (GIST). Considering the good response to imatinib in such patients, therapy with imatinib was attempted in our patient. The treatment tolerance and outcomes were very good, with reduced mast cell infiltration of the bone marrow, normalization of the serum tryptase concentration and resolution of the clinical signs and symptoms. In the absence of the KIT-D816V in systemic forms of mast cell proliferation, a search for other mutations is indicated, preferably by sequencing the entire KIT gene, as this can influence the choice of treatment. The finding of the p.A502_Y503dup in exon 9, a mutation which has been observed in GIST but not previously reported in any form of aggressive mastocytosis, can be associated with a good response to imatinib in both diseases.