Millaray Marincevic

High-density Screening Reveals a Different Spectrum of Genomic Aberrations in Chronic Lymphocytic Leukemia Patients with ‘Stereotyped’ IGHV3-21 and IGHV4-34 B-cell Receptors

Background The existence of multiple subsets of chronic lymphocytic leukemia expressing ‘stereotyped’ B-cell receptors implies the involvement of antigen(s) in leukemogenesis. Studies also indicate that ‘stereotypy’ may influence the clinical course of patients with chronic lymphocytic leukemia, for example, in subsets with stereotyped IGHV3-21 and IGHV4-34 B-cell receptors; however, little is known regarding the genomic profile of patients in these subsets. Design and Methods We applied 250K single nucleotide polymorphism-arrays to study copy-number aberrations and copy-number neutral loss-of-heterozygosity in patients with stereotyped IGHV3-21 (subset #2, n=29), stereotyped IGHV4-34 (subset #4, n=17; subset #16, n=8) and non-subset #2 IGHV3-21 (n=13) and non-subset #4/16 IGHV4-34 (n=34) patients. Results Over 90% of patients in subset #2 and non-subset #2 carried copy-number aberrations, whereas 75–76% of patients in subset #4 and subset #16 showed copy-number aberrations. Subset #2 and non-subset #2 patients also displayed a higher average number of aberrations compared to patients in subset #4. Deletion of 13q was the only known recurrent aberration detected in subset #4 (35%); this aberration was even more frequent in subset #2 (79%). del(11q) was more frequent in subset #2 and non-subset #2 (31% and 23%) patients than in subset #4 and non-subset #4/16 patients. Recurrent copy-number neutral loss-of-heterozygosity was mainly detected on chromosome 13q, independently of B-cell receptor stereotypy. Conclusions Genomic aberrations were more common in subset #2 and non-subset #2 than in subset #4. The particularly high frequency of del(11q) in subset #2 may be linked to the adverse outcome reported for patients in this subset. Conversely, the lower prevalence of copy-number aberrations and the absence of poor-prognostic aberrations in subset #4 may reflect an inherently low-proliferative disease, which would prevent accumulation of genomic alterations.

Distinct gene expression profiles in subsets of chronic lymphocytic leukemia expressing stereotyped IGHV4-34 B-cell receptors

Background Numerous subsets of patients with chronic lymphocytic leukemia display similar immunoglobulin gene usage with almost identical complementarity determining region 3 sequences. Among IGHV4-34 cases, two such subsets with “stereotyped” B-cell receptors were recently identified, i.e. subset #4 (IGHV4-34/IGKV2-30) and subset #16 (IGHV4-34/IGKV3-20). Subset #4 patients appear to share biological and clinical features, e.g. young age at diagnosis and indolent disease, whereas little is known about subset #16 at a clinical level. Design and Methods We investigated the global gene expression pattern in sorted chronic lymphocytic leukemia cells from 25 subset/non-subset IGHV4-34 patients using Affymetrix gene expression arrays. Results Although generally few differences were found when comparing subset to non-subset 4/16 IGHV4-34 cases, distinct gene expression profiles were revealed for subset #4 versus subset #16. The differentially expressed genes, predominantly with lower expression in subset #4 patients, are involved in important cell regulatory pathways including cell-cycle control, proliferation and immune response, which may partly explain the low-proliferative disease observed in subset #4 patients. Conclusions Our novel data demonstrate distinct gene expression profiles among patients with stereotyped IGHV4-34 B-cell receptors, providing further evidence for biological differences in the pathogenesis of these subsets and underscoring the functional relevance of subset assignment based on B-cell receptor sequence features.

Aberrantly activated claudin 6 and 18.2 as potential therapy targets in non-small-cell lung cancer

Claudins (CLDNs) are central components of tight junctions that regulate epithelial‐cell barrier function and polarity. Altered CLDN expression patterns have been demonstrated in numerous cancer types and lineage‐specific CLDNs have been proposed as therapy targets. The objective of this study was to assess which fraction of patients with non‐small‐cell lung cancer (NSCLC) express CLDN6 and CLDN18 isoform 2 (CLDN18.2). Protein expression of CLDN6 and CLDN18.2 was examined by immunohistochemistry on a tissue microarray (n = 355) and transcript levels were supportively determined based on gene expression microarray data from fresh‐frozen NSCLC tissues (n = 196). Both were analyzed with regard to frequency, distribution and association with clinical parameters. Immunohistochemical analysis of tissue sections revealed distinct membranous positivity of CLDN6 (6.5%) and CLDN18.2 (3.7%) proteins in virtually non‐overlapping subgroups of adenocarcinomas and large‐cell carcinomas. Pneumocytes and bronchial epithelial cells were consistently negative. Corresponding to the protein expression, in subsets of non‐squamous lung carcinoma high mRNA levels of CLDN6 (7–16%) and total CLDN18 (5–12%) were observed. Protein expression correlated well with total mRNA expression of the corresponding gene (rho = 0.4–0.8). CLDN18.2 positive tumors were enriched among slowly proliferating, thyroid transcription factor 1 (TTF‐1)‐negative adenocarcinomas, suggesting that isoform‐specific CLDN expression may delineate a specific subtype. Noteworthy, high CLDN6 protein expression was associated with worse prognosis in lung adenocarcinoma in the univariate [hazard ratio (HR): 1.8; p = 0.03] and multivariate COX regression model (HR: 1.9; p = 0.02). These findings encourage further clinical exploration of targeting ectopically activated CLDN expression as a valuable treatment concept in NSCLC.

Distinct transcriptional control in major immunogenetic subsets of chronic lymphocytic leukemia exhibiting subset-biased global DNA methylation profiles.

Chronic lymphocytic leukemia (CLL) can be divided into prognostic subgroups based on the IGHV gene mutational status, and is further characterized by multiple subsets of cases with quasi-identical or stereotyped B cell receptors that also share clinical and biological features. We recently reported differential DNA methylation profiles in IGHV-mutated and IGHV-unmutated CLL subgroups. For the first time, we here explore the global methylation profiles of stereotyped subsets with different prognosis, by applying high-resolution methylation arrays on CLL samples from three major stereotyped subsets: the poor-prognostic subsets #1 (n = 15) and #2 (n = 9) and the favorable-prognostic subset #4 (n = 15). Overall, the three subsets exhibited significantly different methylation profiles, which only partially overlapped with those observed in our previous study according to IGHV gene mutational status. Specifically, gene ontology analysis of the differentially methylated genes revealed a clear enrichment of genes involved in immune response, such as B cell activation (e.g., CD80, CD86 and IL10), with higher methylation levels in subset #1 than subsets #2 and #4. Accordingly, higher expression of the co-stimulatory molecules CD80 and CD86 was demonstrated in subset #4 vs. subset #1, pointing to a key role for these molecules in the crosstalk of CLL subset #4 cells with the microenvironment. In summary, investigation of three prototypic, stereotyped CLL subsets revealed distinct DNA methylation profiles for each subset, which suggests subset-biased patterns of transcriptional control and highlights a key role for epigenetics during leukemogenesis.

Mantle cell lymphoma displays a homogenous methylation profile: A comparative analysis with chronic lymphocytic leukemia

Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) are mature CD5+ B‐cell malignancies with different biological/clinical characteristics. We recently reported an association between different prognostic subgroups of CLL (i.e., IGHV mutated and unmutated) and genomic methylation pattern. However, the relationship between DNA methylation and prognostic markers, such as the proliferation gene expression signature, has not been investigated in MCL. We applied high‐resolution methylation microarrays (27,578 CpG sites) to assess the global DNA methylation profiles in 20 MCL (10 each with high/low proliferation signature) and 30 CLL (15 poor‐prognostic IGHV unmutated subset #1 and 15 good‐prognostic IGHV mutated subset #4) samples. Notably, MCL and each CLL subset displayed distinct genomic methylation profiles. After unsupervised hierarchical clustering, 17/20 MCL cases formed a cluster separate from CLL, while CLL subsets #1 and #4 formed subclusters. Surprisingly, few differentially methylated genes (n = 6) were identified between high vs. low proliferation MCL. In contrast, distinct methylation profiles were demonstrated for MCL and CLL. Importantly, certain functional classes of genes were preferentially methylated in either disease. For instance, developmental genes, in particular homeobox transcription factor genes (e.g., HLXB9, HOXA13), were more highly methylated in MCL, whereas apoptosis‐related genes were enriched among targets methylated in CLL (e.g., CYFIP2, NR4A1). Results were validated using pyrosequencing, RQ‐PCR and reexpression of specific genes. In summary, the methylation profile of MCL was homogeneous and no correlation with the proliferation signature was observed. Compared to CLL, however, marked differences were discovered such as the preferential methylation of homeobox genes in MCL. Am. J. Hematol., 2012.

Prognostic impact of COX-2 in non-small cell lung cancer : a comprehensive compartment-specific evaluation of tumor and stromal cell expression

Cyclooxygenase-2 (COX-2) is an enzyme that has been extensively investigated as a prognostic marker in cancer. In non-small cell lung cancer (NSCLC) previous results regarding the prognostic impact of COX-2 expression are inconsistent. Therefore we evaluated the association between transcript levels and overall survival in nine publicly available gene expression data sets (total n = 1337) and determined in situ compartment-specific tumor and stromal cell protein expression in two independent cohorts (n = 616). Gene expression did not show any correlation with clinical parameters or with overall survival. Protein expression in tumor and stromal cells did not correlate with any clinical parameter or with overall survival in one of the analyzed cohorts, while a significant association of high stromal expression with longer survival was observed in both univariate and multivariate analysis in the other cohort. Stromal expression of COX-2 has not been separately evaluated in NSCLC previously and may be a subject of further investigation, whereas the presented findings from this comprehensive compartment specific evaluation clearly reject the hypothesis of COX-2 tumor cell expression having a prognostic value in NSCLC.

Infrequent occurrence of PIK3CA mutations in chronic lymphocytic leukemia.

The phosphoinositide 3-kinase (PI3K) pathway is central to many different cell signalling pathways associated with cell growth, cell survival, cell movement and apoptosis [1]. PI3Ks are lipid kinases involved in phosphorylation of the inositol ring of phosphatidylinositol, which is a necessary component in signal transduction [2]. Class I PI3Ks are heterodimeric molecules composed of a catalytic subunit (p110a), also known as PIK3CA, together with a regulatory subunit. In recent years, several mutational and functional analyses of PIK3CA have shown that this catalytic subunit may function as an activating oncogene. PIK3CA mutations have frequently been detected in several cancer types, such as breast (8–27%), gastric (6–25%) and colon (11–32%) cancer [3,4]. Less is known about PIK3CA mutations in hematological malignancies, although screening studies in acute myeloid leukemia, acute lymphoblastic leukemia and non-Hodgkin lymphomas (subtypes not specified) have revealed a mutational frequency of 51% [4,5]. In contrast, a recent study on diffuse large B-cell lymphoma detected mutations in 17 out of 215 cases (8%) [6]. AKT is located downstream of PI3K and also serves as an important molecule in cellular signalling. Since several biological events including apoptosis are dependent on the PI3K-AKT signalling pathway, this is of particular interest in chronic lymphocytic leukemia (CLL), a disease where defects in apoptosis are considered to be a main feature. PI3K has been shown to be constitutively activated in CLL and furthermore linked to the expression of PKC isoforms [7,8]. The AKT signalling pathway has also been studied in CLL whereby stronger activation of AKT is associated with progressive disease [9]. Previous studies in solid tumors have identified a point mutation (E17) that leads to the activation of AKT1, however, a similar study in CLL and ALL could not detect any such mutation [10,11]. To this end, we sought to investigate any potential association between PIK3CA mutational status and the constitutive activation of PI3K in CLL and have performed mutational analysis of PIK3CA in 61 Swedish CLL cases (median age of diagnosis 64 years and male:female ratio 1.7:1). Previous sequencing results showed that 27/61 cases (44%) carried unmutated IGHV genes whilst 34/61 (56%) had less than 98% identity and were therefore classified as mutated. Furthermore, FISH analysis on 38/61 cases revealed 18 cases with 13q deletion, 8 with 11q deletion, 7 with 17p deletion (including two cases classified as ‘borderline’, 10–15%), one trisomy 12 case and five cases carrying no genetic aberration. PCR amplification using a proof-reading enzyme (AccuPrime Pfx, Invitrogen, USA) and subsequent sequence analysis of PIK3CA was performed to target two hotspot regions, Exon 9 and Exon 20, which have been reported to contain a high mutational load in various cancer forms. A comparison was made between the sequences and the PIK3CA cDNA sequence (GeneBank accession number MN_006218.2). Overall, only two mutations were detected in Exon 20, i.e. the kinase domain of PIK3CA (corresponding to the catalytic domain), whereas no mutations were observed in Exon 9. The first mutation was found at

Abstract A37: Ectopic claudin 6 and 18.2 expression as potential treatment target in non-small cell lung cancer.

Claudins are central components of tight junctions that regulate epithelial cell barrier function and polarity. Altered claudin expression patterns have been demonstrated in numerous cancer types and lineage-specific claudins have been proposed as therapy targets. The objective of this study was to assess claudin 6 (CLDN6) and claudin 18 isoform 2 (CLDN18.2) expression in human non-small cell lung cancer (NSCLC). Transcript levels were evaluated based on gene expression microarray data from a fresh-frozen NSCLC tissue cohort (n=196). Protein expression levels of CLDN6 and CLDN18.2 were examined by immunohistochemistry on a tissue microarray (n=355), and analyzed with regard to frequency distribution and association with clinical parameters. High mRNA levels of CLDN6 and total CLDN18 were observed in subsets of non-squamous lung carcinomas. Correspondingly, distinct membranous positivity of CLDN6 (6.5%) and CLDN18.2 (3.7%) was displayed by virtually non-overlapping subgroups of adenocarcinomas and large cell carcinomas, while pneumocytes and bronchial epithelial cells were consistently negative. CLDN18.2 positive tumors were enriched among slowly proliferating, TTF1-negative adenocarcinomas, suggesting that isoform-specific claudin expression may delineate a specific subtype. Noteworthy, high CLDN6 mRNA as well as protein expression was associated with worse prognosis in lung adenocarcinoma. In conclusion, distinct CLDN6 and CLDN18.2 positivity was observed in minor subsets of non-squamous NSCLC, indicating that tumor-specific claudin expression may define molecular subtypes associated with clinical parameters. These findings encourage further clinical exploration of targeting ectopic claudin expression as a valuable treatment concept in NSCLC. Citation Format: Johanna SM Mattsson, Patrick Micke, Karolina Edlund, Miriam Lohr, Karin Jirstrom, Anders Berglund, Johan Botling, Jorg Rahnenfuehrer, Millaray Marincevic, Fredrik Ponten, Simon Ekman, Jan G. Hengstler, Stefan Woll, Ugur Sahin, Ozlem Tureci. Ectopic claudin 6 and 18.2 expression as potential treatment target in non-small cell lung cancer. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr A37.