Gain Of 2q Research Articles

An 8q24 Gain in Pancreatic Juice Is a Candidate Biomarker for the Detection of Pancreatic Cancer

Secretin-stimulated pancreatic juice (PJ), collected from the duodenum, presents a valuable biomarker source for the (earlier) detection of pancreatic cancer (PC). Here, we evaluate the feasibility and performance of shallow sequencing to detect copy number variations (CNVs) in cell-free DNA (cfDNA) from PJ for PC detection. First, we confirmed the feasibility of shallow sequencing in PJ (n = 4), matched plasma (n = 3) and tissue samples (n = 4, microarray). Subsequently, shallow sequencing was performed on cfDNA from PJ of 26 cases (25 sporadic PC, 1 high-grade dysplasia) and 19 controls with a hereditary or familial increased risk of PC. 40 of the 45 PJ samples met the quality criteria for cfDNA analysis. Nine individuals had an 8q24 gain (oncogene MYC; 23%; eight cases (33%) and one control (6%), p = 0.04); six had both a 2q gain (STAT1) and 5p loss (CDH10; 15%; four cases (7%) and two controls (13%), p = 0.72). The presence of an 8q24 gain differentiated the cases and controls, with a sensitivity of 33% (95% CI 16-55%) and specificity of 94% (95% CI 70-100%). The presence of either an 8q24 or 2q gain with a 5p loss was related to a sensitivity of 50% (95% CI 29-71%) and specificity of 81% (95% CI 54-96%). Shallow sequencing of PJ is feasible. The presence of an 8q24 gain in PJ shows promise as a biomarker for the detection of PC. Further research is required with a larger sample size and consecutively collected samples in high-risk individuals prior to implementation in a surveillance cohort.

Chromosome 2q gain and epigenetic silencing of GATA3 in microglandular adenosis of the breast.

Microglandular adenosis (MGA) represents a rare neoplasm of the mammary gland, which in a subset of cases may be associated with triple‐negative breast cancer (BC). The biology of MGA is poorly understood. In this study, eight MGA cases (n = 4 with and n = 4 without associated BC) were subjected to a comprehensive characterization using immunohistochemistry, genome‐wide DNA copy number (CN) profiling, fluorescence in situ hybridization (FISH), next‐generation sequencing (NGS), and DNA methylation profiling using 850 K arrays and bisulfite pyrosequencing. Median patient age was 61 years (range 57–76 years). MGA lesions were estrogen receptor (ER)‐negative, progesterone receptor‐negative, HER2‐negative, and S100‐positive. DNA CN alterations (CNAs) were complex or limited to few gains and losses. CN gain on chromosome 2q was the most common CNA and was validated by FISH in five of eight cases. NGS demonstrated an average of two mutations per case (range 0–5) affecting 10 different genes (ARID1A, ATM, CTNNB1, FBXW7, FGFR2, MET, PIK3CA, PMS2, PTEN, and TP53). CNAs and mutations were similar in MGA and adjacent BC, indicating clonal relatedness. DNA methylation profiling identified aberrant hypermethylation of CpG sites within GATA3, a key transcription factor required for luminal differentiation. Immunohistochemistry showed regular GATA3 protein expression in the normal mammary epithelium and in ER‐positive BC. Conversely, GATA3 was reduced or lost in all MGA cases tested (8/8). In conclusion, MGA is characterized by common CN gain on chromosome 2q and loss of GATA3. Epigenetic inactivation of GATA3 may provide a new clue to the peculiar biology of this rare neoplasia.

Papillary Glioneuronal Tumors

This study was designed to evaluate 4 new cases of papillary glioneuronal tumors (PGNTs), 2 of which had atypical histologic features, provides extensive IHC characterization, performed comparative genomic hybridization in 2 of our cases, and reviews the recent literature. The study group comprised 3 women and 1 man, with ages ranging from 12 to 75 years. Patients presented with seizures (n = 3) or muscle spasm (n = 1), and the tumors were located in the supratentorial region of the brain (3 in the frontal and 1 in the parietotemporal lobe). The 2 atypical tumors showed a moderately high mitotic rate (4 and 7/10 HPF, each), vascular endothelial hyperplasia, and necrosis. Tumor cells expressed both neuronal and glial markers, but the degree of neuronal versus glial expression was varied. None of the tumors expressed p53, EGFR wild type/vIII, IDH1, or CD34; however, nestin, galectin-3, and S100 were positive in the tumor cells. No EGFR gene amplification or 1p/19q deletion was found by fluorescence in situ hybridization. Half of the cases revealed PTEN loss by immunohistochemistry, and MGMT methylation was positive in 3 cases by MGMT methylation-specific polymerase chain reaction. Ultrastructurally, either astrocytic or neuronal differentiation was observed, but we could not identify any hybrid cells. An array-based comparative genomic hybridization study revealed loss of 1q, 6p, 8p, 9p, 9q, and 16q and gain of 2q, 3p, 5q, 6p, 7q, 10q, 16q, 19p, and 22q in 2 cases simultaneously. The first patient, who underwent subtotal resection, died because of progression of the tumor within 9 months after surgery; however, 2 patients were symptom free and progression free at 34 and 48 months after gross total resection (the patient 2: plus radiotherapy, the patient 3: no adjuvant chemo- or radiotherapy). The last patient developed seizures after a long symptom-free period (40 mo) with no evidence of tumor recurrence. Our 4 new cases, in conjunction with the literature review, reinforce that PGNTs are tumors that usually occur in young adults (mean age, 24 y); they are most often cystic with a mural nodule or are cystic/solid, supratentorial, closely located with the ventricle, molecularly genetically different from astrocytic or oligodendroglial tumors, and indolent in behavior. Cases (2 of 4 in our study) with atypical histologic features or that occur in advanced age (75 y), and sporadic reports of histologically or biologically aggressive PGNTs, serve to remind pathologists that the full spectrum of PGNTs is as yet unknown.

Abstract B6: Patterns of DNA copy number changes in primary breast cancer and brain metastases

Abstract Metastatic breast cancer remains essentially incurable, with mortality being especially high in patients who develop brain metastases. Approximately 15% of all epithelial tumors metastasize to the brain, with incidence rates highly dependent on the primary tumor type. Whereas colorectal cancer very seldom metastasizes to the brain (1–5%), breast cancer (15–20%) commonly metastasizes to the brain. Apparently, the brain microenvironment is specifically permissive for the growth of disseminated tumor cells from some carcinomas but not others. It is unknown by what mechanism the metastatic tumor cells adapt to the selection pressure exerted by the brain microenvironment. In this study we wanted to identify molecular breast tumor markers associated with metastatic spread to the brain. The CGH array profiles of 30 tumors from primary breast cancer patients were compared to 10 tumors of brain metastases from breast cancer cases. Copy number changes were found in all samples, and every chromosome arm (X chromosome and acrocentric short arms were excluded from the analysis). Overall, the CGH results of the primary breast tumors were in agreement with those described before. The brain metastases showed in general similar aberration patterns as the primary tumors. However, in the brain metastases a notably higher frequency of gains and losses could be found almost at every chromosome locus. Only gain of 1p and loss of 16q described as favorable markers for breast cancer were more common in the primary tumors. Statistically significant (multiarray p-value <0.05) differences were found at 18 different chromosomal loci, nine loci were significantly more often lost and 7 loci gained in the brain metastases. Gains of 2q32-33 and 11q14-q21 and loss of most of chromosome 10q were unique findings to the brain metastases. Furthermore, an amplification of EGFR was found in 30% and a gain in 20% of the brain metastases, whereas 0% had an amplification and only 9.4% of the primary tumors showed a gain of EGFR. The aberration patterns of 10q and EGFR was verified by FISH and LOH analysis. In general, no major difference in the number and distribution of the high-level amplifications (20 loci in primary tumors and 11 in brain metastases) could be found between the primary and metastatic tumors. Most of the amplifications were unique to only one case, with only 5 loci in common for both primary tumors and metastases and 11 loci found in more than one cases. In conclusion, the results show a similarity of genetic aberration patterns between primary and metastatic tumors, i.e. the metastases carried the majority of the genetic alterations present in the corresponding primary tumors but with a significantly higher frequency. This would imply that brain metastases have occurred at a late stage of cancer progression. However, specific aberrations like gain of 2q and loss of 10q could almost exclusively be found in the metastases. Some of these markers could be useful to define a subgroup of high-risk patients and might have implications for their treatment management. Citation Information: Clin Cancer Res 2010;16(7 Suppl):B6

Cytogenetic evaluation of neuroblastoma using fine needle aspiration cultures

Open surgical biopsies of the primary tumor and bone marrow sampling in metastatic disease are the major sources of tumor material for genetic studies in neuroblastoma. The possibility of using cultures of material procured through Fine Needle Aspiration (FNA) for upfront cytogenetic studies of neuroblastomas is studied. From January 2006 to April 2008, 18 patients were diagnosed as neuroblastoma on fine needle aspiration cytology (FNAC). FNA material was cultured and processed to obtain metaphases and chromosomal analysis was performed. Minimum of five selected metaphases were analyzed and changes were documented. The diagnosis of neuroblastoma was confirmed by FNAC in all. Among the 18 cases, 9 cases (50%) showed metaphases on cultures. Four cultures did not grow and five cultures got contaminated. Out of the nine cases that showed metaphases, four cases showed cytogenetic abnormalities. Near triploidy and Double minutes (Dmins) were seen in one case and tetraploidy in another case. 1q gain and 2q gain were observed respectively in third and fourth cases. FNA provides viable single cell suspension suitable for cultures. This may be a good alternative strategy to acquire material for cytogenetic studies in neuroblastoma.

Gain of the Short Arm of Chromosome 2 Is a New Recurring Chromosome Aberration in CLL Which Is Significantly Associated with an Unmutated IgGVH Status and a Complex Aberrant Karyotype and Leads to a Higher Expression of Genes Located on 2P.

CLL is a genetically heterogeneous disease. Genetic aberrations allow to distinguish different biological subgroups within CLL. Based on chromosome banding analysis we identified complete and partial gain of the short arm of chromosome 2 always including 2p23 to 2p25 in 28/1051 CLL cases (2.7%) as a new recurring chromosome aberration. Recurring aberrations accompanying gain of 2p were: loss of 1p (n=3), 1q (n=3), 2q (n=3), 6q (n=3), 8p (n=7), 11q (n=10), 12q (n=4), 13q (n=21), 17p (n=7), 17q (n=3), 18p (n=5), and gain of 2q (n=3), 3q (n=3), 13q (n=3), 21q (n=3). In 24/28 cases the mutational status of the immunoglobulin variable heavy chain gene (IgVH) was available. 20 cases showed an unmutated and only 4 a mutated IgVH status. Thus, 2p gain is significantly associated with an unmutated IgVH status as compared to the non 2p group (83% vs 51%, p=0.002). In 8 cases an ATM deletion (29%) and in 5 cases a TP53 deletion (18%) (1 case showed both) were observed (frequency in non 2p+ cohort: 12%; p=0.036 and 7%; p=0.031). A median number of 4 chromosome aberrations per case was observed in 2p+ CLL (range: 1–16, mean=5.2) as compared to only 1 abnormality per case in the non 2p+ cohort (range: 0–10, mean 1.7) (p<0.0001). In 4 cases the rearrangement leading to 2p+ was the sole abnormality. In 6 cases 2p gain was found in a subclone only. Gene expression analysis (Affymetrix, HG U133 Plus 2.0) was performed in 25 cases with gain of 2p and compared to 11 cases with CLL and normal karyotype. Using 10fold cross validation (10f CV) resulted in an assignment of all 25 cases with 2p into the correct class, however 4 cases with normal karyotype were misclassified into the 2p group (accuracy (a) 89%, sensitivity (s) 100%, specificity (sp) 64% for 2p+). Classification based on an independent test set led to comparable results (median a: 83%, s: 100%, sp: 50% for 2p+). In addition cases with gain of 2p were compared to 48 CLL cases comprising various chromosome aberrations excluding gain of 2p. Using 10f CV resulted in an assignment of only 13 of 25 cases with 2p into the correct class (a: 75%, s: 52%, sp: 88%). Classification based on an independent test set led to comparable results (median a: 79%, s: 50%, sp: 94%). However, focussing on the top 100 differentially expressed probe sets comprising 88 genes revealed that 10 of 28 genes with a higher expression in the 2p+ group as compared to the non 2p+ group were located on 2p (LOC56902, PPP3R1, MSH6, RTN4, COX7A2L, HADHA, TTC32, ACP1, CPSF3, PDIA6). These are involved in mismatch repair and in negative regulation of anti-apoptosis. In contrast only one of the top 60 genes showing a lower expression in the 2p+ group was located on 2p. Interestingly, ATM showed a significantly lower expression in the 2p+ group. In conclusion:1.Gain of 2p is a new recurrent chromosome abnormality in CLL. It occurs both as the sole abnormality and as a secondary event during clonal evolution.2.Gain of 2p is associated with an unmutated IgVH status, a complex aberrant karyotype, and a high frequency of ATM- and TP53-deletion.3.Comparable to other unbalanced chromosome abnormalities gain of 2p is not associated with a distinct gene expression profile sufficient for classification but leads to a higher expression of genes located on 2p.

Genomic copy number alterations in non-small cell lung cancers identified using CBS and MSA

7695 Background: Lung cancer is the leading cause of cancer death in both men and women with a 5-yr survival rate of 15.5%. Previous studies have begun to characterized genomic copy number alterations of non-small-cell lung CA using array CGH and SNP arrays. We have used aCGH using our 1MB BAC arrays and two algorthims for making copy number alteration (CNA) determinations. We have also pursued the exact copy number gains and losses of several genes using Q-PCR. Methods: Genomic DNA from fresh frozen tumors of 27 patients with NSCLC. We performed aCGH using 1MB Arrays. We used CBS, and MSA to identify regions of CNA. We further pursued several genes of interest (including HRAS, CRK, and CDC42) identified using Q-PCR. Unsupervised hierarchical clustering was performed to look for distinct subgroups. Significant Analysis of Microarrays (SAM) was applied to identify the association between CNAs clinical parameters including tumor subtype, gender, lymph node involvement, tumor stage, and overall survival. Results: 240 regions of amplification and 181 regions of deletions were found, and included all previously published regions implicated in lung cancer. CNAs in &gt; 70% of tumors included amplifications in 1q, 3q, 5p, 6p, 11p, 16q, 20q, and Xq, and deletions in 1p, 8p and 13q. We verified CNAs of HRAS, CRK and CDC42 using Q-PCR. Hierarchical clustering revealed 2 subgroups: one with amplifications in 2q, 4p, 4q, 8q, 21q, 15q, and 16p, and the other with amplifications in 3q, and 5q. These were confirmed by supervised SAM analysis. Using SAM we found that gain of 2q, 4p and 10q, and loss of 16p and 19q were significantly present in adenocarcinomas. (q = 0, FDR = 0%). Gain of 10q, and loss of 6p and 14q were associated with female gender. (q = 0, FDR = 0%). Conclusions: We used aCGH to identify CNAs that characterize non-small cell lung CA tumors with the aim of finding key regions which may harbor important oncogenes and tumor suppressors. Several regions of CNA have been identified, several of which have been associated with clinical parameters. Because much heterogeneity exists in non-small-cell lung tumors, we have demonstrated that clustering analysis is useful in identifying subtypes which may possess prognostic and therapeutic significance. No significant financial relationships to disclose.

Genome‐wide array‐based comparative genomic hybridization of natural killer cell lymphoma/leukemia: Different genomic alteration patterns of aggressive NK‐cell leukemia and extranodal Nk/T‐cell lymphoma, nasal type

Natural killer (NK) cell lymphomas/leukemias are highly aggressive lymphoid malignancies, but little is known about their genomic alterations, and thus there is an urgent need for identification and analysis of NK cell lymphomas/leukemias. Recently, we developed our own array-based comparative genomic hybridization (array CGH) with an average resolution of 1.3 Mb. We performed an array CGH analysis for 27 NK-cell lymphoma/leukemia cases that were classified into two disease groups based on the World Health Organization Classification (10 aggressive NK-cell leukemia cases and 17 extranodal NK/T-cell [NK/T] lymphomas, nasal type). We identified the differences in the genomic alteration patterns of the two groups. The recurrent regions characteristic of the aggressive NK-cell leukemia group compared with those of the extranodal NK/T lymphoma, nasal-type group, were gain of 1q and loss of 7p15.1-p22.3 and 17p13.1. In particular, gain of 1q23.1-24.2 (P = 0.041) and 1q31.3-q44 (P = 0.003-0.047), and loss of 7p15.1-p22.3 (P = 0.012-0.041) and 17p13.1 (P = 0.012) occurred significantly more frequently in the former than in the latter group. Recurrent regions characteristic of the extranodal NK/T lymphoma, nasal-type group, compared with those of the other group were gain of 2q, and loss of 6q16.1-q27, 11q22.3-q23.3, 5p14.1-p14.3, 5q34-q35.3, 1p36.23-p36.33, 2p16.1-p16.3, 4q12, and 4q31.3-q32.1. Our results can be expected to provide further insights into the genetic basis of lymphomagenesis and the clinicopathologic features of NK-cell lymphomas/leukemias.

Mitoxantrone resistance in a small cell lung cancer cell line is associated with ABCA2 upregulation.

The aim of this study was to find factors that could explain the accumulation difference of mitoxantrone in the BCRP1-negative GLC4-MITO cell line compared to GLC4. Comparative genomic hybridisation (CGH) was applied to determine chromosomal differences between GLC4 and GLC4-MITO. Comparative genomic hybridisation analysis revealed gain of 2q, 6p, 9q, 13q, 14q, 15q, 19q and Xp and loss of 1p, 2q, 3p, 3q, 4q, 6q, 8q, 11p, 16p, 17q, 18p, 20p and Xq. In the over-represented chromosomal areas, seven transporter genes were identified: ABCB6, ABCB2 (TAP1), ABCB3 (TAP2), ABCF1 (ABC50), ABCC10 (MRP7), ABCA2 (ABC2) and ABCC4 (MRP4). No RNA or protein upregulation was observed for ABCB6, ABCF1, ABCC10, ABCC4, ABCB2 and ABCB3, but an increased expression was detected for ABCA2 mRNA in GLC4-MITO. ABCA2 is known to be involved in resistance to estramustine. In the MTT assay, GLC4-MITO was two-fold resistant to estramustine compared to GLC4. Coincubation with estramustine and mitoxantrone increased mitoxantrone accumulation in GLC4-MITO, while this was not affected in GLC4. This suggests that estramustine is able to block mitoxantrone efflux in GLC4-MITO cells. These data reveal that cellular reduction of mitoxantrone in a mitoxantrone-resistant cell line is associated with overexpression of ABCA2.

Cytogenetic findings in two new cases of hepatoblastoma

We describe two cases of hepatoblastoma occurring in an 18-month-old boy and a 3-month-old girl. Cytogenetic analysis of the primary tumors revealed gain of 2q and 20 in both cases. In case 1, t(7;8;11) was seen as a secondary abnormality. Other chromosomal aberrations seen in case 2 were unbalanced t(1;1) and t(2;11), resulting in partial gains of 1q and 2q. These results support previous reports that gains of 2q and 20 and rearrangement of chromosome 1 are strongly associated with hepatoblastoma and may be essential for establishing this neoplasm. The 11q and 7q abnormalities may represent a pathway of genetic evolution associated with hepatoblastoma progression.

Lymphocyte predominance Hodgkin disease is characterized by recurrent genomic imbalances

Single-cell polymerase chain reaction (PCR) has been used as a tool to demonstrate clonality and B-cell origin of Reed-Sternberg (RS) cells in Hodgkin disease (HD). An analogous approach was used to investigate genomic imbalances in a (cyto)genetically poorly characterized subentity: lymphocyte predominance Hodgkin disease (LPHD). Nineteen cases of LPHD were selected for a comparative genomic hybridization (CGH) study. CGH was performed with degenerate oligonucleotide primed-PCR (DOP-PCR)-amplified DNA from 4-5 microdissected CD20+ malignant cells. All analyzed cases revealed a high number of genomic imbalances (average 10.8 per case), involving all chromosomes but the excluded 19, 22, and Y, indicating a high complexity of LPHD. The majority of detected aberrations were recurrent. Gain of 1, 2q, 3, 4q, 5q, 6, 8q, 11q, 12q, and X, and loss of chromosome 17 were identified in 36.8% to 68.4% of the analyzed cases. Some of them have also been found in non-Hodgkin lymphoma (NHL), and possibly represent secondary changes associated with disease progression. Gain of 2q, 4q, 5q, 6, 11q, however, are much more rarely observed in NHL and could be more specifically associated with LPHD. Particularly interesting is a frequent overrepresentation of chromosome arm 6q, a region usually deleted in NHL. Rearrangement of the BCL6 gene (3q27) demonstrated by cytogenetics and fluorescence in situ hybridization in 2 cases in this study suggests its contribution in pathogenesis of LPHD. In conclusion, the data show a consistent occurrence of genomic alterations in LPHD and highlight genomic regions that might be relevant for development and/or progression of this lymphoma entity.

Cytogenetic findings in 31 papillary thyroid carcinomas

Chromosome studies performed on 31 papillary thyroid carcinomas (PTCs) revealed clonal numerical and structural abnormalities in 12 tumors. The numerical clonal aberrations found were trisomy 2, trisomy 7, and loss of the Y chromosome. A nonrandom telomeric association, tas(15;16)(p13;p13), was observed in one carcinoma. Structural alterations with a breakpoint at 10q11.2 were detected in two tumors. Other chromosomes involved in rearrangements were chromosomes 1, 2, 3, 5, 7, 9, 11, 12, and 14. The observation of clonal changes of chromosome 2 [i(2)(q10) and trisomy 2] in two tumors, which were both histologically classified as tall-cell PTC variants, suggests that gain of 2q may be important in the development of this morphological variant.