Soili Kytölä

Thymic carcinoids in multiple endocrine neoplasia type 1

Thymic carcinoid is a rare malignancy with about 150 cases reported to date. It is associated with multiple endocrine neoplasia type 1 (MEN-1), but compared with other MEN-1-related neoplasia little is known about it. We have recently described and studied 20 MEN-1-related cases and found that up to 25% of all reported thymic carcinoids are MEN-1 related. It is an insidious tumour not associated with Cushings syndrome or carcinoid syndrome. Local invasion, recurrence and distant metastasis are common with no known effective treatment. Its male predominance, the absence of loss of heterozygosity (LOH) in the MEN1 region, clustering in some MEN-1 families and the findings of different MEN1 mutations in these clustered families suggest the involvement of additional aetiological factors. We propose that computed tomography (CT) or magnetic resonance imaging (MRI) of the chest should be included as part of the clinical workup for all MEN-1 patients. Prophylactic thymectomy should be considered during subtotal or total parathyroidectomy on MEN-1 patients to reduce the risk of this malignancy.

Comparative genomic hybridization identifies loss of 18q22-qter as an early and specific event in tumorigenesis of midgut carcinoids.

Carcinoid tumors are rare neuroendocrine tumors occurring in the lung or in the digestive tract where they are further subclassified as foregut, midgut, or hindgut carcinoids. To gain a better understanding of the genetic basis of the different types of carcinoid tumors, we have characterized numerical imbalances in a series of midgut carcinoids, and compared the results to previous findings in carcinoids from the lung. Numerical imbalances were revealed in 16 of the 18 tumors, and the most commonly detected aberrations were losses of 18q22-qter (67%), 11q22-q23 (33%), and 16q21-qter (22%), and gain of 4p14-qter (22%). The total number of alterations found in the metastases was significantly higher than in the primary tumors, indicating the accumulation of acquired genetic changes in the tumor progression. Losses of 18q and 11q were present both in primary tumors and metastases, whereas loss of 16q and gain of 4 were only detected in metastases. Furthermore, the pattern of comparative genomic hybridization alterations varied depending on the total number of detected alterations. Taken together, the findings would suggest a progression of numerical imbalances, in which loss of 18q and 11q represent early events, and loss of 16q and gain of 4p are late events in the tumor progression of midgut carcinoids. When compared to previously published comparative genomic hybridization abnormalities in lung carcinoids, loss of 11q was found to occur in both tumor types, whereas loss of 18q and 16q and gain of 4 were not revealed in lung carcinoids. The results indicate that inactivation of a putative tumor suppressor gene in 18q22-qter represents a frequent and early event that is specific for the development of midgut carcinoids.

Chromosomal Alterations in 15 Breast Cancer Cell Lines by Comparative Genomic Hybridization and Spectral Karyotyping

Breast cancer cell lines have been widely used as models in functional and therapeutical studies, but their chromosomal alterations are not well known. We characterized the chromosomal aberrations in 15 commonly used human breast carcinoma cell lines (BT‐474, BT‐549, CAMA‐1, DU4475, MCF7, MDA‐MB‐134, MDA‐MB‐157, MDA‐MB‐361, MDA‐MB‐436, MPE600, SK‐BR‐3, T‐47D, UACC‐812, UACC‐893, and ZR‐75‐1) by comparative genomic hybridization (CGH) and spectral karyotyping (SKY). By CGH the most frequent gains were detected at 1q, 8q, 20q, 7, 11q13, 17q, 9q, and 16p, whereas losses were most common at 8p, 11q14–qter, 18q, and Xq. SKY revealed a multitude of structural and numerical chromosomal aberrations. Simple translocations, typically consisting of entire translocated chromosome arms, were the most common structural aberrations. Complex marker chromosomes included material from up to seven different chromosomes. Evidence for a cytogenetic aberration not previously described in breast cancer, the isoderivative chromosome, was found in two cell lines. Translocations t(8;11), t(12;16), t(1;16), and t(15;17) were frequently found, although the resulting derivative chromosomes and their breakpoints were strikingly dissimilar. The chromosomes most frequently involved in translocations were 8, 1, 17, 16, and 20. An excellent correlation was found between the number of translocation events found by SKY in the individual cell lines, and the copy number gains and losses detected by CGH, indicating that the majority of translocations are unbalanced. Genes Chromosomes Cancer 28:308–317, 2000.

Patterns of Chromosomal Imbalances in Parathyroid Carcinomas

In this study we have characterized chromosomal imbalances in a panel of 29 parathyroid carcinomas using comparative genomic hybridization (CGH). The most frequently detected imbalances were losses of 1p and 13q that were seen in >40% of the cases. The commonly occurring regions of loss were assigned to 1p21-p22 (41%), 13q14-q31 (41%), 9p21-pter (28%), 6q22-q24 (24%), and 4q24 (21%), whereas gains preferentially involved 19p (45%), Xc-q13 (28%), 9q33-qter (24%), 1q31-q32 (21%) and 16p (21%). The distribution of CGH alterations supports the idea of a progression of genetic events in the development of parathyroid carcinoma, where gains of Xq and 1q would represent relatively early events that are followed by loss of 13q, 9p, and 1p, and by gain of 19p. A sex-dependent distribution was also evident for two of the common alterations with preferential gain of 1q in female cases and of Xq in male cases. When the CGH profiles for the 29 carcinomas were compared with our previously published results for sporadic parathyroid adenomas, highly significant differences were revealed. Loss of 1p, 4q, and 13q as well as gains of 1q, 9q, 16p, 19p and Xq were significantly more common in the carcinomas than in the adenomas. In contrast, loss of the 11q13 region, which is the most common CGH abnormality in sporadic adenomas, was not detected in any of the carcinomas. Taken together, the findings identify several candidate locations for tumor suppressor genes and oncogenes that are potentially involved in parathyroid carcinogenesis.

Primary Cutaneous T-Cell Lymphomas Show a Deletion or Translocation Affecting NAV3, the Human UNC-53 Homologue

Multicolor fluorescent in situ hybridization (FISH) was used to identify acquired chromosomal aberrations in 12 patients with mycosis fungoides or Sézary syndrome, the most common forms of primary cutaneous T-cell lymphoma (CTCL). The most frequently affected chromosome was 12, which showed clonal deletions or translocations with a break point in 12q21 or 12q22 in five of seven consecutive Sézary syndrome patients and a clonal monosomy in the sixth patient. The break point of a balanced translocation t(12;18)(q21;q21.2), mapped in the minimal common region of two deletions, fine mapped to 12q2. By locus-specific FISH, the translocation disrupted one gene, NAV3 (POMFIL1), a human homologue of unc-53 in Caenorhabditis elegans. A missense mutation in the remaining NAV3 allele was found in one of six cases with a deletion or translocation. With locus-specific FISH, NAV3 deletions were found in the skin lesions of four of eight (50%) patients with early mycosis fungoides (stages IA-IIA) and in the skin or lymph node of 11 of 13 (85%) patients with advanced mycosis fungoides or Sézary syndrome. Preliminary functional studies with lentiviral small interfering RNA-based NAV3 silencing in Jurkat cells and in primary lymphocytes showed enhanced interleukin 2 expression (but not CD25 expression). Thus, NAV3 may contribute to the growth, differentiation, and apoptosis of CTCL cells as well as to the skewing from Th1-type to Th2-type phenotype during disease progression. NAV3, a novel putative haploinsufficient tumor suppressor gene, is disrupted in most cases of the commonest types of CTCL and may thus provide a new diagnostic tool.

Alterations of the SDHD gene locus in midgut carcinoids, Merkel cell carcinomas, pheochromocytomas, and abdominal paragangliomas.

Several types of endocrine tumors show frequent somatic deletions of the distal part of chromosome arm 11q, where the tumor‐suppressor gene SDHD (succinate‐ubiquinone oxidoreductase subunit D), constitutionally mutated in paragangliomas of the head and neck, is located. In this study, we screened 18 midgut carcinoids, 7 Merkel cell carcinomas, 46 adrenal pheochromocytomas (37 sporadic and 9 familial), and 7 abdominal paragangliomas for loss of heterozygosity (LOH) and/or mutations at the SDHD gene locus. LOH was detected in 5 out of 8 (62%) informative midgut carcinoids, in 9 out of 30 (30%) sporadic pheochromocytomas, in none of the familial pheochromocytomas (0%), and in 1 out of 6 (17%) abdominal paragangliomas. No sequence variants were detected in the pheochromocytomas or paragangliomas. However, two constitutional putative missense mutations, H50R and G12S, were detected in two midgut carcinoids, which were both associated with LOH of the other allele. The same sequence variants were also detected in two Merkel cell carcinomas. In addition, the S68S polymorphism was found to coexist with the G12S sequence variant in both cases. In conclusion, we show that alterations of the SDHD gene seem to be involved in the tumorigenesis of both midgut carcinoids and Merkel cell carcinomas.

Characterization of a Novel Breast Carcinoma Xenograft and Cell Line Derived from a BRCA1 Germ-Line Mutation Carrier

A human tumor xenograft (L56Br-X1) was established from a breast cancer axillary lymph node metastasis of a 53-year-old woman with a BRCA1 germ-line nonsense mutation (1806C>T; Q563X), and a cell line (L56Br-C1) was subsequently derived from the xenograft. The xenograft carries only the mutant BRCA1 allele and expresses mutant BRCA1 mRNA but no BRCA1 protein as determined by immunoprecipitation or Western blotting. The primary tumor, lymph node metastasis, and xenograft were hypodiploid by DNA flow cytometry, whereas the cell line displayed an aneuploidy apparently developed via polyploidization. Cytogenetic analysis, spectral karyotyping, and comparative genomic hybridization of the cell line revealed a highly complex karyotype with numerous unbalanced translocations. The xenograft and cell line had retained a somatic TP53 missense mutation (S215I) originating from the primary tumors, as well as a lack of immunohistochemically detectable expression of steroid hormone receptors, epidermal growth factor receptor, human epidermal growth factor receptor 2 (HER-2), and keratin 8. Global gene expression analysis by cDNA microarrays supported a correlation between the expression profiles of the primary tumor, lymph node metastasis, xenograft, and cell line. We conclude that L56Br-X1 and L56Br-C1 are useful model systems for studies of the pathogenesis and new therapeutic modalities of BRCA1-induced human breast cancer.

Aberrations of chromosome 8 in 16 breast cancer cell lines by comparative genomic hybridization, fluorescence in situ hybridization, and spectral karyotyping

Comparative genomic hybridization (CGH) studies have shown that chromosome 8 is a frequent target for chromosomal aberrations in breast cancer. We characterized these aberrations of chromosome 8 in 16 breast cancer cell lines (BT-474, BT-549, CAMA-1, DU-4475, MCF-7, MDA-MB-134, MDA-MB-157, MDA-MB-361, MDA-MB-415, MDA-MB-436, MPE600, SK-BR-3, T-47D, UACC-812, UACC-893 and ZR-75-1) by CGH, fluorescence in situ hybridization (FISH) with arm- and locus-specific probes, and spectral karyotyping (SKY). Chromosome 8 was structurally abnormal in 13 of 16 cell lines. Loss of 8p was detected in nine cell lines, gain of entire 8q in six cell lines, 8q21-qter in three, 8q23-qter in two, and 8q12-qter and 8p21-q21 in one cell line. Extra copies of the C-MYC oncogene were found in 11 cell lines, but high-level amplification only in SK-BR-3. Derivative chromosomes including material from chromosomes 8 were complex, and the breakpoints were strikingly dissimilar. Chromosome 11 was the most frequent translocation partner with chromosome 8 (in 7 cell lines). Isochromosomes and/or isoderivative 8q were found in four cell lines. The high frequency and complexity of alterations at 8q indicate a significant pathogenetic role in breast cancer. The high-level amplification of c-myc is less common than previously thought.

Amplification of c- myc by Fluorescence In Situ Hybridization in a Population-Based Breast Cancer Tissue Array

A total of 261 primary breast carcinomas were analyzed for amplification of the c-myc oncogene by fluorescence in situ hybridization performed on tumor tissue array samples. Results were compared with individual clinicopathologic and follow-up data. Thirty-eight (14.6%) of the tumors showed c-myc gene amplification (defined as two or more additional copies of c-myc gene in relation to the number of chromosome 8 centromere). The reproducibility of fluorescence in situ hybridization assay (defined by hybridization with two different myc probes) was good (kappa coefficient 0.402). Statistically significant associations were found between c-myc amplification and DNA aneuploidy (P = .0011), and progesterone receptor negativity (P = .0071), and c-myc amplification also tended to be associated with high histologic grade (P = .064), positive axillary nodal status (P = .080), and a high S-phase fraction (P = .052). c-myc amplification was not significantly associated with overall survival of patients with invasive cancer (P = .32). These data from a population-based tumor material suggest that c-myc amplification is a feature of aggressive breast cancers, but that it is unlikely to be a clinically useful prognostic factor.

Malignant melanoma in patients with multiple endocrine neoplasia type 1 and involvement of the MEN1 gene in sporadic melanoma.

Multiple endocrine neoplasia type 1 (MEN 1) is a familial cancer syndrome associated primarily with endocrine tumors of the parathyroids, enteropancreas and anterior pituitary. However, tumors of mesenchymal origin such as angiofibroma and collagenoma of the skin have also been associated with the syndrome. This highlights the possibility of an association between MEN 1 and some other types of tumors. Here we report 7 cases of primary malignant melanoma occurring in 7 MEN 1 families, all patients exhibiting classic features of MEN 1. Based on these findings and the previous implication of multiple melanoma tumor suppressor(s) in 11q, including the MEN1 region, we have investigated the involvement of the MEN1 gene in melanoma tumorigenesis. Mutation analysis was performed on a panel of 39 sporadic metastatic melanomas, 13 melanoma cell lines and 20 melanoma families without CDKN2A or CDK4 germline mutations. In addition, 19 sporadic metastatic tumors were screened for loss of heterozygosity (LOH) in 11q13. LOH was detected in 6 tumors (32%), and in 4 of the tumors the pattern of LOH suggested that the deletion included the MEN1 gene locus. A novel somatic nonsense mutation in exon 7 (Q349X) was identified in 1 sporadic tumor which also showed loss of the wild‐type allele. We conclude that the MEN1 gene plays a role in the tumorigenesis of a small subgroup of melanoma. Int. J. Cancer 87:463–467, 2000.