Distinct gene expression profiling in chronic lymphocytic leukemia with 11q23 deletion.

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with regard to its clinical course. The limitations of the methods currently available for prognostic assessment in CLL do not allow accurate prediction of the risk of disease progression in individual patients. The recently developed cDNA array technique provides a unique opportunity to study gene expression in various malignancies. To identify new molecular markers for prognostication of CLL patients, we analyzed cDNA arrays by using hierarchical clustering and standard statistic t-test on 34 CLL patients. We found significant expression differences in 78 genes compared to the reference tonsillar B lymphocytes. A cluster of genes, LCP1, PARP, BLR1, DEK, NPM, MCL1, SLP76, STAM, HIVEP1, EVI2B, CD25, HTLF, HIVEP2, BCL2, MNDA, PBX3, EB12, TCF1, CGRP, CD14, ILB, GZMK, GPR17 and CD79B, was associated (P < 0.05) with the unfavorable 11q deletion and also with the unfavorable Binet stages B and C. We present here gene expression profiling that is associated with CLL patients with the 11q23 deletion. Many of the genes in the cluster have not previously been shown to be related to the initiation or progression of CLL. These novel findings provide fundamental information for further attempts to understand the interaction of the clustered genes in the leukomogenesis of CLL in order to better design treatments aimed at specific molecular target(s).

Expression of myeloid-specific genes in childhood acute lymphoblastic leukemia - a cDNA array study.

Several specific cytogenetic changes are known to be associated with childhood acute lymphoblastic leukemia (ALL), and many of them are important prognostic factors for the disease. Little is known, however, about the changes in gene expression in ALL. Recently, the development of cDNA array technology has enabled the study of expression of hundreds to thousands of genes in a single experiment. We used the cDNA array method to study the gene expression profiles of 17 children with precursor-B ALL. Normal B cells from adenoids were used as reference material. We discuss the 25 genes that were most over-expressed compared to the reference. These included four genes that are normally expressed only in the myeloid lineages of the hematopoietic cells: RNASE2, GCSFR, PRTN3 and CLC. We also detected over-expression of S100A12, expressed in nerve cells but also in myeloid cells. In addition to the myeloid-specific genes, other over-expressed genes included AML1, LCP2 and FGF6. In conclusion, our study revealed novel information about gene expression in childhood ALL. The data obtained may contribute to further studies of the pathogenesis and prognosis of childhood ALL.

Establishment and characterisation of human papillomavirus type 16 DNA immortalised human tonsillar epithelial cell lines.

Oncogenic human papillomavirus (HPV) plays a possible aetiological role in a subset of head and neck cancers, particularly in tonsillar carcinomas. For establishing a model to study mechanisms involved in HPV-associated tonsillar carcinogenesis, normal human tonsillar epithelial (HTE) cells were transfected with full-length HPV-16 DNA. The transfections produced four immortalised cell lines, designated HTE-114/K1, HTE-114/K2, HTE-114/K3 and HTE-114/B. All transfected HTE cell lines were cytogenetically abnormal. They exhibited altered morphology and impaired expression of cytokeratins in organotypic cultures. They failed to form colonies in soft agarose and formed no tumours in nude mice within 6 months. Each of them contained integrated viral DNA in a distinctive pattern as shown by Southern blot hybridisation. Early viral transcripts containing the E7 gene were detected by northern blot hybridisation. In conclusion, primary HTE cells can be immortalised following transfection with full-length HPV-16 DNA; the immortalised cell lines had partially retained epithelial characteristics in their morphology and function. They seem to represent early stages of premalignant epithelial cells and thus provide a useful model for studying further the multistep molecular events of HPV-16-associated tonsillar carcinogenesis.

Time- and dose-related changes in the expression of substance P in salivary glands in response to fractionated irradiation.

PURPOSE: The expression of different neuropeptides in the innervation of submandibular and parotid glands of the rats was examined 2 and 5 days after initiation of radiation treatment as well as 10 and 180 days following the termination of irradiation. METHODS AND MATERIALS: The irradiation was given on 2 or 5 consecutive days with daily doses of 4-8 Gy up to a total dose of 20-40 Gy. Immunohistochemical methods were used for the demonstration of substance P (SP), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and, the rate-limiting enzyme in the catecholamine synthetic pathway, tyrosine hydroxylase (TH). The content of SP was also analyzed by the use of radioimmunoassay (RIA). RESULTS: In the parenchyma of both the submandibular and the parotid glands of control animals as well as after 2 days of irradiation treatment, a few nerve fibers showing SP-like immunoreactivity (LI) were observed. A marked increase in the expression of SP in the innervation of the parenchyma in both glands was observed 10 days after cessation of radiation treatment. The number of stained nerve fibers and the intensity of fluorescence in the fibers seemed to be dose dependent because the group subjected to a total dose of 40 Gy displayed a more pronounced staining intensity than that treated with 30 Gy. These results were supported by the RIA analysis. One hundred eighty days after treatment no obvious differences in SP-expression were seen between control and irradiated animals. No acute and long-term alterations were seen with regard to the other peptides and TH. CONCLUSIONS: These results suggest that specific dose- and time-dependent changes in the expression of SP in the parenchyma of both submandibular and parotid glands occur in response to fractionated irradiation. The observations add further aspects to the tissue differences in physiological response and sensitivity to irradiation.

Concomitant loss of chromosome 3 and whole arm losses and gains of chromosome 1, 6, or 8 in metastasizing primary uveal melanoma.

PURPOSE: To elucidate the genetic differences between metastasizing and nonmetastasizing primary tumors, uveal melanoma samples were screened for DNA copy number alterations by comparative genomic hybridization (CGH). METHODS: DNA copy number changes were studied on 14 primary uveal melanomas that had not metastasized, 15 primary uveal melanomas that had metastasized, and on 6 metastases that were available from 6 primary uveal melanomas. CGH is based on quantitation of the fluorescence intensity of differentially labeled DNAs. Tumor DNA labeled with FITC dCTP and dUTP and normal DNA labeled with Texas red dCTP and dUTP were hybridized to normal metaphase chromosomes. The hybridizations were analyzed using an Olympus fluorescence microscope and the ISIS digital image analysis system to identify gain or loss of genetic material. RESULTS: Primary uveal melanomas that had metastasized and metastases had significantly more changes than primary uveal melanomas that had not metastasized. Comparison between primary nonmetastasizing tumors, metastasizing tumors, and metastases showed that the most common DNA copy number changes were -3 (21%, 73%, 67%, respectively), -6q (7%, 40%, 83%), -1p (0, 33%, 33%), -13q (14%, 13%, 50%), -8p (14%, 27%, 0), -18 (7%, 13%, 33%), +8q (14%, 53%, 100%), +6p (29%, 20%, 17%), +1q (0, 7%, 33%), and +16p (0, 7%, 33%). CONCLUSIONS: Loss of chromosome 3, loss of 6q, and gain of 8q were significantly associated with poor overall survival. In addition, losses of 1p were only found in primary uveal melanomas that had metastasized and in metastases, which suggests that this region may harbor a tumor suppressor gene important in the tumor progression. Finally, loss of chromosome 3 may be associated with isochromosome formation of 1q, 6p, 8q, 16p, 20q, and 22q.

Intra-and inter-individual heterogeneity in exon 2 of the MDR1 gene in primary breast carcinoma and healthy individuals.

Increased expression of P-glycoprotein, encoded by the MDR1 gene, is considered to be responsible for chemotherapy failure in a number of human cancers. Although it is clear that mutations in the MDR1 gene affect substrate specificity of the transporter in multidrug-resistant cell lines, scant interest has been directed at whether mutations have a unique clinical presentation. To address this question, we studied exon 2 of the MDR1 gene in 9 patients with primary breast carcinoma and 9 healthy controls using PCR and DNA sequence analysis. In order to reduce the possibility of nucleotide misincorporations introduced by Tag polymerase, sequencing of six subclones of each DNA specimen was performed. A mutation was seen as a substitution from G to A at position -1 in two patients and one control. An A to G nucleotide substitution giving rise to an amino acid substitution (Asn-->Asp) in codon 21 at the first potential N-glycosylation site of the P-glycoprotein was seen in primary tumors from four patients and in an axillar lymph node metastases from one of these patients. This mutation was also seen in two healthy individuals, which similar to the patients, both seem to be heterozygous for this MDR1 exon 2 allele. Three other mutations were also found in the patients; a substitution of A to G at position 23 and A to G at position 52 in the same patient and in another patient, G at position 42 was changed to A. However, the last three mutations were not confirmed by repeating analysis of the original genomic sample. The results revealed different distribution of a point mutation between various parts of the same primary tumor and between a lymph node metastasis and the primary tumor tissue. Thus, demonstrating both intra-and inter-tumor heterogeneity. The results also emphasized constitutional allelic variation in the MDR1 gene. Whether this might affect sensitivity to chemotherapy has to be further evaluated.

Enhanced expression of neuropeptides in human breast cancer cell lines following irradiation.

Previously, we have observed that the expression of the neuropeptides bombesin (BN-), the mammalian counterpart being gastrin-releasing peptide (GRP), and substance P (SP) in intact normal tissues, such as salivary and laryngeal glands, increases in response to irradiation. In the present study, the aim was to evaluate whether irradiation can have effects on individual cells that normally synthesize neuropeptides. In addition, since these neuropeptides are potentially mitogenic, we studied tumor cells. Therefore, the estrogen receptor-negative human breast cancer cell line MDA-MB-231 and its subline, with acquired doxorubicin resistance, MDA-MB-231 Dox were examined before irradiation and 4, 10, and 15 days after irradiation with 4 Gy (195 kV, 2 Gy fractions with 4 hours interval). Potential dose related changes were studied by delivering single doses of 2 or 9 Gy with the same technique. Immunohistochemical and radioimmunoassay (RIA) methods were used for detection of the SP and BN/GRP. Before, and at all time points following irradiation, a subpopulation in both cell lines displayed an intense immunostaining of SP and BN/GRP. A partial reorganization of the immunoreactive material was observed 10 days after irradiation. The RIA-analyses displayed signs of a dose-related increase, and a time-dependent transient and significant increase in the content of both peptides. The pattern of changes differed between the two peptides, and was especially pronounced in the doxorubicin resistant cells with regard to SP. Another neuropeptide, calcitonin gene related peptide (CGRP), was not detected in the cells used. The results suggest that irradiation has effects on a population of cultured neuropeptide-synthesizing cells. The occurrence and the specific changes obtained in the levels of neuropeptides, in response to irradiation, might imply an importance in the growth of breast cancer cells and in explaining repair processes following irradiation.

Bombesin-like peptide is present in duct cells in salivary glands: studies on normal and irradiated animals.

Bombesin (BN) and its mammalian counterpart gastrin-releasing peptide act as neuroregulatory hormones and tissue-specific growth factors, and have been implicated as peripheral and central satiety-inducing agents. In the present study, the immunohistochemical expression of BN in submandibular, sublingual and parotid glands of rats was examined 10 days after 5 consecutive days with daily doses of 6-8 Gy irradiation. Radioimmunoassay (RIA) methods were also used. Immunoreactive granular structures were observed within duct cells of both controls and irradiated animals. In the parenchyma of irradiated animals, very few nerve fibres showing BN-like immunoreactivity were observed. The RIA analysis showed that the content of BN-like material significantly increased in submandibular and parotid glands in response to irradiation. The results suggest that mainly a non-neural form of BN is detected in the salivary glands in the immunohistochemical analysis. Thus, the immunohistochemical observations suggest that BN-like peptides may be present in the duct system, where they may be constituents of the saliva. The observations of an increase in BN content in response to irradiation are of interest as BN has mitogenic effects, may stimulate secretion and contributes to satiety.

Comparative genomic hybridization reveals changes in DNA-copy number in poor-risk neuroblastoma.

Aggressive neuroblastoma remains a therapeutic challenge, and additional understanding of its biology is of paramount importance. Changes in DNA-copy number were analysed in the neuroblastoma cells of 27 patients using comparative genomic hybridization (CGH). Eighteen of the patients had a poor risk disease (16/18 stage IV) and 9 had a non-poor-risk disease (3/9 stage I-II, 2/9 stage III, and 4/9 stage IVS). Changes in DNA-copy number were detected in 72% of the poor-risk and 22% of the non-poor-risk tumors with gains of chromosomal material being more prevalent than losses. Gains were most common in chromosomes 2, 7, and 17 and losses in chromosome 11. Changes in DNA-copy number were multiple in all but one of the patients with poor-risk disease. The applicability of CGH in studies on the genomic changes in pediatric malignancies is demonstrated by our data also adding weight to the argument of multiple elements with oncogenic and/or tumor suppressor potential being involved in the aggressive phenotype of poor-risk neuroblastoma.

DNA copy number changes in familial malignant mesothelioma.

Malignant mesothelioma (MM) is predominantly a sporadic malignancy linked to exposure to asbestos. Clustering of MM in families suggests genetic susceptibility as a contributing factor. We performed comparative genomic hybridization (CGH) analysis on tumor samples from members of a family with MM of the pleura and a history of parental cancer. Our specific aim was to find a recurrent copy number loss indicating the chromosomal area to which a gene underlying the development of MM could be assigned according to the Knudson two-hit hypothesis. We found losses at 1p, 6q, 9p, 13q, and 14q. The copy number changes were very similar to those reported in sporadic cases. Our findings and results from sporadic cases highlight the importance of cloning the genes in the loss sites at 1p, 6q, 14q, and 22q.

DNA copy number losses are more frequent in primary larynx tumors with lymph node metastases than in tumors without metastases.

Comparative genomic hybridization was performed on 38 primary laryngeal carcinomas divided into two groups according to the metastatic phenotype. DNA copy number changes were detected in 22 of the 38 cases (57.9%). Gains were most frequently observed at 3q, 8q, and 9q, and losses were found in decreasing order at 18q, 3p, and 4. The mean value of losses was 2.5 times as high in metastasizing primary tumors (23/38) as in nonmetastasizing tumors. The most frequent losses in metastasizing tumors were at 18q, 3p, and 5q.

Gain of chromosome 3 and loss of 13q are frequent alterations in pituitary adenomas.

Genetic changes underlying the tumorigenesis of pituitary adenomas (PA) are poorly characterized. To search for characteristic genomic imbalances involved in PA, we examined 38 cases: 12 hormone-secreting (HS) and 26 non-functioning (NF) PA, by comparative genomic hybridization. The most frequent DNA copy number change in both kinds of tumors was loss of 13q. Gains of chromosomes 3, 7 and 14, 6p, and 20q were more frequent in HSPA than in NFPA. These data indicate that the 13q region may harbor tumor suppressor genes determining the tumorigenesis of PA and gain in chromosome 3 may be related to hormone secretion. These findings provide a basis to search for candidate diagnostic markers of HSPA.

Clinical importance of genomic imbalances in synovial sarcoma evaluated by comparative genomic hybridization.

The t(X;18)(p11.2;q11.2) (SYT/SSX1 or SSX2) is represented in more than 95% of synovial sarcoma. Even if recent data has implicated that the type of fusion gene (SYT/SSX1 or SYT/SSX2) can be of prognostic importance, the cellular and molecular mechanisms underlying the clinical behavior of synovial sarcoma are still poorly understood. To approach this issue, we investigated whether secondary genetic aberrations may influence the clinical outcome of synovial sarcoma. Clinical outcome with reference to comparative genomic hybridization (CGH) findings (losses or gains of genetic material) were analyzed for a uniquely large modern material of 69 synovial sarcomas. Thirty-five of 69 specimens showed DNA sequence copy number changes. The frequency of aberrations/tumor were higher (mean 4.7) for monophasic tumors than for biphasic tumors (mean 2.1). Gains of the whole or parts, including the long arm, of chromosome 8 were significantly overrepresented in large tumors (> 5 cm), suggesting that tumors with this genetic abnormality have an increased growth rate. No difference regarding metastasis-free or overall survival was seen between patients with or without tumors containing secondary copy number changes. No specific copy number change was linked to a significantly improved or impaired metastasis-free survival.

MSX1 gene is deleted in Wolf-Hirschhorn syndrome patients with oligodontia.

Abnormalities of the short arm of chromosome 4 cause multiple congenital malformations, including craniofacial, oral, and dental manifestations. A candidate gene for oral defects in this region is MSX1, which is mandatory for normal oral and tooth development. We examined the dentition and the presence of MSX1 in eight Finnish patients with abnormalities of 4p, including seven cases of Wolf-Hirschhorn syndrome. Five of the Wolf-Hirschhorn syndrome patients presented with agenesis of several teeth, suggesting that oligodontia may be a common (even though previously not well-documented) feature in Wolf-Hirschhorn syndrome. In fluorescence in situ hybridization (FISH) analysis, the five patients with oligodontia lacked one copy of MSX1, while the other three had two hybridization signals. One of these presented with the only case of cleft palate among the patients. Our result confirms that haploinsufficiency for MSX1 serves as a mechanism that causes selective tooth agenesis but, alone, is not enough to cause oral clefts.

Gene expression profiling of ameloblastoma and human tooth germ by means of a cDNA microarray.

The molecular and genetic characteristics of ameloblastoma are still poorly understood. We analyzed gene expression in fresh-frozen ameloblastomas and human fetal tooth germs, using a cDNA microarray. Thirty-four genes exhibited significant changes in expression levels in the ameloblastoma. Eleven genes were overexpressed more than three-fold, and 23 genes were underexpressed to below 0.4 of the control level. The oncogene FOS was the most overexpressed gene (from eight- to 14-fold), followed by tumor-necrosis-factor-receptor 1 (TNFRSF1A). Genes for sonic hedgehog (SHH), TNF-receptor-associated-factor 3 (TRAF3), rhoGTP-ase-activating protein 4 (ARHGAP4), deleted in colorectal carcinoma (DCC), cadherins 12 and 13 (CDH12 and 13), teratocarcinoma-derived growth-factor-1 (TDGF1), and transforming growth-factor-beta1 (TGFB1) were underexpressed in all tumors. In selected genes, a comparison between cDNA microarray and real-time RT-PCR confirmed similar relative gene expression changes. The gene expression profile identifies candidate genes that may be involved in the origination of ameloblastoma and several genes previously unidentified in relation to human tooth development.

Is radiation-induced degranulation of mast cells in salivary glands induced by substance P?

Although DNA is the critical target for the lethal effects of irradiation, the precise mechanisms by which irradiation causes damage in tissues and biological systems is not fully understood. In the present study, the number of mast cells and the expression of the neuropeptide substance P (SP) in salivary glands were examined 10 days after a regimen of irradiation. The irradiation was given as a single dose or 5 consecutive days with daily doses of 7 Gy up to a total dose of 35 Gy. In addition, the number of mast cells and the expression of SP were examined 2 and 24 h after a single dose of 7 Gy. Immunohistochemical staining for 5-hydroxytryptamine (5-HT) and staining with avidin peroxidase and toluidine blue were used to detect mast cells. At examination 2 and 24 h after irradiation treatment, no change in the number of mast cells and the pattern of SP expression was observed. Ten days after irradiation there was a remarkable reduction in the number of mast cells in all the three glands, but there was a marked increase in the number of nerve fibers showing SP-like immunoreactivity in the parenchyme. The results show that early time-dependent alterations in the density of mast cells occur in response to irradiation, and that these changes occur concomitantly with changes in the expression of SP. Since the peripheral nervous system is a main regulator of salivary gland function, it is tempting to speculate that the nervous system interacts with mast cells via SP in modulating irradiation provoked tissue responses in salivary glands.

Amplification at 9p in cervical carcinoma by comparative genomic hybridization.

DNA copy number changes were studied by comparative genomic hybridization on 10 tumor specimens of squamous cell carcinoma of cervix obtained from Korean patients. DNA was extracted from paraffin-embedded sections after removal of non-malignant cells by microdissection technique. Copy number changes were found in 8/10 tumors. The most frequent changes were chromosome 19 gains (n=6) and losses on chromosomes 4 (n=4), 5 (n=3), and 3p (n=3). A novel finding was amplification in chromosome arm 9p21-pter in 2 cases. Gains in 1, 3q, 5p, 6p, 8q, 16p, 17, and 20q and losses at 2q, 6q, 8p, 9q, 10p, 11, 13, 16q, and 18q were observed in at least one of the cases.

Among numerous DNA copy number changes, losses of chromosome 13 are highly recurrent in plasmacytoma.

Chromosomal imbalances were studied by comparative genomic hybridization (CGH) on 27 specimens from 24 patients with plasmacytoma. All the specimens exhibited DNA copy number changes (mean, 7.7 aberrations/tumor; range, 2-15). The most recurrent change involved losses at 13q, found in 19 out of 24 patients. Other frequent losses were at 1p (42%), 14q (33%), X (33%), 8p (25%), and 6q (25%). Gains were frequent at 19p (58%), 9q (58%), 1q (58%), 7p (42%), 11q (38%), 15 (33%), 6p (25%), 8q (25%), and 5p (21%). High-level copy number increases were found at 1q, 5, 7, 8q, 9q, 11q, 15, and 19. The findings of highly recurrent chromosomal imbalances in plasmacytomas confirm the analytical power of CGH to detect chromosomal abnormalities in malignancies characterized by low mitotic activity. Our most striking finding, the losses in chromosome 13, provides a basis to investigate the role of the 13q loss in the tumorigenesis and progression of plasmacytoma and to evaluate the prognostic significance of this loss.

Does bombesin-like peptide mediate radiation-induced anorexia and satiety?

Bombesin (BN) and its mammalian counterpart gastrin-releasing peptide (GRP) act as neuroregulatory hormones and peripheral and central satiety-inducing agents. Previously, we demonstrated that irradiation induces an increase in the expression of BN/GRP in the innervation of the salivary glands in rats. We therefore carried out a study using radioimmunoassay (RIA) analysis and immunohistochemistry to examine whether saliva contains BN and whether irradiation affects the BN release to saliva in rats. Immunoreactivity for BN was detected not only in the innervation of the parenchyma but also in the duct cells and in the lumina of the ducts, suggesting entrance of BN into saliva. The RIA analysis confirmed that rat saliva contains a BN-like peptide. The observation shows that saliva contains this peptide but that there is no significant increase following the radiation schedule used. Nevertheless, the occurrence of an enhanced expression of BN in different peripheral tissues such as the salivary and laryngeal glands should be taken into consideration when discussing the clinically important problem of reduced food intake and anorexia in cancer patients.

Effects of fractionated irradiation on the cytoskeleton and basal lamina in parotid glands--an immunohistochemical study.

Cytoskeletal, cytocontractile and basement membrane proteins were studied using the immunofluorescence technique in the parotid gland in female rats after half-side fractionated megavoltage irradiation. The non-irradiated parallel-handled parotid glands served as controls. The qualitative expression of cytoskeletal proteins remained unchanged 10 days following irradiation compared to controls, i.e. cytokeratin was observed but not vimentin, desmin or GFAP (glial fibrillary acidic proteins). Six months after irradiation the cytokeratin expression adjacent to duct lumina was clearly stronger. Actin staining was more pronounced in the periphery of the acini. Ten days after irradiation no alterations of the basal lamina proteins, laminin and fibronectin, were detected. Six months post-irradiation laminin deposits were detected in areas where the entire acini had degenerated and had been replaced by fibrosis. An increased expression of fibronectin was also observed in the stroma at that time, reflecting an increased fibrosis. In areas where the acini remained, laminin immunofluorescence was mainly found in basal laminae of normal thickness, but the mean diameter of the acini seemed to have increased. This indicates a regeneration of acini and a restructuring of the basal lamina of the parenchyma.