Molecular genetic aspects of renal cell carcinoma development
Author: Alimov, Andrei
Date: 2003-05-16
Location: Föreläsningssalen, Cancercentrum R8 plan 00, Karolinska Sjukhuset
Time: 9.30
Department: Institutionen för onkologi-patologi / Department of Oncology-Pathology
Abstract
Renal cell carcinoma (RCC) is the most common tumor of the renal parenchyma. The aim of this study was to further characterize known as well as new genetic abnormalities in a clinical material of RCC, as well as in established cell lines to gain more information about etiological and progression factors. In particular, chromosome 3p, 10q and 14q have been studied, which according to chromosome imbalance maps has shown loss in 70%, 6% and 30% of the tumors, respectively.
Paired normal and tumor tissue samples, as well as established cell lines and cultured tumors, were analyzed by genotyping with microsatellite markers, DNA sequencing, comparative genomic hybridization (CGH) and spectral karyotyping (SKY). In order to approach the genes in 3p relevant for RCC development NotI-linking clones were isolated from a commonly deleted region. Assignment and ordering of NotI-linking clones showed that the clones were localized fairly even on chromosome 3 (paper I, II). In order to locate the minimal deleted region on 3p the loss of heterozygosity (LOH) strategy was selected (paper III). Thirty-seven RCC tumors were screened for deletions of different regions on 3p to clarify their respective roles in the development of conventional RCC. Twelve cases were selected for more detailed analyses of 3p by comparing LOH and CGH data. In three RCC cell lines and three primary tumors interstitial deletions were detected by both methods. Our results suggest that only a fraction of clear cell RCCs have terminal deletions and 3p carries at least four frequently affected regions (paper III).
In order to locate the minimal target region for 10q deletions a panel of fifty-four cases of RCCs was screened for 10q LOH. In addition, selected cases were screened for mutations in the PTEN tumor suppressor gene. Two smallest deleted regions were observed. The five tumors with LOH covering 1Oq23 were selected for mutation analysis of the PTEN/MMAC1 gene. From the direct sequencing of the nine exons, we found three different base pair changes in three tumors with LOH. Homozygous deletion of the PTEN gene was found in one RCC cell line (paper IV).
In approaching the genetic background of RCC development we have performed a detailed mapping of 14q deletions in RCC tumors. Five minimal regions of overlapping deletions were identified, three of which were defined from the primary RCCs. Losses in telomeric regions were each significantly associated with high tumor grade (i.e. Grade 3; p<0.05). Further-more, LOH within the region between D14S617 and D14S260 was also associated with a significantly shorter survival (p=0.02). In conclusion, the high frequency of distal 14q LOH supports the relevance of this alteration for the development of RCC (paper V).
Finally, primary RCC tumors were obtained at surgery and short term cultured for subsequent cytogenetic characterization by CGH and SKY. Overall numerical alterations were more frequent than structural changes. The two structural alterations identified constituted of a deletion of 3p in conventional RCC, and a ring chromosome derived from chromosome 8 in a papillary RCC. Gain of copy number were revealed on chromosomes 3, 5, 7, 8q, and 20, while the losses encompassed 3p and 17p. The SKY and CGH data suggest that the conventional RCCs are genetically more homogenous than the other types of kidney cancer (paper VI).
In conclusion, comparisons between deletion mapping of 3p, 1Oq, and 14q suggested that discontinous LOH represent a general mechanism of RCC development. A subset of RCC appears to constitute of heterogenous tumor cell populations. Furthermore, in sporadic cases, the variations in chromosome copy number changes seen between subgroups of tumor cells, is one dominant mechanism for tumor cell evolution in the kidney.
Paired normal and tumor tissue samples, as well as established cell lines and cultured tumors, were analyzed by genotyping with microsatellite markers, DNA sequencing, comparative genomic hybridization (CGH) and spectral karyotyping (SKY). In order to approach the genes in 3p relevant for RCC development NotI-linking clones were isolated from a commonly deleted region. Assignment and ordering of NotI-linking clones showed that the clones were localized fairly even on chromosome 3 (paper I, II). In order to locate the minimal deleted region on 3p the loss of heterozygosity (LOH) strategy was selected (paper III). Thirty-seven RCC tumors were screened for deletions of different regions on 3p to clarify their respective roles in the development of conventional RCC. Twelve cases were selected for more detailed analyses of 3p by comparing LOH and CGH data. In three RCC cell lines and three primary tumors interstitial deletions were detected by both methods. Our results suggest that only a fraction of clear cell RCCs have terminal deletions and 3p carries at least four frequently affected regions (paper III).
In order to locate the minimal target region for 10q deletions a panel of fifty-four cases of RCCs was screened for 10q LOH. In addition, selected cases were screened for mutations in the PTEN tumor suppressor gene. Two smallest deleted regions were observed. The five tumors with LOH covering 1Oq23 were selected for mutation analysis of the PTEN/MMAC1 gene. From the direct sequencing of the nine exons, we found three different base pair changes in three tumors with LOH. Homozygous deletion of the PTEN gene was found in one RCC cell line (paper IV).
In approaching the genetic background of RCC development we have performed a detailed mapping of 14q deletions in RCC tumors. Five minimal regions of overlapping deletions were identified, three of which were defined from the primary RCCs. Losses in telomeric regions were each significantly associated with high tumor grade (i.e. Grade 3; p<0.05). Further-more, LOH within the region between D14S617 and D14S260 was also associated with a significantly shorter survival (p=0.02). In conclusion, the high frequency of distal 14q LOH supports the relevance of this alteration for the development of RCC (paper V).
Finally, primary RCC tumors were obtained at surgery and short term cultured for subsequent cytogenetic characterization by CGH and SKY. Overall numerical alterations were more frequent than structural changes. The two structural alterations identified constituted of a deletion of 3p in conventional RCC, and a ring chromosome derived from chromosome 8 in a papillary RCC. Gain of copy number were revealed on chromosomes 3, 5, 7, 8q, and 20, while the losses encompassed 3p and 17p. The SKY and CGH data suggest that the conventional RCCs are genetically more homogenous than the other types of kidney cancer (paper VI).
In conclusion, comparisons between deletion mapping of 3p, 1Oq, and 14q suggested that discontinous LOH represent a general mechanism of RCC development. A subset of RCC appears to constitute of heterogenous tumor cell populations. Furthermore, in sporadic cases, the variations in chromosome copy number changes seen between subgroups of tumor cells, is one dominant mechanism for tumor cell evolution in the kidney.
List of papers:
I. Alimov AA, Rodova MA, Gizatullin RZ, Kost-Alimova MY, Fedorova LI, Barsky VE, Kashuba V, Klein G, Zabarovsky ER, Zelenin AV (1997). A micro-dissection approach for isolation of NotI linking clones from regions frequently deleted in RCC and SCLC. Genet Anal. 14(1): 21-3.
Pubmed
II. Fedorova L, Kost-Alimova M, Gizatullin RZ, Alimov A, Zabarovska VI, Szeles A, Protopopov AI, Vorobieva NV, Kashuba VI, Klein G, Zelenin AV, Sheer D, Zabarovsky ER (1997). Assignment and ordering of twenty-three unique NotI-linking clones containing expressed genes including the guanosine 5-monophosphate synthetase gene to human chromosome 3. Eur J Hum Genet. 5(2): 110-6.
Pubmed
III. Alimov A, Kost-Alimova M, Liu J, Li C, Bergerheim U, Imreh S, Klein G, Zabarovsky ER (2000). Combined LOH/CGH analysis proves the existence of interstitial 3p deletions in renal cell carcinoma. Oncogene. 19(11): 1392-9.
Pubmed
IV. Alimov A, Li C, Gizatullin R, Fredriksson V, Sundelin B, Klein G, Zabarovsky E, Bergerheim U (1999). Somatic mutation and homozygous deletion of PTEN/MMAC1 gene of 10q23 in renal cell carcinoma. Anticancer Res. 19(5B): 3841-6.
Pubmed
V. Alimov A, Sundelin B, Wang N, Larsson C, Bergerheim U (2003). Loss of 14q31-q32.2 in renal cell carcinoma is associated with high malignancy grade and poor survival. [Submitted]
VI. Alimov A, Sundelin B, Bergerheim U, Pavlenko M, Pisa P, Zetterberg A, Larsson C, Lagercrantz S (2003). Molecular cytogenetic characterization of kidney tumors by CGH and SKY. [Submitted]
I. Alimov AA, Rodova MA, Gizatullin RZ, Kost-Alimova MY, Fedorova LI, Barsky VE, Kashuba V, Klein G, Zabarovsky ER, Zelenin AV (1997). A micro-dissection approach for isolation of NotI linking clones from regions frequently deleted in RCC and SCLC. Genet Anal. 14(1): 21-3.
Pubmed
II. Fedorova L, Kost-Alimova M, Gizatullin RZ, Alimov A, Zabarovska VI, Szeles A, Protopopov AI, Vorobieva NV, Kashuba VI, Klein G, Zelenin AV, Sheer D, Zabarovsky ER (1997). Assignment and ordering of twenty-three unique NotI-linking clones containing expressed genes including the guanosine 5-monophosphate synthetase gene to human chromosome 3. Eur J Hum Genet. 5(2): 110-6.
Pubmed
III. Alimov A, Kost-Alimova M, Liu J, Li C, Bergerheim U, Imreh S, Klein G, Zabarovsky ER (2000). Combined LOH/CGH analysis proves the existence of interstitial 3p deletions in renal cell carcinoma. Oncogene. 19(11): 1392-9.
Pubmed
IV. Alimov A, Li C, Gizatullin R, Fredriksson V, Sundelin B, Klein G, Zabarovsky E, Bergerheim U (1999). Somatic mutation and homozygous deletion of PTEN/MMAC1 gene of 10q23 in renal cell carcinoma. Anticancer Res. 19(5B): 3841-6.
Pubmed
V. Alimov A, Sundelin B, Wang N, Larsson C, Bergerheim U (2003). Loss of 14q31-q32.2 in renal cell carcinoma is associated with high malignancy grade and poor survival. [Submitted]
VI. Alimov A, Sundelin B, Bergerheim U, Pavlenko M, Pisa P, Zetterberg A, Larsson C, Lagercrantz S (2003). Molecular cytogenetic characterization of kidney tumors by CGH and SKY. [Submitted]
Issue date: 2003-04-25
Publication year: 2003
ISBN: 91-7349-559-X
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