Mutations in the p16INK4/MTS1/CDKN2, p15INK4B/MTS2, and p18 genes in primary and metastatic lung cancer

We examined the genomic status of cyclin-dependent kinase-4 and -6 inhibitors, p16INK4,p15INK4B, and p18, in 40 primary lung cancers and 31 metastatic lung cancers. Alterations of the p16INK4 gene were detected in 6 (2 insertions and 4 homozygous deletions) of 22 metastatic non-small cell lung cancers (NSCLCs; 27%), but none were detected in 25 primary NSCLCs, 15 primary small cell lung cancers (SCLCs), or 9 metastatic SCLCs, indicating that mutation in the p16INK4 gene is a late event in NSCLC carcinogenesis. Although three intragenic mutations of the p15INK4B gene were detected in 25 primary NSCLCs (12%) and five homozygous deletions of the p15INK4B gene were detected in 22 NSCLCs (23%), no genetic alterations of the p15INK4B gene were found in primary and metastatic SCLCs. The p18 gene was wild type in these 71 lung cancers, except 1 metastatic NSCLC which showed loss of heterozygosity. We also examined alterations of these three genes and expression of p16INK4 in 21 human lung cancer cell lines. Alterations of the p16INK4 and p15INK4B genes were detected in 71% of the NSCLC cell lines (n = 14) and 50% of the NSCLC cell lines (n = 14), respectively, but there were none in the 7 SCLC cell lines studied. No p18 mutations were detected in these 21 cell lines. These results indicate that both p16INK4 and p15INK4B gene mutations are associated with tumor progression of a subset of NSCLC, but not of SCLC, and that p15INK4B mutations might also be an early event in the molecular pathogenesis of a subset of NSCLC.     

Homozygous deletions at chromosome 9p21 and mutation analysis of p16 and p15 in microdissected primary non-small cell lung cancers

Loss of heterozygosity on chromosome 9p has been detected in many primary human tumors and cell lines, suggesting that this chromosomal arm harbors one or more tumor suppressor genes. The recently cloned p16 and p15 genes, mapped to 9p21, are likely candidates for such tumor suppressors. To map the deletion at chromosome 9p21 in non-small cell lung tumors, we analyzed DNA from 25 tumors and matching normal DNAs at six microsatellite markers that flank the region occupied by the p16 and p15 genes. Loss of heterozygosity of at least one microsatellite marker on chromosome 9p21 was detected in 13 (52%) of 25 tumors, including one tumor that exhibited homozygous deletion of both human IFNalpha and D9S171. Six tumors analyzed by a comparative multiplex PCR technique showed homozygous deletions of the sequence tag site marker c5.1 (within p16). Screening for mutations in p16 and p15 revealed one tumor with a non-sense mutation in exon 2 of p16, but no mutations were detected in p15 in any of the tumors. Thus, in these analyses approximately one-half of the non-small cell lung tumors had loss of heterozygosity at chromosome 9p21, and of these tumors, one-half had homozygous deletions of the region that includes p16. This appears to confirm the importance of a locus in this region critical to growth control in lung. The apparent lack of other mutations in p16 and p15 in the tumors with loss of heterozygosity leaves open the possibility of an unidentified gene in this region that may function as a tumor suppressor.     

High effectiveness and safety of one-week antibiotic regimen in Helicobacter pylori eradication

Forty-three sporadic gastric cancers were analyzed with regard to whether mutations of simple repeated sequences in the transforming growth factor beta type II receptor (TbetaR-II) gene are associated with microsatellite instability (MSI) and gastric carcinogenesis. In 12 of the 43 cancers (28%), MSI was observed at least at 1 of the 2 microsatellite loci. Frameshift mutations of the TbetaR-II gene, all of which were 1 base deletion of 10 adenine repeats, were detected in 3 of 6 cancers, with MSI at 2 loci. However, mutations were not detected in 6 cancers, with MSI only at 1 locus and 31 cancers without MSI. Moreover, microanalysis in these cases revealed that the mutant-type alleles of TbetaR-II were invariably common in different areas within the tumor, in contrast to the markedly variable alleles of microsatellite loci. Our results suggest that frameshift mutation of the TbetaR-II gene may be a critical event associated with MSI and may contribute to carcinogenesis of the stomach. One of the possible mechanisms of escape from growth control by TGFbeta during gastric carcinogenesis could involve frameshift mutations of the TbetaR-II gene caused by DNA replication errors.     

Lack of ETV6 (TEL) gene rearrangements or p16INK4A/p15INK4B homozygous gene deletions in infant acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) occurring in infants less than 1 year of age differs clinically and biologically from that observed in older children. Cytogenetically, 11q23 translocations are detected in approximately 50% of infant ALLs and fuse the 11q23 gene HRX with a variety of partner chromosomal loci. Overall, HRX rearrangements are detected molecularly in 70-80% of infant ALLs as compared to 5-7% of ALLs arising in older children. Two recently described molecular abnormalities in childhood ALL are ETV6 gene rearrangements and homozygous deletions of p16(INK4A) and/or p15(INK4B). Each of these abnormalities occurs in 15-20% of all childhood ALLs, and neither can be accurately identified by routine cytogenetic analyses. The incidence of these genetic abnormalities and their potential relationship to HRX gene status in infant ALL is unknown. Using Southern blot analyses, we determined ETV6 and p16(INK4A)/p15(INK4B) gene status in a cohort of infant ALLs. No ETV6 rearrangements or homozygous deletions (n=69) or homozygous p16(INK4A) and/or p15(INK4B) gene deletions (n=54) were detected in any of the infant ALLs. Therefore, ETV6 and p16(INK4A)/p15(INK4B) do not play a significant role in the pathogenesis of infant ALL, further emphasizing the distinctive biology of this subset of leukemias.     

p16(INK4a) gene inactivation by deletions, mutations, and hypermethylation is associated with transformed and aggressive variants of non-Hodgkin's lymphomas

The molecular mechanisms underlying the pathogenesis of aggressive lymphomas and the histological transformation of indolent variants are not well known. To determine the role of p16(INK4a) gene alterations in the pathogenesis of non-Hodgkin's lymphomas (NHLs) and the histological progression of indolent variants, we have analyzed the expression, deletions, and mutations of this gene in a series of 112 NHLs. Hypermethylation of the gene was also examined in a subset of tumors with lack of protein expression but without mutations or deletions of the gene. p16(INK4a) gene alterations were detected in 3 out of 64 (5%) indolent lymphomas but in 16 out of 48 (33%) primary or transformed aggressive variants. In the low-grade tumors, p16(INK4a) alterations were detected in 1 (4%) chronic lymphocytic leukemia (hemizygous missense mutation), 1 (6%) follicular lymphoma (homozygous deletion), and 1 (5%) typical mantle cell lymphoma (homozygous deletion). The two later cases followed an aggressive clinical evolution. In the aggressive tumors, p16(INK4a) gene alterations were observed in 2 (29%) Richter's syndromes (2 homozygous deletions), 3 (33%) transformed follicular lymphomas (1 homozygous deletion and 2 nonsense mutations), 3 (43%) blastoid mantle cell lymphomas (2 homozygous and 1 hemizygous deletions), 5 (28%) de novo large-cell lymphomas (1 homozygous deletion and 4 hypermethylations), 2 lymphoblastic lymphomas (2 homozygous deletions), and 1 of 2 anaplastic large cell lymphomas (hypermethylation). Protein expression was lost in all tumors with p16(INK4a) alterations except in the typical chronic lymphocytic leukemia (CLL) with hemizygous point mutation. Sequential samples of the indolent and transformed phase of three cases showed the presence of p16(INK4a) deletions in the Richter's syndrome but not in the CLL component of two cases, whereas in a follicular lymphoma the deletion was present in both the follicular tumor and in the diffuse large-cell lymphoma. In conclusion, these findings indicate that p16(INK4a) gene alterations are a relatively infrequent phenomenon in NHLs. However, deletions, mutations, and hypermethylation of the gene with loss of protein expression are associated with aggressive tumors and they may also participate in the histological progression of indolent lymphomas.     

Analyses of mutation and loss of heterozygosity of coding sequences of the entire transforming growth factor beta type II receptor gene in sporadic human gastric cancer

Mutations in the transforming growth factor beta type II receptor (TGFbetaRII) gene have been detected in several human cancer types exhibiting microsatellite instability. Using intron primers previously reported for examination of the entire coding region of the TGFbetaRII gene, 29 sporadic gastric cancers were screened with non-radioactive single strand conformation polymorphism and subsequent DNA sequencing analysis. Mutations of the TGFbetaRII gene were detected in three out of 29 tumors (10%). Two cases showed deletions in a polyadenine tract in both alleles and was positively associated with replication error. One case had an insertion of GA dinucleotide sequence in one allele. Mutations of the TGFbetaRII gene were restricted to exon 3 and other coding regions were not affected. Loss of heterozygosity was detected by analyzing a polymorphic site in intron 2. Three out of nine (33%) informative cases, which were all of intestinal type and advanced cases, showed loss of heterozygosity but neither TGFbetaRII mutation nor replication error was found in these cases. Immunoreactivity of TGFbetaRII in tumor tissues was reduced to a different extent in the gastric cancer with genetically abnormal transforming growth factor. Although the numbers studied are small, homozygous (A)10 deletion or loss of heterozygosity of TGFbetaRII is involved in tumorigenesis and progression of at least some part of sporadic gastric cancer.     

Mutations of the transforming growth factor-beta type II receptor gene are strongly related to sporadic proximal colon carcinomas with microsatellite instability

BACKGROUND: Mutations of the transforming growth factor-beta type II receptor gene (TGF-beta RII) have been found in several replication error-positive sporadic colorectal carcinomas and hereditary nonpolyposis colorectal carcinoma cell lines. The aim of this study was to clarify the role of TGF-beta RII in sporadic colorectal carcinogenesis. METHODS: The authors screened for mutations at simple repeated sequences in the TGF-beta RII gene by polymerase chain reaction-single strand conformation polymorphism. They also examined genomic instability, using five microsatellite DNA markers in 69 sporadic colorectal carcinomas. When the carcinomas exhibited the TGF-beta RII mutations, the authors screened further for mutations in two DNA mismatch repair genes, hMSH2 and hMLH1. RESULTS: Seven of the 69 cancers (10%) showed one or two A deletions in TGF-beta RII and resultant frameshift mutations in nucleotide positions 709-718 containing a (A) 10 repeated sequence; but none of these appeared in the corresponding normal DNA, indicating a somatic mutation. All of the seven cancers were located in the proximal colon; there were none in the distal colon (P < 0.01). On the other hand, 22 of the 69 carcinomas (32%) showed the replication error-positive phenotype. The frequency of replication errors in proximal colon carcinomas was higher than that in distal colon carcinomas (P < 0.05). All 7 cancers with TGF-beta RII mutations showed replication errors. One of them revealed a nonsense mutation at codon 413, and 1 revealed a loss of heterozygosity in hMSH2. CONCLUSIONS: These data indicate that mutations of TGF-beta RII are strongly related to proximal colon carcinomas with microsatellite instability and that the mechanism of carcinogenesis in some proximal colon carcinomas is similar to that in hereditary nonpolyposis colorectal carcinoma.     

Frequency of mutation and deletion of the tumor suppressor gene CDKN2A (MTS1/p16) in hepatocellular carcinoma from an Australian population

The tumor suppressor gene CDKN2A (MTS1/p16), located on chromosome 9p21, is inactivated in a variety of tumors including melanomas and tumors of the biliary tract, pancreas, and stomach. The aim of the present study was to determine whether this gene is inactivated in hepatocellular carcinoma (HCC). Twenty-three primary HCCs and four HCC cell lines were examined. Loss of heterozygosity (LOH) analysis was performed using eight polymorphic markers immediately surrounding CDKN2A, and showed a contiguous region of loss, with the two most commonly deleted markers being D9S1604, located between the p16 and p15 genes, at which 7 of 13 informative tumors (54%) showed loss, and D9S171, with 4 of 14 LOH (29%). Exons 1, 2, and 3 of CDKN2A were amplified by polymerase chain reaction to detect homozygous deletions, and single-strand conformation polymorphism (SSCP) analysis was performed to screen for mutations. No homozygous deletions were detected in any sample. SSCP and sequence analysis showed the same nucleotide change at codon 148 in four tumors. This has been reported elsewhere as a polymorphism. One of these four tumors also contained a mutation at codon 119, resulting in the substitution of an acidic amino acid for a basic one. It is concluded that CDKN2A is infrequently deleted or mutated in HCC. The region of allelic loss upstream from CDKN2A might result in inactivation of regulatory sequences important in the expression of this gene; alternatively, a second tumor suppressor gene may be present in the region 9p21-22, proximal to CDKN2A. These possibilities require further investigation.     

Hepatocyte growth factor/scatter factor and its receptor c-Met are overexpressed and associated with an increased microvessel density in malignant pleural mesothelioma

The product of the p16/INK4a/CDKN2/MTS1 tumor-suppressor gene acts as a negative cell cycle regulator by inhibiting G1 cyclin-dependent kinases that phosphorylate the retinoblastoma protein. p16 is inactivated in a wide range of human malignancies, including familial melanoma. However, its expression and function in sporadic melanoma has not been extensively investigated. We studied p16 expression in 62 archival melanomas and 30 archival nevi and lentigines by immunohistochemistry. Eighteen of 26 (69%) superficial spreading melanomas, 17 of 28 (61%) nodular melanomas, all of three lentigo maligna melanomas, and all of five melanoma metastases were found to harbor less than 10% p16-positive tumor cells. In contrast, only six of 24 (25%) nevi had less than 10% positive cells. No correlation between tumor thickness and loss of p16 expression was found. Using DNA from micro-dissected tumor and matched normal tissues, five of seven (71%) p16-negative melanoma cases had 9p21 loss of heterozygosity (LOH), and one of these 9p21 LOH cases had promoter region hypermethylation of the remaining p16 allele. These data demonstrate that partial or complete loss of p16 expression is prevalent in sporadic melanoma and is frequently associated with 9p21 LOH.     

Production of macrocyclic ellagitannin oligomers by Oenothera laciniata callus cultures

Melanocyte-stimulating hormone (MSH) has been reported to enhance the experimental metastatic behaviour of melanoma cells in the mouse model. alpha-MSH production and MSH receptor (melanocortin 1 receptor gene) expression have been detected in cultured normal human melanocytes and metastasized melanomas. The exact role of MSH in the metastatic behaviour of human melanoma cells is, however, not yet known. To clarify a possible role of proopiomelanocortin (POMC)-derived peptides, including alpha-MSH, in melanoma development and progression, we analysed immunohistochemically the localization of alpha-MSH adrenocorticotrophic hormone (ACTH) and beta-endorphin in various kinds of benign pigmented naevocytic lesions and malignant melanomas. Three of 21 samples of common and dysplastic naevi showed detectable alpha-MSH staining in naevus cells, and five and six of 15 samples were weakly positive for ACTH and beta-endorphin staining, respectively. In melanoma samples, 24 of 45, 23 of 39 and 30 of 42 samples showed positive staining with alpha-MSH, ACTH and beta-endorphin antibodies, respectively. Furthermore, staining for all three antibodies was noted to be more intense and diffuse in samples of nodular melanoma, vertically growing acral lentiginous melanoma and superficial spreading melanoma as well as metastatic lesions compared with those of naevi. Although it is yet to be determined whether or not this strong staining for POMC-derived peptides in advanced melanoma cells indicates a role of autocrine or paracrine regulation, our results suggest a possible involvement of POMC gene products in melanoma progression.     

Identification of various medically important Candida species in clinical specimens by PCR-restriction enzyme analysis

It is still unclear whether the sporadic form of dysplastic nevi (SDN) represents a premalignant lesion of malignant melanoma and whether genetic alterations are involved in the development of SDN. To determine whether p16INK4a and p53 genetic abnormalities could be associated with development of SDN, nevus cell nests were procured selectively from H & E-stained slide sections by using a modified microdissection technique and were screened for the presence of mutations and loss of heterozygosity (LOH) of p16INK4a and p53 genes using a polymerase chain reaction-based LOH, single-strand conformation polymorphism, and direct DNA sequencing analyses. Hemizygous deletion was detected in 9 of 12 informative cases (75%) for 9p21-22 (p16INK4a) at one or more loci and 60% (6/10) for 17p13 (p53). As for mutation, we found 3 missense mutations and 1 mutation in the first intron in p16INK4a and 2 missense mutations in p53. Among these mutations in p16INK4a and p53, 5 of 6 mutations were of the C:G to T:A transitional type; this is known to be related to ultraviolet radiation as previously confirmed in other skin cancers. This indicates that p16INK4a and p53 genetic alterations may play an important role in the evolution of SDN and may represent an early event in the development of malignant melanoma. Furthermore, ultraviolet radiation might be the predominant etiologic agent in the development of SDN.     

A novel missense mutation and frameshift mutations in the type II receptor of transforming growth factor-beta gene in sporadic colon cancer with microsatellite instability

Microsatellite instability of DNA samples of 79 sporadic colon cancer patients were analyzed. These samples were also screened to search mutations in the repeat sequences in the gene for the type II receptor of transforming growth factor-beta (TGF-beta RII) using polymerase chain reaction (PCR), electrophoresis with urea gel, and PCR-single strand conformation polymorphism (PCR-SSCP) method. The incidence of microsatellite instability, defined as severe replication error phenotype (RER) with microsatellite alterations in more than three loci, was 6%. Deletion and insertion of an A residue in the (A)10 region, which cause frameshift mutation, were found in four samples and their incidence in the samples with microsatellite instability was 80%. A novel nucleotide substitution of T for G at 1918, which causes missense mutation of arginine to leucine at codon 528, was found in a sample with microsatellite instability. The mutation at 1918 was in highly conservative amino acid residue.     

p16INK4 gene mutations are relatively frequent in ampullary carcinomas

A high incidence of gene mutations or deletions of p16INK4, a cell cycle regulator which inhibits the activity of cyclin-dependent kinase 4/cyclin D complex and blocks the G1-to-S transition, has been reported in pancreato-biliary tract cancers. In order to investigate p16INK4 gene alterations in sporadic ampullary carcinomas, 17 sporadic ampullary carcinomas were examined. After histological diagnosis, DNA samples extracted separately from both cancerous and normal paraffin-embedded tissues were investigated. Loss of heterozygosity (LOH) was investigated utilizing 3 microsatellite markers on 9p21-22, and a mutational analysis was performed by cloning and sequencing. LOH was observed in 3 cases (17.6%) and somatic mutations with retention of heterozygosity were found in 7 cases (41.2%). Of note was that two mutations resulted in truncated incomplete proteins and one was a point mutation at the consensus site in the conserved ankyrin repeats, which would be crucial for function. Although two-hit inactivation was not evident in any of the mutation cases and further investigation would be needed to elucidate the role of altered p16INK4, these results suggest that the p16INK4 gene mutations are relatively frequent and its inactivation might be important in ampullary carcinogenesis.     

Detection of loss of heterozygosityat microsatellite loci in esophageal squamous-cell carcinoma

Microsatellite alterations at 3 genetic loci (chromosomes 2p, 3p and 17p) were analyzed in 25 tumors (20 primary tumors and 5 metastatic lymph nodes) from 20 patients after surgical treatment for esophageal cancer. DNA samples from tumors were compared with control DNA from lymphocytes obtained from the peripheral blood of the individual patients. Microsatellite alterations [microsatellite instability (MSI) and loss of heterozygosity (LOH)] were detected in 15% of 20 primary tumors with marker D2S123 (chromosome 2p), 55% with marker D3S1067 (chromosome 3p) and 50% with marker TP53 (chromosome 17p). The 3-year disease-free survival rate of the 10 patients who had tumors without alterations or with an alteration at only 1 of 3 microsatellite loci was 75% and it was better than that of the 10 patients who had tumors with alterations at 2 or 3 microsatellite loci (48%, p = 0.049). This finding suggests that esophageal cancer with alterations at multiple microsatellite loci might have strong malignant potential. However, MSI was only detected in one of 20 patients, which suggests that MSI might not play an important role in the development of this cancer. Three of 5 metastatic lymph nodes showed no LOH even though primary tumors of these patients exhibited LOH with 1 or 2 markers, and 1 metastatic lymph node had LOH that was detected with D3S1067 even though the primary tumor of this patient had no LOH with all markers. Thus, clonal heterogeneity might exist in esophageal squamous-cell carcinomas.     

Molecular genetics of familial cutaneous melanoma

PURPOSE: A family history of melanoma is a significant risk factor for the disease, and recently several loci that determine susceptibility to the development of melanoma have been identified. The most important of these is p16/CDKN2A. We attempted to determine the degree to which the p16/CDKN2A gene has been implicated in the development of melanoma, and to identify other genetic factors that play a role as well. METHODS: We reviewed the literature published since the isolation of p16/CDKN2A and identified 13 studies that report the status of the gene in melanoma samples and 12 reports that examine p16/CDKN2A in melanoma kindreds. We also reviewed associated studies on CDK4 and RB1 involvement in melanoma, and examined the role of p16/CDKN2A in other inherited cancers. RESULTS: The evidence strongly implicates p16/CDKN2A in determining predisposition to malignant melanoma. Overall, approximately 20% of families that have been studied show mutations in the gene. However, because of clustering of sporadic cases in families, and potentially because of technical factors, this is likely an underestimate of the proportion of the genetic predisposition for melanoma that is due to p16/CDKN2A mutation. Rare families carry a mutated CDK4 gene that is also responsible for inherited melanoma. CONCLUSION: The gene p16/CDKN2A is an important determinant of melanoma risk. A commercial test is presently available to assess the status of this locus. However, because of uncertainties regarding the interpretation of the results of p16/CDKN2A genetic testing, we do not recommend routine clinical use of this test at this time.     

Allelic losses and DNA methylation at DNA mismatch repair loci in sporadic colorectal cancer

Normal and tumor DNA samples of 35 patients with sporadic colorectal carcinoma were analyzed for microsatellite alterations at 12 markers linked to mismatch repair loci: hMLH1, hMSH2, hMSH3, hMSH6, hPMS1 and hPMS2. Remarkably, no correlation was observed between the replication error phenotype (RER+) and allelic losses at these loci. Hemizygous deletions, seen in 6/35 (17%) informative cases at hMLH1, 4/27 (15%) at hMSH2/hMSH6 and 6/34 (18%) at hMSH3, were rarely found in RER+ tumors. Since mismatch repair protein components act in molecular complexes of defined stoichiometry we propose that hemizygous deletion of the corresponding loci may be involved in colorectal tumorigenesis through defects in cellular functions other than replication error correction. The analysis of the methylation status of the promoter region of hMLH1 revealed that methylation might be an important mechanism of this locus inactivation in RER+ sporadic colorectal cancer.     

Identification of two distinct deletion targets at 11q23 in cutaneous malignant melanoma

Karyotypic and molecular data indicate that genetic alterations of the long arm of chromosome 11 (11q) are involved in the pathogenesis of malignant melanoma as well as of other malignancies. We have shown previously, by analysis of loss of heterozygosity (LOH), that a tumor-suppressor gene playing an important role in malignant melanoma is likely to be located within a 51-cM region at 11q23. Its loss appeared to be a late event in tumor progression and an indicator of a less favorable clinical outcome. To further test this hypothesis on a larger set of tumors and to refine the region(s) of common allelic loss, we analyzed 21 polymorphic microsatellite repeats on 11q. A PCR-based assay for LOH was used to study normal and tumor tissues from 53 individuals with primary cutaneous malignant melanoma or metastatic disease. Our findings indicate that in cutaneous malignant melanoma there are at least 2 distinct regions of common allelic loss on 11q, one of them centered around marker APOC3 at 11q23.1-q23.2 delineated by markers D11S1347 and D11S4142 and spanning approximately 5 Mb and a second 3-Mb region around marker D11S925 at 11q23.3 delineated by markers D11S528 and D11S1345. Both regions have been described as deletion targets or as being included within larger allelic deletions detected in several other common tumor types. Thus, these 2 putative melanoma-suppressor loci are likely to harbor tumor-suppressor genes relevant to tumorigenesis of melanoma and a number of other common human malignancies.