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Loss of Heterozygosity on Chromosome 11q22-23 in Melanoma Is Associated with Retention of the Insertion Polymorphism in the Matrix Metalloproteinase-1 Promoter

2001; Elsevier BV; Volume: 158; Issue: 2 Linguagem: Inglês

10.1016/s0002-9440(10)64011-4

1525-2191

Walter W. Noll, Dorothy R. Belloni, Joni L. Rutter, Craig A. Storm, Alan R. Schned, Linda Titus‐Ernstoff, Marc S. Ernstoff, Constance Brinckerhoff,

Peptidase Inhibition and Analysis

Matrix metalloproteinase-1 (MMP-1, collagenase-1), which degrades interstitial collagen, is expressed at high levels by some tumor cells and is thought to enhance their invasiveness and metastatic potential. We recently described a common single nucleotide insertion polymorphism (2G allele) at −1,607 bp in the promoter of the MMP-1 gene that creates a binding site for the ETS family of transcription factors, and that is associated with enhanced transcription of this gene and increased enzyme activity. Allelic loss at the MMP-1 locus on chromosome 11 occurs in many tumors including melanoma, an invasive and aggressive cancer. We hypothesized that although loss of either the 1G or 2G allele from 1G/2G heterozygotes is random, retention of the transcriptionally more active 2G allele would favor tumor invasion and metastasis. As a result, a higher proportion of metastases would contain the 2G genotype than the 1G genotype. We report here the development of quantitative methods for assessing allelic loss at the MMP-1 locus, and demonstrate that 83% of the metastatic melanomas with loss of heterozygosity at this locus retained the 2G allele. This supports the hypothesis that retention of the 2G allele favors tumor invasion and metastasis in melanoma. Matrix metalloproteinase-1 (MMP-1, collagenase-1), which degrades interstitial collagen, is expressed at high levels by some tumor cells and is thought to enhance their invasiveness and metastatic potential. We recently described a common single nucleotide insertion polymorphism (2G allele) at −1,607 bp in the promoter of the MMP-1 gene that creates a binding site for the ETS family of transcription factors, and that is associated with enhanced transcription of this gene and increased enzyme activity. Allelic loss at the MMP-1 locus on chromosome 11 occurs in many tumors including melanoma, an invasive and aggressive cancer. We hypothesized that although loss of either the 1G or 2G allele from 1G/2G heterozygotes is random, retention of the transcriptionally more active 2G allele would favor tumor invasion and metastasis. As a result, a higher proportion of metastases would contain the 2G genotype than the 1G genotype. We report here the development of quantitative methods for assessing allelic loss at the MMP-1 locus, and demonstrate that 83% of the metastatic melanomas with loss of heterozygosity at this locus retained the 2G allele. This supports the hypothesis that retention of the 2G allele favors tumor invasion and metastasis in melanoma. Matrix metalloproteinase-1 (MMP-1, collagenase-1), a member of a family of MMPs that degrades most components of the extracellular matrix, breaks down the interstitial collagens types I, II, and III.1Vincenti MP White LA Schroen DJ Benbow U Brinckerhoff CE Regulating expression of the gene for matrix metalloproteinase-1 (collagenase): mechanisms that control enzyme activity, transcription and mRNA stability.Crit Rev Eucaryotic Gene Express. 1996; 6: 391-411Crossref PubMed Scopus (243) Google Scholar, 2Borden P Heller RA Transcriptional control of matrix metalloproteinases and the tissue inhibitors of matrix metalloproteinases.Crit Rev Eukaryotic Gene Express. 1997; 7: 159-178Crossref PubMed Scopus (291) Google Scholar, 3Nagase H Woessner Jr, JF Matrix metalloproteinases.J Biol Chem. 1999; 274: 21491-21492Crossref PubMed Scopus (3916) Google Scholar MMP-1 is ubiquitously expressed by most normal cell types, usually at low levels. 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Indeed, evidence suggests that expression of MMP-1 is linked to poor outcome in several cancers, including colon carcinoma, esophageal carcinoma, pancreatic carcinoma, and melanoma.12Murray GI Duncan ME O'Neil P Melvin WT Fothergill JE Matrix metalloproteinase-1 is associated with poor prognosis in colorectal cancer.Nat Med. 1996; 2: 461-462Crossref PubMed Scopus (440) Google Scholar, 13Murray GI Duncan ME O'Neil P McKay JA Melvin WT Fothergill JE Matrix metalloproteinase-1 is associated with poor prognosis in oesophageal cancer.J Pathol. 1998; 185: 256-261Crossref PubMed Scopus (268) Google Scholar, 14Ito T Ito M Shiozawa J Naito S Kanematsu T Sekine I Expression of the MMP-1 in human pancreatic carcinoma: relationship with prognostic factor.Mod Pathol. 1999; 12: 669-674PubMed Google Scholar, 15Airola K Karonen T Vaalamo M Lehti K Lohi J Kariniemi AL Keski-Oja J Saarialho-Kee UK Expression of collagenases-1 and -3 and their inhibitors TIMP-1 and -3 correlates with the level of invasion in malignant melanomas.Br J Cancer. 1999; 80: 733-743Crossref PubMed Scopus (206) Google Scholar Recently we described a common single nucleotide polymorphism (SNP) in the promoter of MMP-1, in which one allele is associated with enhanced transcription of this gene.8Rutter JL Mitchell TI Buttice G Meyers J Gusella JF Ozelius LJ Brinckerhoff CE A single nucleotide polymorphism in the matrix melloproteinase-1 promoter creates an Ets binding site and augments transcription.Cancer Res. 1998; 58: 5321-5325PubMed Google Scholar The SNP is located at −1,607 bp and is represented by the presence or absence of a guanine (G) at this position. In the absence of the G, the sequence reads 5′-GAA-3′, whereas the presence of an extra G gives the sequence 5′-GGAA-3′, a consensus binding site for the ETS family of transcription factors. Indeed, MMP-1 genes with promoters carrying the 2G allele are transcribed at a significantly higher rate in both normal fibroblasts and in tumor cells than are MMP-1 genes with promoters carrying the 1G allele.8Rutter JL Mitchell TI Buttice G Meyers J Gusella JF Ozelius LJ Brinckerhoff CE A single nucleotide polymorphism in the matrix melloproteinase-1 promoter creates an Ets binding site and augments transcription.Cancer Res. 1998; 58: 5321-5325PubMed Google Scholar The frequencies of the 1G and 2G alleles appear to be approximately equal in normal populations, whereas in a small sample of tumor cell lines tested, the frequency of the 2G allele was increased to 60%.8Rutter JL Mitchell TI Buttice G Meyers J Gusella JF Ozelius LJ Brinckerhoff CE A single nucleotide polymorphism in the matrix melloproteinase-1 promoter creates an Ets binding site and augments transcription.Cancer Res. 1998; 58: 5321-5325PubMed Google Scholar In addition, there was a higher than expected proportion of apparent 2G homozygotes in these cell lines, suggesting that the 2G allele may be associated with aggressive and invasive cancers.8Rutter JL Mitchell TI Buttice G Meyers J Gusella JF Ozelius LJ Brinckerhoff CE A single nucleotide polymorphism in the matrix melloproteinase-1 promoter creates an Ets binding site and augments transcription.Cancer Res. 1998; 58: 5321-5325PubMed Google Scholar This concept is supported by a significant association of the 2G allele with ovarian16Kanamori Y Matsushima M Minaguchi T Kobayashi K Sagae S Kudo R Terakawa N Nakamura Y Correlation between expression of the matrix metalloproteinase-1 gene in ovarian cancers and an insertion/deletion polymorphism in its promoter region.Cancer Res. 1999; 59: 4225-4227PubMed Google Scholar and endometrial cancer,17Nishioka Y Kobayashi K Sagae S Ishioka S Nishikawa A Matsushima M Kanamori Y Minaguchi T Nakamura Y Tokino T Kudo R A single nucleotide polymorphism in the matrix metalloproteinase-1 promoter in endometrial carcinomas.Jpn J Cancer Res. 2000; 91: 612-615Crossref PubMed Scopus (90) Google Scholar and with increased expression of MMP-1 protein in these diseases.16Kanamori Y Matsushima M Minaguchi T Kobayashi K Sagae S Kudo R Terakawa N Nakamura Y Correlation between expression of the matrix metalloproteinase-1 gene in ovarian cancers and an insertion/deletion polymorphism in its promoter region.Cancer Res. 1999; 59: 4225-4227PubMed Google Scholar, 17Nishioka Y Kobayashi K Sagae S Ishioka S Nishikawa A Matsushima M Kanamori Y Minaguchi T Nakamura Y Tokino T Kudo R A single nucleotide polymorphism in the matrix metalloproteinase-1 promoter in endometrial carcinomas.Jpn J Cancer Res. 2000; 91: 612-615Crossref PubMed Scopus (90) Google Scholar Although the findings in tumor cell lines may simply reflect a greater proportion of tumors derived from homozygous 2G hosts, it is also possible that these aggressive tumors result from the loss of the 1G allele in tumors from heterozygous hosts [loss of heterozygosity (LOH)]. LOH is a common event in many tumors and is often associated with advancing disease, presumably because of loss of tumor suppressor gene(s) at the deleted locus.18Driouch K Briffod M Bieche I Champeme M-H Lidereau R Location of several putative genes possibly involved in human breast cancer progression.Cancer Res. 1998; 58: 2081-2086PubMed Google Scholar, 19Laake K Launonen B Niederacher D Gudlaugsdottir S Seitz S Rio P Champeme M-H Bieche I Birnbaum D White G Sztan M Sever N Plummer S Osorio A Broeks A Huusko P Spurr N Borg A Cleton-Jansen A-M van't Veer L Benitez J Casey G Peterlin B Olah E Varley J Bignon Y-J Scherneck S Sigurdardottir V Lidereau R Eyfjord J Beckmann MW Winqvist R Skovlund E Borresen-Dale A-L Breast Cancer Somatic Genetics Consortium Loss of Heterozygosity at 11q23.1 and survival in breast cancer: results of a large European study.Genes, Chromosom Cancer. 1999; 25: 212-221Crossref PubMed Scopus (34) Google Scholar, 20Wang SS Esplin ED Li JL H L G A Minna J Evans GA Alterations of the PPP2R1B gene in human lung and colon cancer.Science. 1998; 282: 284-287Crossref PubMed Scopus (328) Google Scholar, 21Herbst RA Larson A Weiss J Cavenee WK Hampton GM Arden KC A defined region of loss of heterozygosity at 11q23 in cutaneous malignant melanoma.Cancer Res. 1995; 55: 2494-2496PubMed Google Scholar, 22Herbst RA Gutzmer R Matiaske F Mommert S Casper U Kapp A Weiss J Identification of two distinct deletion targets at 11q23 in cutaneous malignant melanoma.Int J Cancer. 1999; 80: 205-209Crossref PubMed Scopus (33) Google Scholar, 23Launonen V Stenback F Puistola U Bloigu R Huuski P Kytola K Kauppila A Winqvist R Chromosome 11q22.3-q25 LOH in ovarian cancer: association with a more aggressive disease course and involved subregions.Gynecol Oncol. 1998; 71: 299-304Abstract Full Text PDF PubMed Scopus (41) Google Scholar, 24Lazar AD Winter MR Nogueira CP Larson PS Finnemore EM Dolan RW Fuleihan N Chakravarti A Zietman A Rosenberg CL Loss of heterozygosity at 11q23 in squamous cell carcinoma of the head and neck is associated with recurrent disease.Clin Cancer Res. 1998; 4: 2787-2793PubMed Google Scholar, 25Morita R Fujimoto A Hatta N Takehara K Takata M Comparison of genetic profiles between primary melanoma and their metastases reveals genetic alterations and clonal evolution during progression.J Invest Dermatol. 1998; 111: 919-924Crossref PubMed Scopus (52) Google Scholar, 26Phillips KK Welch DR Miele ME Lee J-H Wei LL Weissman BE Suppression of MDA-MB-435 breast carcinoma cell metastasis following the introduction of human chromosome 11.Cancer Res. 1996; 56: 1222-1227PubMed Google Scholar LOH occurs at several chromosomal loci, including the MMP-1 locus at 11q22.23.27Pendas AM Balbin M Llano E Jimenez MG Lopez-Otin C Structural analysis and promoter characterization of the human collagenase-3 gene (MMP-13).Genomics. 1997; 40: 222-233Crossref PubMed Scopus (188) Google Scholar We tested the hypothesis that if metastatic tumors were derived from cells that retained the 2G allele, they would have a selective invasive advantage and would, therefore, be overrepresented in heterozygous hosts compared to tumors that had retained the 1G allele. Accordingly, we genotyped normal and metastatic tumor tissues from patients with melanoma to determine the frequency of the 2G allele and the occurrence of LOH. To perform LOH studies with a relatively large number of patient samples, we developed a rapid method to screen samples for heterozygosity and precise quantitative techniques to objectively assess LOH. Samples were available from a total of 61 patients: frozen tumor and peripheral blood leukocytes from 12 patients; formalin-fixed, paraffin-embedded normal and tumor tissue from 44 patients; and both frozen and paraffin-embedded samples from five patients. The frozen tissues had been stored for up to 3 years at −70OC; the paraffin blocks had been stored for up to 7 years at room temperature. In addition to these paired normal tissue and tumor samples, control germline DNA samples from known heterozygotes were run with each experiment to provide additional data for calculation of interassay variability. Genomic DNA was isolated from frozen peripheral blood lymphocytes or from frozen single-cell tumor suspensions using Gentra’s Puregene (Minneapolis, MN) kit according to the manufacturer’s instructions. These tumor samples had been collected for the purpose of vaccine production and had been trimmed, originally, to be free of normal tissue; however, this was not confirmed at the time of this study. One 5-μm section and three adjacent 10-μm serial sections of formalin-fixed, paraffin-embedded tumor and normal tissue were mounted on glass slides. The 5-μm section was stained with hematoxylin and eosin. Under a dissecting microscope, normal and tumor tissue were identified by a pathologist on unstained sections by comparing these to the adjacent stained section, and were scraped separately with a sterile pin into a microfuge tube. The pathologist performing the dissections estimated that no more than 20% of the cells dissected from tumor were contaminating normal cells. Digestion buffer, consisting of 25 μl of 50 mmol/L Tris-HCl, pH 8.5, 2% Laureth-12, 400 μg/ml proteinase K, was added, and the samples were incubated overnight in a 55°C oven to minimize evaporation. An additional 25 μl of digestion buffer was added and the samples were incubated at 55°C for an additional 4 to 5 hours. The proteinase K was inactivated by heating to 95°C for 10 minutes. The samples were spun at 13,000 × g in a microcentrifuge for 2 minutes and the supernatant was used for polymerase chain reaction (PCR). DNA from some tumor samples could not be amplified using this lysis method, perhaps because of high melanin concentrations that may have interfered with the PCR reaction.28Price K Linge C The presence of melanin in genomic DNA isolated from pigmented cell lines interferes with successful polymerase chain reaction: a solution.Melanoma Res. 1999; 9: 5-9Crossref PubMed Scopus (24) Google Scholar Those samples were recut and DNA was isolated using the Puregene kit. An RFLP method to distinguish the SNP was developed to screen samples rapidly and to identify heterozygotes for LOH studies. Restriction enzyme sites were introduced into the PCR amplicon at the SNP locus by making a single base change in the downstream primer, such that the 1G allele digested with Bgl II and the 2G allele digested with Alw I (Figure 1A). Purified DNA (100 ng) or lysate (5 μl) from the paraffin-embedded samples was amplified using 50 pmol of each primer (RFLP forward: GTC TTC CCA TTC TTC TTA CC; RFLP reverse: ATT GAT TTG AGA TAA GTC AGA TC) in 1× Qiagen (Valencia, CA) PCR buffer (Tris-Cl, pH 8.7, at 20°C, KCl, (NH4)2SO4,1.5 mmol/L MgCl2), 200 μmol/L each dNTP, 1.5 mmol/L additional MgCl2, for a total of 3 mmol/L MgCl2, 2.5 U HotStarTaq (Qiagen) in a 100 μl reaction. Cycling conditions were 95°C for 15 minutes followed by 35 cycles of 95°C for 30 seconds, 50°C for 30 seconds, and 72°C for 30 seconds with a final 72°C extension for 2 minutes. Samples (20 μl) were digested using 10 U of Alw I or Bgl II (New England Biolabs, Beverly, MA) at 37°C for 2 to 3 hours. The digests were electrophoresed on 3% SFR Agarose (Amresco, Solon, OH) and visualized with ethidium bromide. Tumor and normal tissue samples from heterozygotes were analyzed by two quantitative PCR assays, using sets of nonoverlapping primer pairs, that generated either a 72-bp amplicon or an 82-bp amplicon. This dual amplification with no overlap between the primer sets controlled for the possibility that a point mutation was present under either of the primer binding sites in the tumor DNA (that might alter the relative amplification of the 1G and 2G alleles), thereby giving a false indication of LOH. The primers for the 72-bp product were: forward: GTT ATG CCA CTT AGA TGA GG, end-labeled with γ-32P ATP (3,000 Ci/mmol); reverse: CTT GGA TTG ATT TGA GAT AAG (Figure 1B). The primers for the 82-bp product were: forward: GAA ATT GTA GTT AAA TAA TTA G, end-labeled with γ-32P ATP; reverse: GCT TAC TCA TAA ACA ATA CTT CAG (Figure 1B). The reverse primer of the 82-bp set was modified by the addition of a 5′ terminal G that, along with the cycling conditions, helped drive the PCR reaction to allele + A.29Magnuson VL Ally DS Nylund SJ Rayman JB Knapp JI Lowe AL Ghosh S Collins FS Substrate nucleotide-determined non-templated addition of adenine by Taq DNA polymerase: implications for PCR-based genotyping and cloning.Biotechniques. 1996; 21: 700-709Crossref PubMed Scopus (142) Google Scholar This modification eliminated shadow bands seen in previous studies using a different primer set.8Rutter JL Mitchell TI Buttice G Meyers J Gusella JF Ozelius LJ Brinckerhoff CE A single nucleotide polymorphism in the matrix melloproteinase-1 promoter creates an Ets binding site and augments transcription.Cancer Res. 1998; 58: 5321-5325PubMed Google Scholar, 30Dunleavey L Seyyare B Ye S Rapid genotype analysis of the matrix metalloproteinase-1 gene 1G/2G polymorphism that is associated with risk of cancer.Matrix Biol. 2000; 19: 175-177Crossref PubMed Scopus (46) Google Scholar For both PCR reactions 100 ng of purified DNA or 5 μl lysate from the paraffin-embedded samples was amplified using 50 pmol of each primer in 1× Qiagen PCR buffer (Tris-Cl, pH 8.7, at 20°C, KCl, (NH4)2SO4,1.5 mmol/L MgCl2), 200 μmol/L each dNTP, 1.5 mmol/L additional MgCl2, for a total of 3 mmol/L MgCl2, 2.5 U HotStarTaq (Qiagen) in a 100 μl reaction. Samples containing known ratios of 1G and 2G DNA were prepared for a standard curve (see Results) using purified peripheral blood DNA from 1G and 2G homozygotes. For the 72-bp product, cycling conditions were 95°C for 15 minutes, followed by 30 cycles of 95°C for 30 seconds, 50°C for 30 seconds, and 72°C for 30 seconds with a final 72°C extension for 2 minutes. For the 82-bp product, cycling conditions were 94°C for 15 minutes, followed by 10 cycles of 94°C for 15 seconds, 50°C for 30 seconds, and 72°C for 60 seconds, then 20 cycles of 89°C for 15 seconds, 50°C for 30 seconds, and 72°C for 60 seconds with a final 72°C extension for 30 minutes. Five μl of PCR product from each reaction was then mixed with 5 μl of formamide loading buffer and denatured at 95°C for 5 minutes. Five μl aliquots of the sample mixtures were loaded on an 8% denaturing acrylamide gel and run at 55 W constant power until the xylene cyanol was ∼6 cm from the bottom of the gel. Under these conditions there was excellent resolution of the 1G and 2G alleles for both the 72/73-bp and 82/83-bp products (see Results). Gels were exposed to a phosphorimager screen (Molecular Dynamics, Sunnyvale, CA) for 15 to 60 minutes and to film for 30 minutes to several hours. For quantitative analysis of allele intensity, the phosphorimager screens were scanned and the band intensities quantified using ImageQuant v. 1.2 (Molecular Dynamics) by drawing a 2 by 1 matrix around each lane, placing the 2G allele in the upper box and the 1G allele in the lower box. The percent 2G was calculated by dividing the signal in the upper box by the total signal in both boxes and multiplying by 100. The signals were not corrected for background. Under the null hypothesis, assuming that allele retention is a random event, we would expect the 1G or 2G allele to be retained in an equal number of tumors (ie, a genotype ratio of 1:1). We calculated an exact P value using a binomial distribution31Conover WJ Practical Nonparametric Statistics. 2nd ed. Wiley, New York1980Google Scholar for the likelihood that the observed genotype ratio in the 12 tumors with LOH was a chance finding. Normal DNA from 61 patients was genotyped using the RFLP method, yielding 102- or 103bp products. There were 31 1G/2G heterozygotes, 16 1G/1G homozygotes, and 14 2G/2G homozygotes (allele frequencies: 1G = 0.52, 2G = 0.48). Figure 2 demonstrates the RFLP pattern after digestion with Bgl II (the 102-bp 1G allele is cleaved) and after digestion with Alw I (the 103-bp 2G allele is cleaved). This method is rapid and does not require radioactivity, and the use of separate restriction enzymes to recognize each of the two alleles serves as an internal control. Of the 31 heterozygous patients identified by RFLP analysis, one patient had three metastases available for study, one patient had two metastases available for study (both of which were refractory to DNA amplification), and 29 had one metastasis available for study (one of which was refractory to amplification). The three samples refractory to DNA amplification were from paraffin blocks. In summary, 31 tumors and normal tissue from 29 patients were tested successfully for LOH at the SNP locus with the quantitative 72-bp and 82-bp amplicon assays. The most sensitive means of detecting LOH was by visual inspection of exposed film, the unaided eye readily seeing small deviations from the pattern of equal allele intensity seen with normal DNA from heterozygotes. However, at the outset of the study it was decided that an objective, quantitative method of assessing allelic imbalance was preferable to visual inspection to eliminate any possibility of observer bias. Accordingly, each experiment included seven standards, composed of various proportions of DNA from 1G and 2G homozygotes (0/100, 25/75, 40/60, 50/50, 60/40, 75/25, 100/0) expressed as “percent 2G” (Figure 3). A standard curve (x axis = actual percent 2G, y axis = observed percent 2G) was plotted and analyzed by linear regression, and the regression equation was used to calculate the actual percent 2G in the unknown samples. Because the phosphorimager data used to determine observed percent 2G were not corrected for background (see Materials and Methods), the regression line has a positive intercept on the y axis. The standard curves from successive experiments with each primer pair were remarkably reproducible and linear (Figure 4).Figure 4Standard curves for the 72-bp amplicon assay (A) and the 82-bp amplicon assay (B). Standards were composed of mixtures of DNA from 1G and 2G homozygous controls that were analyzed concurrently with patient samples. The actual composition of the standards is plotted on the x axis as “Actual %2G.” Data from quantitative phosphorimager analysis of the standards (see text) is plotted on the y axis as “Observed %2G.” Data from multiple experiments are shown, plotted as mean ± SD. Standard curves were used to convert “Observed %2G” of tumor samples to “Actual %2G.”View Large Image Figure ViewerDownload Hi-res image Download (PPT) The interassay variability of the percent 2G for heterozygous samples in the 72-bp amplicon assay was calculated from data obtained in multiple experiments from 94 normal tissue samples and control blood samples; 33 samples provided similar data for the 82-bp amplicon assay. The calculated percent 2G of heterozygous samples using the 72-bp amplicon assay was 50.5 ± 6.7 (mean, ±2 SD); for the 82-bp amplicon assay it was 49.9 ± 4.3 (mean, ±2 SD). These ranges are indicated as dashed lines in Figure 5. Tumor samples were classified as having undergone LOH if the calculated percent 2G exceeded the 2 SD range for heterozygotes in each of the two assays (Figure 5). If the deviation from heterozygosity was large, single concordant results from each assay were accepted as sufficient. If the deviation from heterozygosity was small or borderline, consistent reproducible findings with both primer pairs were required to classify the sample as having undergone LOH. In general, differences between replicate samples were small and the results of the two assays were in close agreement (Figure 5). Twelve of the 31 tumors (39%) showed marked or consistent deviation from the 1G/2G ratio characteristic of normal heterozygous tissue and were classified as having undergone LOH. Each of these tumors was from a different patient. Ten (83%) of the tumors underwent LOH with retention of the 2G allele, and two (17%) tumors underwent LOH with retention of the 1G allele (Figure 5). This departure from the expected 1:1 ratio was statistically significant (P = 0.04). Table 1 lists some of the clinical characteristics of the patients whose tumors had undergone LOH, the tissue source of the metastatic tumors that were analyzed, and the likely mode of spread (lymphatic or hematogenous) of each of the metastases.Table 1Summary of Heterozygous Patients with Metastases Demonstrating LOH at the MMP-1 LocusPatient no.AgeSexLocation of metastasisMechanism of spread*Mechanism of spread was scored as lymphatic if the metastasis was in a lymph node or in soft tissue in the path of lymphatic drainage of the primary tumor; other metastases were scored as hematogenous.Loss of 1G allele in metastasis854FSoft tissueLymphatic1464FLymph nodeLymphatic3475MLungHematogenous3842FLungHematogenous475FLymph nodeUnknown†This was scored as unknown because the location of the primary tumor in this patient was unknown.4871MLymph nodeLymphatic5339MLymph nodeLymphatic5477FSoft tissueLymphatic6167MSubcutaneousLymphatic6564MLymph nodeHematogenousLoss of 2G allele in metastasis138MLymph nodeLymphatic2253MSoft tissueHematogenous* Mechanism of spread was scored as lymphatic if the metastasis was in a lymph node or in soft tissue in the path of lymphatic drainage of the primary tumor; other metastases were scored as hematogenous.† This was scored as unknown because the location of the primary tumor in this patient was unknown. Open table in a new tab Some of the tumors displayed marked allelic imbalance, suggesting that the tissue samples that were analyzed consisted almost entirely of tumor cells, all of which had undergone LOH, and few nonneoplastic stromal or inflammatory cells. Other tumors had a lesser degree of allelic imbalance, suggesting the presence of a larger fraction of nonneoplastic cells in the sampled tissue or heterogeneity within the tumor cells, with only a fraction having undergone LOH. In the simplest model of LOH, loss of one allele with no other confounding genetic alterations, this study was capable of detecting LOH if at least 20 to 25% of the total cells in the sampled tissue had undergone allelic loss. In this report we describe methods for the rapid detection of a SNP in the MMP-1 (collagenase 1) promoter that can increase transcription of this gene, and thus enhance the destruction of the extracellular matrix necessary for tumor invasion and metastasis. In addition, we developed rapid and quantitative techniques for assessing LOH at this locus. We chose metastatic melanoma for this study because expression of col