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Clinical Cancer Research Vol. 5, 2431-2437, September 1999
© 1999 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Genetic Alterations of the Tumor Suppressor Gene PTEN/MMAC1 in Human Brain Metastases

Matthias Hahn1, Ivonne Wieland, Olga N. Koufaki, Heike Görgens, Stephan B. Sobottka, Gabriele Schackert and Hans K. Schackert

Departments of Surgical Research [M. H., I. W., O. N. K., H. G., H. K. S.] and Neurosurgery [S. B. S., G. S.], Technical University of Dresden, D-01307 Dresden, Germany


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The high mutation rate in advanced brain tumors, recent functional studies, and the high frequency of mutations in prostate metastases all strongly suggest that PTEN/MMAC1 alterations are involved in the formation of metastases. We searched for genetic alterations in the PTEN/MMAC1 gene in 56 consecutive brain metastases from various primary tumors by loss of heterozygosity (LOH), direct sequence analysis, and differential PCR analysis. The highest LOH rates were detected in metastases deriving from lung (67%) and breast (64%) cancers. Three (25%) of the eight detected inactivating mutations (one nonsense mutation, one splice-site mutation, one 11-bp deletion, and five homozygous deletions) were found in metastases originating from 12 different lung carcinomas, suggesting that PTEN/MMAC1 alterations may play a role in the progression of this tumor. With the exception of lung carcinomas, our findings indicate that genetic abnormalities of the PTENM/MMAC1 gene are only involved in a relatively small subset of brain metastases. However, the discrepancy between the high overall LOH rate (50%) and the low frequency of PTEN/MMAC1 mutation detection rate (14%) suggests the presence of one or more additional tumor suppressor genes on chromosome 10q.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
One frequently observed genetic alteration in a variety of advanced tumor entities is LOH2 at chromosome 10q23. Through the mapping of homozygous deletions on 10q23, a candidate tumor suppressor gene has been identified, and genetic alterations have been documented in brain, breast, kidney, and prostate cancer cell lines or tumor specimens (1 , 2) . Extensive further studies on this candidate tumor suppressor gene, which has been designated PTEN (phosphatase and tensin homolog deleted on chromosome 10) or MMAC1 (mutated in multiple advanced cancers 1), validated and extended initial observations in a variety of different tumor entities. Somatic mutations were frequently identified in glioblastoma (3, 4, 5, 6, 7, 8, 9) , endometrial (10, 11, 12) , and prostate (13, 14, 15) cancers and, to a lesser extent, in some additional tumors.

Extensive PTEN/MMAC1 homologies to the cytoskeletal protein tensin gave rise to the speculation that the gene of interest might be involved in tumor cell invasion and metastasis through tensin interactions with actin filaments at focal adhesions (1) . This hypothesis was recently supported by transfection experiments conducted by Tamura et al. (16 , 17) showing that overexpression of PTEN/MMAC1 inhibited cell migration, whereas antisense PTEN/MMAC1 enhanced migration. Furthermore, it has been demonstrated that only wild-type PTEN/MMAC1 down-regulates integrin-mediated cell spreading and the formation of focal adhesions. An unaltered PTEN/MMAC1 gene may, thus, play a role in negatively regulating dynamic cell surface interactions with the extracellular matrix. Numerous studies on metastatic tissue of different primary tumors seem to support these functional findings. Guldberg et al. (18) found PTEN/MMAC1 mutations in some of the metastases but not in the matching primary melanomas. A series of studies on prostate cancer tissues revealed a correlation between chromosome 10 inactivation and metastatic phenotype. Nihei et al. (19) demonstrated metastasis suppression by introduction of human chromosome 10 transfer in a rat prostate cancer cell line. Komiya et al. (20) studied 10q alterations in both metastatic and primary tumors and found 10q abnormalities in metastatic tissue but not in corresponding primary cancer foci in five of nine cases. Allelic loss at one or more loci on 10q was observed in all metastatic tissues. Suzuki et al. (15) detected PTEN/MMAC1 gene alterations in at least one metastatic site in 12 of 19 prostate cancer patients studied, suggesting that PTEN/MMAC1 gene alterations occur frequently in lethal prostate cancer. Petersen et al. (21) recently showed that LOH on chromosome 10q is associated with the metastatic phenotype of squamous cell carcinomas of the lung.

The high mutation rate in advanced brain tumors, recent functional studies, and the findings of frequent PTEN/MMAC1 mutations in metastatic tissue all raise the question of whether PTEN/MMAC1 may also be involved in the acquisition of metastatic ability in brain metastases. To test this hypothesis, we performed LOH analysis and subsequent PTEN/MMAC1 sequence analysis in 56 consecutive brain metastases from various primary tumors.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tumor Specimens and DNA Extraction.
Tumor and corresponding blood samples were obtained from 56 patients undergoing surgical removal of brain metastases at the Department of Neurosurgery, Technical University of Dresden. Metastases were derived from the following primary tumors: 14 breast cancers, 12 lung cancers (11 non-small cell and 1 small cell), 9 colorectal cancers, 7 kidney cancers, 6 malignant melanomas, 2 thyroid cancers, 1 endometrial cancer, 1 esophageal cancer, 1 liposarcoma, 1 leiomyosarcoma, 1 chondrosarcoma, and 1 germ cell tumor. Tumor samples were collected fresh at the time of surgery, snap-frozen in liquid nitrogen, and kept frozen at -80°C. Paraffin-embedded tissues of brain metastases were histopathologically diagnosed by the Institute of Pathology, Technical University of Dresden, according to WHO criteria. For preparation of tumor DNA, microdissection techniques were applied to minimize contamination with surrounding normal tissue DNA. High molecular weight DNA from tumor tissue and corresponding leukocyte DNA were extracted and purified with the QIAamp blood and tissue kit (Qiagen, Inc., Hilden, Germany) following standard procedures.

LOH Analysis.
LOH analysis was performed on all 56 consecutive surgically removed brain metastases of different primary tumors (Fig. 1)Citation . Two microsatellite markers, D10S215 and D10S541, are closely flanking markers to PTEN/MMAC1; two others, AFMa086wg9 (1) and PTENCA (5) , are intragenetically localized. To determine LOH extension, we used two microsatellite markers, one centromeric (D10S532) and one telomeric (D10S583), spanning a genetic distance of 10 cM around the locus of interest. Each forward primer was end-labeled with Cy-5 by the manufacturer (Amersham-Pharmacia Biotech Europe GmbH). After amplification through 24 cycles, PCR products were resolved on 6.5% Long Ranger polyacrylamide gels on Automated Laser Fluorescence Sequencers (model A.L.F.; Amersham-Pharmacia). This technical approach enabled plotting of microsatellite alleles with direct calculation of peak areas using the Fragment Manager Version 1.1 software (Pharmacia Biotech, Freiburg, Germany). For evaluation of LOH, we used a semiquantitative approach, as proposed by Cawkwell et al. (22) . Briefly, the ratio of both microsatellite alleles (allele kept/allele lost) in the tumor DNA was divided by the corresponding ratio found in blood leukocyte DNA. Resulting values of >1.5 (imbalance factor) were classified as LOH, consistent with >33% allele reduction in tumor DNA.



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  		Fig. 1. Results of the LOH and sequence analysis in 56 brain metastases. The microsatellite markers D10S215 (centromeric) and D10S541 (telomeric) closely flank PTEN/MMAC1, whereas AFMa086wg9 and PTENCA are intragenetically localized. To assess the extension of allelic losses, we used D10S532 and D10S583. , LOH. {square}: HET, retention of heterozygosity; NI, noninformative marker; X, allele intensity not consistent with homozygous deletion [more than the value of (x - 3 SDs)/2]. {blacksquare}, homozygous deletions; and •, homozygous deletions with apparent retention of heterozygosity flanked by LOH, both confirmed by differential PCR analysis. {circ}, retention of heterozygosity flanked by LOH at markers both telomeric and centromeric of PTEN/MMAC1 coding regions but no verified homozygous deletion. *, LOH with amplified retained allele (data not shown).

 
RNA Extraction and RT-PCR.
mRNA was extracted and purified from tumor tissue cells by use of QuickPrep Micro mRNA Purification Kit (Amersham-Pharmacia) and stored at -80°C. cDNA was generated using random hexamer primers supplied by the First Strand cDNA Synthesis Kit (Amersham-Pharmacia), according to the manufacturer’s protocol. The complete coding region was amplified from 1.5 µl of the cDNA reaction using the following primers: sense, 5'-atg aca gcc atc atc aaa g-3'; and antisense, 5'-tca gac ttt tgt aat ttg tgt a-3'. PCR was performed in a final volume of 22 µl containing 0.18 µM each primer and 0.8 units of Taq polymerase. The PCR consisted of 35 cycles of 1 min at 94°C, 1 min at 58°C, and 1 min at 72°C, with an initial denaturation step of 5 min at 94°C and a final elongation step of 7 min at 72°C. The PCR-amplified PTEN/MMAC1 cDNAs were subjected to electrophoresis on a 1% agarose gel and visualized by ethidium bromide. Glyceraldehyde-3-phosphate dehydrogenase-specific primers were used as a positive control under the same PCR conditions mentioned above (23) .

Sequence Analysis.
All tumors that exhibited LOH in at least one of the four markers within or closely flanking the PTEN/MMAC1 gene or MSI were subjected to sequence analysis. Genomic tumor DNAs from all nine exons including intron/exon boundaries were amplified. For exons 1–4, we used the primer set described by Steck et al. (2) , modified by removal of the nonspecific 5' sequence tails from the nested primers. For the forward nested primers, the nonspecific primer sequence at the 5' end was exchanged by an alternative primer sequence (5'-CGACGTTGTAAAACGACGGCCAGT-3'). Accordingly, cyanine-labeled primers with this sequence were used for the cycle-sequencing reaction. For exons 5–9, primers were used as described by Liaw et al. (24) with the difference that the sequencing primers were labeled with Cy-5. Each PCR mix consisted of 50 ng of genomic tumor DNA, 0.1 µM each primer, 1x PCR buffer, 2.0 mM MgCl2, 200 µM dNTPs, and 0.75 unit of Taq polymerase (Perkin-Elmer Corp., Foster City, CA) in a total volume of 25 µl. PCRs were performed in a GeneAmp 9700 thermocycler (Perkin-Elmer) at 94°C for 1 min, 58°C for 1 min, and 72°C for 1 min for a total of 35 cycles, with an initial denaturation step (95°C for 4 min) and a final extension step (72°C for 7 min). PCR-amplified products were electrophoresed on agarose gels, excised, purified by MicroSpin S-200 HR columns (Amersham-Pharmacia), and subjected to cycle sequencing reactions for 21 cycles using the Thermo Sequenase dye-primer cycle sequencing kit (Amersham-Pharmacia). After a denaturation step (94°C for 5 min), cycle sequencing products were resolved on an Automated Laser Sequencer (A.L.F.; Amersham-Pharmacia) using 6.5% Long Ranger gels at 40 W, 900 V, and 40 mA.

Differential PCR Analysis.
To test for homozygous deletions, we established comparative multiplex PCRs, each consisting of one standard primer pair outside the region of suspected homozygous deletions (Mfd26 on 18q12.2–12.3; sense, 5'-Cy-5-cag aaa att ctc tct ggc ta-3'; antisense, 5'-ctc atg ttc ctg gca aga at-3'), and two test primer pairs that were specific for exons 3 (sense, 5'-Cy-5-ata gaa ggg gta ttt gtt gga-3'; antisense, 5'-cct cac tct aac aag cag ata-3') and 7 (sense, 5'-Cy-5-cag tta aag gca ttt cct gtg-3'; antisense, 5'-gga tat ttc tcc caa tga aag-3') of the PTEN/MMAC1 gene. If one of the two exons tested positive for homozygous deletions, four additional microsatellites (D10S215, D10S541, AFMa086wg9, and PTENCA) were used to confirm and determine the extension of the respective homozygous deletion. The PCR mix consisted of 30 ng of genomic tumor DNA, 0.12 µM Mfd26, 0.20 µM exon 3 primers and 0.16 µM exon 7 primers, 1.2x PCR buffer, 2.25 mM MgCl2, 200 µM dNTPs, and 0.5 units of Taq polymerase (Perkin-Elmer) in a total volume of 12.5 µl. PCRs were performed in a GeneAmp 9700 thermocycler (Perkin-Elmer) at 94°C for 1 min; 56°C (Mfd26/D10S215, Mfd26/D10S541), 58°C (Mfd26/AFMa086wg9, Mfd26/PTENCA), or 59°C (Mfd26/exon 3, Mfd26/exon 7) for 1 min; and 65°C for 1 min, for a total of 24 cycles with an initial denaturation step (95°C for 4 min) and a final extension step (70°C for 7 min). Amplified multiplex products were loaded on an Automated Laser Sequencer (A.L.F.; Amersham-Pharmacia) using a 6.5% Long Ranger gel at 40 W, 1200 V, and 40 mA. Resulting peak areas were calculated using the Fragment Manager Version 1.1 software (Amersham-Pharmacia). To determine the standard ratio between the test (exon 3, exon 7, AFMa086wg9, PTENCA, D10S215, and D10S541) and standard marker (Mfd26) three leukocyte DNA samples of six healthy individuals were coamplified in each of the multiplex PCRs. The mean values (x) and SDs were calculated (test peak/standard peak). Following the method of Duerr et al. (8) , peak reductions in exons 3 or 7, AFMa086wg9, PTENCA, D10S215, and D10S541 were scored as homozygous deletions if the individual ratio (exon 3/Mfd26, exon 7/Mfd26, AFMa086wg9/Mfd26, PTENCA/Mfd26, D10S215/Mfd26, or D10S541/Mfd26) was less than the following value: (x - 3 SDs)/2). Potential homozygous deletions or amplifications of the standard marker Mfd26 were excluded by performing a multiplex PCR with two additional microsatellite markers (D3S1300 and D21S1414) on different chromosomes.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
LOH Analysis.
A total of 56 brain metastases deriving from a variety of primary tumors were analyzed for LOH using a panel of four microsatellite markers (D10S215, PTENCA, AFMa086wg9, and D10S541) within or in close proximity to the PTEN/MMAC1 genomic locus. All tumors were informative in at least one of the four markers studied. LOH was found in 28 cases (50%) in at least one marker, and 18 (32%) tumors demonstrated allelic losses at all informative loci (Fig. 1Citation and Table 1Citation ). The highest LOH rates were seen in lung (67%) and breast (64%) cancers.


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  		Table 1 Number and percentage of each type of tumor that showed LOH involving PTEN/MMAC1a

 
To determine the specificity of LOH to PTEN/MMAC1, we used four additional microsatellite markers (D10S532, D10S1687, D10S579, and D10S583), spanning a genomic distance of {approx}10 cM around the genomic locus of interest, for breakpoint analysis. Eighteen (64%) of all tumors that were LOH positive within the PTEN/MMAC1 gene (PTENCA and AFMa086wg9) or in the closest flanking markers (D10S215 to D10S541, <=1 cM) also exhibited allelic losses in at least one of the most distant flanking markers, D10S532 (2 cM, centromeric) or D10S583 (8 cM, telomeric). Only seven (25%) cases with LOH exclusively targeted one or more loci within or in close proximity to PTEN/MMAC1 (D10S215 to D10S541, <=1 cM), suggesting that the vast majority of LOHs do not exclusively target PTEN/MMAC1.

Differential PCR Analysis.
To assess the incidence of homozygous deletions, we subjected a commonly observed genetic alteration at 10q23 (1 , 2) in all 56 brain tumors to differential PCR analysis, using the microsatellite Mfd26 (D18S34) as a standard marker to determine the individual gene dosage of PTEN/MMAC1. Five tumors (cases 4, 9, 29, 31, and 56; 9%) repeatedly presented with substantial reductions in band intensities in exon 3 or 7 of PTEN/MMAC1, which are compatible with loss of both alleles [less than the value of (x - 3 SDs)/2; Figs. 2Citation and 3Citation ). All brain metastases with reduction in band intensity consistent with homozygous deletions in exon 3 or 7 were subjected to an additional differential PCR analysis using the markers D10S215, AFMa086wg9, PTENCA, and D10S541. In all five cases with homozygous deletions in exon 3 or 7, at least one adjoining marker exhibited a value in band reduction that is in accordance with loss of both alleles and, thus, confirmed our initial findings. All five obviously homozygously deleted tumors were subjected to RT-PCR and showed no visible PTEN/MMAC1 RT-PCR product. In the same RT-PCR, five randomly chosen brain metastases without evidence of homozygous deletion were RT-PCR positive for PTEN/MMAC1.



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  		Fig. 2. Summary of deletion mapping for each tumor that showed homozygous deletion. The relative orientation of the loci tested are represented by circles. Homozygous, hemizygous, or no deletion of the locus in a specific tumor are indicated by black, gray, and open circles, respectively. X, allele intensity not consistent with homozygous deletion [more than the value of (x - 3 SDs)/2]; NI, noninformative marker. Double circles, homozygous deletions with apparent retention of heterozygosity flanked by LOH. *, LOH with amplified retained allele.

 


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  		Fig. 3. Representative example of a differential PCR analysis (case 56) showing a homozygous deletion of the intragenic marker AFMa086wg9 in tumor DNA. The peaks are compared with a standard microsatellite marker Mfd26 on chromosome 18q. Three samples of normal DNA of six healthy individuals were coamplified in each of the multiplex PCRs. The mean ratios (x) and SDs were calculated (test marker/standard marker). Following the method of Duerr et al. (8) , peak reductions were scored as homozygous deletions if the individual ratio (AFMa086wg9/Mfd 26) was less than the value of (x - 3 SDs)/2. The calculation of the ratio of peak areas of AFMa086wg9 and Mfd26 showed an allele reduction of less than the value of (x - 3 SDs)/2. Thus, AFMa086wg9 was classified as homozygously deleted. The residual peak in tumor DNA is due to contaminating normal DNA. Amplifications or homozygous deletions of the standard marker (Mfd26) were excluded in each case by comparison with coamplified markers D3S1300 and D21S1414 (data not shown).

 
Retention of Heterozygosity Flanked by LOH.
Four tumors showed retention of heterozygosity flanked by LOH at markers both telomeric and centromeric of PTEN/MMAC1 coding regions. This observation is in accordance with LOH patterns frequently found at the PTEN/MMAC1 locus in various tumors (5 , 13 , 15 , 25 , 26) . On the basis of fluorescence in situ hybridization and Southern analyses, retention of heterozygosity embedded within an area of LOH has been shown to correlate significantly with homozygous deletions (13 , 15 , 26 , 27) , a comparatively common mutational event in PTEN/MMAC1 (1 , 2) . This retention of heterozygosity obviously reflects the amplification of DNA deriving from contaminating normal cells on the background of loss of both alleles in tumor tissue. In this tumor panel, three (cases 29, 31, and 56) of five homozygously deleted tumors exhibited retention of heterozygosity at the PTEN/MMAC1 locus with telomeric and centromeric LOH, additionally supporting our findings of homozygous deletions by differential PCR analysis. However, one tumor (case 16), presenting with apparent retention of heterozygosity at the intragenic marker PTENCA with LOH at flanking markers, failed to show a significant reduction in band intensity [more than the value of (x - 3 SDs)/2] in exons 3 and 7 and in PTENCA. Furthermore, the RT-PCR yielded a strong and clearly visible PTEN/MMAC1 product. Therefore, the LOH pattern at this locus obviously reflects no homozygous deletion at 10q23.

MSI.
Three brain metastases exhibited MSI in at least one microsatellite marker. All markers with LOH and/or MSI were included in the subsequent sequence analysis.

Sequence Analysis.
According to Knudson’s two-hit hypothesis (28), inactivation of the second allele because of LOH is commonly associated with a point mutation of the remaining alleles in tumor suppressor genes. This raises the question of whether the high frequency (50%) of allelic losses at the PTEN/MMAC1 locus is reflected by numerous inactivating PTEN/MMAC1 point mutations. To address this issue, we subjected each of the 28 brain tumors that exhibited LOH in at least one marker and 3 tumors that exhibited MSI to complete sequence analysis of all nine exons, including intron/exon boundaries (Table 1)Citation .

One nonsense mutation was detected in codon 245 (CAG->TAG) in exon 7. Subsequent sequence analysis showed that this mutation was absent in the corresponding primary tumor that was a non-small cell lung carcinoma.

An 11-bp deletion (codons 338–341) in exon 8 was found in a brain metastasis deriving from a stage IV breast carcinoma. Due to an alteration of the reading frame, this deletion results in a premature stop at codon 338. No deletion at this location was detected in the primary tumor.

One of three tumors showing MSI exhibited a nucleotide substitution at the +3 splice donor site of exon 6 (A->T). By RT-PCR of tumor mRNA, two distinct bands were found, the size of the shorter PCR product being consistent with lack of exon 6 as a result of aberrant splicing. Bidirectional sequence analysis confirmed the absence of exon 6 in the shorter fragment. The lack of exon 6 results in an alteration of the reading frame and creates a premature stop codon in exon 7 (codon 220). Because most markers at this genomic locus were unstable, there was no clear indication of LOH. Therefore, a single-cell PCR was performed using a sample of the corresponding primary endometrial carcinoma that showed the identical mutation in a nonheterozygous state (data not shown), indicating a hemizygous situation, which is compatible with the absence of the second allele.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Despite the high LOH rate (64%) in metastases deriving from breast cancers, only one point mutation was detected in the tumor panel studied. This observation supports the results of other studies that found few or no somatic PTEN/MMAC1 alterations in breast cancers (29, 30, 31) , despite commonly observed LOH at the PTEN/MMAC1 locus (32 , 33) . Singh et al. (33) found LOH in the region near D10S215 in most of the poorly differentiated breast cancers, an observation that, taken together with the data presented here, suggests that LOH at the PTEN/MMAC1 locus is associated with poor prognosis. However, our LOH breakpoint analysis provides evidence that LOH is not exclusively restricted to the PTEN/MMAC1 locus and might involve the alteration of tumor suppressors other than PTEN/MMAC1.

In contrast to 1 of 14 breast carcinomas, 12 different brain metastases deriving from lung carcinomas harbored 3 (25%) somatic gene alterations, including 1 nonsense mutation and 2 homozygous deletions. These data underscore the significance of PTEN/MMAC1 alterations in the progression of lung cancers, paralleled by the observation that a substantial proportion (67%) of lung carcinomas exhibited LOH at the PTEN/MMAC1 locus. The particular significance of losses on chromosome 10q in tumor progression and metastasis formation in lung cancer has been recently demonstrated by Petersen et al. (21) , who found that advanced tumor stages in carcinomas of the lung correlated significantly with the presence of LOH of polymorphic markers located between 10q21 and 10qter. Furthermore, for metastatic squamous cell carcinomas of the lung, a positive association has been found between LOH and metastasis formation. In another study, Petersen et al. (34) identified three regions of allelic losses in squamous cell carcinomas of the lung that were clustered at the loci AFMa086wg9/D10S541, D10S185, and D10S1782/D10S169. In contrast to our data, no PTEN/MMAC1 point mutations were detected in those tumors. This led to the assumption that additional loci on chromosome 10q may harbor tumor suppressor genes that contribute to metastasis formation. Our data, however, provide evidence that PTEN/MMAC1 is inactivated in at least a subset of lung carcinomas (25%) and may contribute to the progression of lung carcinoma to more advanced tumor stages or the acquisition of metastatic potential. Previous studies, particularly in lung cancer cell lines, revealed that point mutations and homozygous deletions are genetic events in the progression of this tumor (35 , 36) . Our sequence analyses of lung cancer metastases support this hypothesis and, thus, suggest that PTEN/MMAC1 point mutations and allelic losses at 10q23 are useful genetic markers in assessment of malignant or even metastatic potential of lung cancers.

The only brain metastasis originating from an endometrial carcinoma exhibited MSI and harbored a splice site variation, resulting in a truncated protein. The identical mutation was found in the tissue sample of the primary tumor in a heterozygous state. The detection of a mutation in the only metastasis deriving from an endometrial carcinoma exhibiting MSI reflects the high proportion of PTEN/MMAC1 alterations that have been observed to range from 32 to 55% in endometrial cancers in numerous studies (10, 11, 12 , 37, 38, 39, 40, 41, 42) as well as the high frequency of PTEN/MMAC1 mutations in endometrial cancers exhibiting MSI (10 , 12 , 38) .

Metastases from two melanomas (two of six) and one ovarian leiomyosarcoma (one of one) each exhibited a homozygous deletion. However, sample sizes appear to be too small to reach any conclusions about the true frequency and relevance of these inactivating mutations according to tumor progression.

The low mutation frequency detected suggests that PTEN/MMAC1 is unlikely to be the primary target in the general pathogenesis of brain metastases. Although it has not yet been reported to be a common inactivation mechanism for PTEN/MMAC1 methylation or other forms of transcriptional inactivation of the retained allele in the cases with LOH cannot be excluded. Furthermore, because of contamination of normal cells in the metastasis tissue, small homozygous deletions that involve one or more complete exons would escape both sequence analysis and the differential PCR analysis used in this study.

Despite the high LOH rate (28 of 56, 50%) observed, 18 metastases (18 of 28, 64%) that were LOH-positive in microsatellite markers located within or in close proximity to PTEN/MMAC1 also showed LOH in at least one marker that was localized at a distance {approx}2 or 8 cM from the gene of interest. The wide extension of genetic losses in conjunction with rare mutational events suggests that one or two additional loci may harbor genes that are relevant for metastasis formation, especially in these tumor entities.

A recent study on metastasizing and nonmetastasizing head and neck squamous cell carcinoma suggested that the chromosome 10q25–q26 is significantly associated with metastasis formation (43) . One additional candidate tumor suppressor gene located on chromosome 10q25.3–26.1 (44) , DMBT1, is suspected to contribute to tumor progression. DMBT1, however, has been shown to be frequently homozygously deleted in medulloblastomas and glioblastomas (44) and seems to be a strong candidate for further analyses.

The data presented here provide evidence that the PTEN/MMAC1 tumor suppressor gene inactivation is not necessarily a prerequisite for the formation of brain metastases. Furthermore, the high rate of LOH on chromosome 10q in a wide range of advanced cancers suggests that one or more additional tumor suppressor genes are localized on chromosome 10q and that their inactivation may play a key role in the progression of advanced tumors. However, the high proportion of inactivating mutations in lung cancer (25%) suggests that PTEN/MMAC1 inactivation contributes to tumor progression in a subgroup of metastases.


   ACKNOWLEDGMENTS
 
We thank U. Neumeister and M. Reichmann for their excellent technical assistance.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Abteilung Chirurgische Forschung, Technische Universität Dresden, Fetscherstrasse 74, D-01307 Dresden, Germany. Phone: 49-351-458-3873; Fax: 49-351-458-4350; E-mail: Matthias.Hahn{at}mailbox.tu-dresden-de Back

2 The abbreviations used are: LOH, loss of heterozygosity; Cy-5, cyanine-5; RT-PCR, reverse transcription-PCR; MSI, microsatellite instability. Back

Received 5/10/99; revised 6/30/99; accepted 6/30/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Li J., Yen C., Liaw D., Podsypanina K., Bose S., Wang S. I., Puc J., Miliaresis C., Rodgers L., McCombie R., Bigner S. H., Giovanella B. C., Ittmann M., Tycko B., Hibshoosh H., Wigler M. H., Parsons R. PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science (Washington DC), 275: 1943-1947, 1997.[Abstract/Free Full Text]
  2. Steck P. A., Pershouse M. A., Jasser S. A., Yung W. K., Lin H., Ligon A. H., Langford L. A., Baumgard M. L., Hattier T., Davis T., Frye C., Hu R., Swedlund B., Teng D. H., Tavtigian S. V. Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23 that is mutated in multiple advanced cancers. Nat. Genet., 15: 356-362, 1997.[Medline]
  3. Liu W., James C. D., Frederick L., Alderete B. E., Jenkins R. B. PTEN/MMAC1 mutations and EGFR amplification in glioblastomas. Cancer Res., 5: 5254-5257, 1997.
  4. Rasheed B. K., Stenzel T. T., McLendon R. E., Parsons R., Friedman A. H., Friedman H. S., Bigner D. D., Bigner S. H. PTEN gene mutations are seen in high-grade but not in low-grade gliomas. Cancer Res., 57: 4187-4190, 1997.[Abstract/Free Full Text]
  5. Wang S. I., Puc J., Li J., Bruce J. N., Cairns P., Sidransky D., Parsons R. Somatic mutations of PTEN in glioblastoma multiforme. Cancer Res., 57: 4183-4186, 1997.[Abstract/Free Full Text]
  6. Boström J., Cobbers J. M., Wolter M., Tabatabai G., Weber R. G., Lichter P., Collins V. P., Reifenberger G. Mutation of the PTEN (MMAC1) tumor suppressor gene in a subset of glioblastomas but not in meningiomas with loss of chromosome arm 10q. Cancer Res., 58: 29-33, 1998.[Abstract/Free Full Text]
  7. Chiariello E., Roz L., Albarosa R., Magnani I., Finocchiaro G. PTEN/MMAC1 mutations in primary glioblastomas and short-term cultures of malignant gliomas. Oncogene, 16: 541-545, 1998.[Medline]
  8. Duerr E. M., Rollbrocker B., Hayashi Y., Peters N., Meyer-Puttlitz B., Louis D. N., Schramm J., Wiestler O. D., Parsons R., Eng C., von Deimling A. PTEN mutations in gliomas and glioneuronal tumors. Oncogene, 16: 2259-2264, 1998.[Medline]
  9. Tohma Y., Gratas C., Biernat W., Peraud A., Fukuda M., Yonekawa Y., Kleihues P., Ohgaki H. J. PTEN (MMAC1) mutations are frequent in primary glioblastomas (de novo) but not in secondary glioblastomas. Neuropathol. Exp. Neurol., 57: 684-899, 1998.[Medline]
  10. Kong D., Suzuki A., Zou T. T., Sakurada A., Kemp L. W., Wakatsuki S., Yokoyama T., Yamakawa H., Furukawa T., Sato M., Ohuchi N., Sato S., Yin J., Wang S., Abraham J. M., Souza R. F., Smolinski K. N., Meltzer S. J., Horii A. PTEN1 is frequently mutated in primary endometrial carcinomas. Nat. Genet., 17: 143-144, 1997.[Medline]
  11. Risinger J. I., Hayes A. K., Berchuck A., Barrett J. C. PTEN/MMAC1 mutations in endometrial cancers. Cancer Res., 57: 4736-4738, 1997.[Abstract/Free Full Text]
  12. Tashiro H., Blazes M. S., Wu R., Cho K. R., Bose S., Wang S. I., Li J., Parsons R., Ellenson L. H. Mutations in PTEN are frequent in endometrial carcinoma but rare in other common gynecological malignancies. Cancer Res., 57: 3935-3940, 1997.[Abstract/Free Full Text]
  13. Cairns P., Okami K., Halachmi S., Halachmi N., Esteller M., Herman J. G., Jen J., Isaacs W. B., Bova G. S., Sidransky D. Frequent inactivation of PTEN/MMAC1 in primary prostate cancer. Cancer Res., 57: 4997-5000, 1997.[Abstract/Free Full Text]
  14. Pesche S., Latil A., Muzeau F., Cussenot O., Fournier G., Longy M., Eng C., Lidereau R. PTEN/MMAC1/TEP1 involvement in primary prostate cancers. Oncogene, 16: 2879-2883, 1998.[Medline]
  15. Suzuki H., Freije D., Nusskern D. R., Okami K., Cairns P., Sidransky D., Isaacs W. B., Bova G. S. Interfocal heterogeneity of PTEN/MMAC1 gene alterations in multiple metastatic prostate cancer tissues. Cancer Res., 58: 204-209, 1998.[Abstract/Free Full Text]
  16. Tamura M., Gu J., Matsumoto K., Aota S., Parsons R., Yamada K. M. Inhibition of cell migration, spreading, and focal adhesions by tumor suppressor PTEN. Science (Washington DC), 280: 1614-1617, 1998.[Abstract/Free Full Text]
  17. Tamura M., Gu J., Takino T., Yamada K. M. Tumor suppressor PTEN inhibition of cell invasion, migration, and growth: differential involvement of focal adhesion kinase and p130Cas. Cancer Res., 59: 442-449, 1999.[Abstract/Free Full Text]
  18. Guldberg P., thor Straten P., Birck A., Ahrenkiel V., Kirkin A. F., Zeuthen J. Disruption of the MMAC1/PTEN gene by deletion or mutation is a frequent event in malignant melanoma. Cancer Res., 57: 3660-3663, 1997.[Abstract/Free Full Text]
  19. Nihei N., Ichikawa T., Kawana Y., Kuramochi H., Oshimura M., Killary A. M., Rinker-Schaeffer C. W., Barrett J. C., Isaacs J. T., Shimazaki J. Localization of metastasis suppressor gene(s) for rat prostatic cancer to the long arm of human chromosome 10. Genes Chromosome Cancer, 14: 112-119, 1995.[Medline]
  20. Komiya A., Suzuki H., Ueda T., Yatani R., Emi M., Ito H., Shimazaki J. Allelic losses at loci on chromosome 10 are associated with metastasis and progression of human prostate cancer. Genes Chromosomes Cancer, 17: 245-253, 1996.[Medline]
  21. Petersen S., Wolf G., Bockmuhl U., Gellert K., Dietel M., Petersen I. Allelic loss on chromosome 10q in human lung cancer: association with tumour progression and metastatic phenotype. Br. J. Cancer, 77: 270-276, 1998.[Medline]
  22. Cawkwell L., Lewis F. A., Quirke P. Frequency of allele loss of DCC, p53, RB1, WT1, NF1, NM23 and APC/MCC in colorectal cancer assayed by fluorescent multiplex polymerase chain reaction. Br. J. Cancer, 70: 813-818, 1994.[Medline]
  23. Wong H., Anderson W. D., Cheng T., Riabowol K. T. Monitoring mRNA expression by polymerase chain reaction: the "primer-dropping" method. Anal. Biochem., 223: 251-258, 1994.[Medline]
  24. Liaw D., Marsh D. J., Li J., Dahia P. L., Wang S. I., Zheng Z., Bose S., Call K. M., Tsou H. C., Peacocke M., Eng C., Parsons R. Germline mutations of the PTEN gene in Cowden disease, an inherited breast and thyroid cancer syndrome. Nat. Genet., 16: 64-67, 1997.[Medline]
  25. Cairns P., Evron E., Okami K., Halachmi N., Esteller M., Herman J. G., Bose S., Wang S. I., Parsons R., Sidransky D. Point mutation and homozygous deletion of PTEN/MMAC1 in primary bladder cancers. Oncogene, 16: 3215-3218, 1998.[Medline]
  26. Wang S. I., Parsons R., Ittmann M. Homozygous deletion of the PTEN tumor suppressor gene in a subset of prostate adenocarcinomas. Clin. Cancer Res., 4: 811-815, 1998.[Abstract]
  27. Cairns P., Polascik T. J., Eby Y., Tokino K., Califano J., Merlo A., Mao L., Herath J., Jenkins R., Westra W., Rutter J. L., Buckler A., Gabrielson E., Tockman M., Cho K. R., Hedrick L., Bova G. S., Isaacs W., Schwab D., Sidransky D. Frequency of homozygous deletion at p16/CDKN2 in primary human tumours. Nat. Genet., 11: 210-212, 1995.[Medline]
  28. Knudson A. G. Hereditary cancer: two hits revisited. J. Cancer Res. Clin. Oncol., 122: 135-140, 1996.[Medline]
  29. Rhei E., Kang L., Bogomolniy F., Federici M. G., Borgen P. I., Boyd J. Mutation analysis of the putative tumor suppressor gene PTEN/MMAC1 in primary breast carcinomas. Cancer Res., 57: 3657-3659, 1997.[Abstract/Free Full Text]
  30. Ueda K., Nishijima M., Inui H., Watatani M., Yayoi E., Okamura J., Yasutomi M., Nakamura Y., Miyoshi Y. Infrequent mutations in the PTEN/MMAC1 gene among primary breast cancers. Jpn. J. Cancer Res., 89: 17-21, 1998.[Medline]
  31. Sakurada A., Suzuki A., Sato M., Yamakawa H., Orikasa K., Uyeno S., Ono T., Ohuchi N., Fujimura S., Horii A. Infrequent genetic alterations of the PTEN/MMAC1 gene in Japanese patients with primary cancers of the breast, lung, pancreas, kidney, and ovary. Jpn. J. Cancer, 88: 1025-1028, 1997.
  32. Bose S., Wang S. I., Terry M. B., Hibshoosh H., Parsons R. Allelic loss of chromosome 10q23 is associated with tumor progression in breast carcinomas. Oncogene, 17: 123-127, 1998.[Medline]
  33. Singh B., Ittmann M. M., Krolewski J. J. Sporadic breast cancers exhibit loss of heterozygosity on chromosome segment 10q23 close to the Cowden disease locus. Genes Chromosomes Cancer, 21: 166-171, 1998.[Medline]
  34. Petersen S., Rudolf J., Bockmuhl U., Gellert K., Wolf G., Dietel M., Petersen I. Distinct regions of allelic imbalance on chromosome 10q22–q26 in squamous cell carcinomas of the lung. Oncogene, 17: 449-454, 1998.[Medline]
  35. Forgacs E., Biesterveld E. J., Sekido Y., Fong K., Muneer S., Wistuba I. I., Milchgrub S., Brezinschek R., Virmani A., Gazdar A. F., Minna J. D. Mutation analysis of the PTEN/MMAC1 gene in lung cancer. Oncogene, 17: 1557-1565, 1998.[Medline]
  36. Kohno T., Takahashi M., Manda R., Yokota J. Inactivation of the PTEN/MMAC1/TEP1 gene in human lung cancers. Genes Chromosomes Cancer, 22: 152-156, 1998.[Medline]
  37. Levine R. L., Cargile C. B., Blazes M. S., van Rees B., Kurman R. J., Ellenson L. H. PTEN mutations and microsatellite instability in complex atypical hyperplasia, a precursor lesion to uterine endometrioid carcinoma. Cancer Res., 58: 3254-3258, 1998.[Abstract/Free Full Text]
  38. Risinger J. I., Hayes K., Maxwell G. L., Carney M. E., Dodge R. K., Barrett J. C., Berchuck A. PTEN mutation in endometrial cancers is associated with favorable clinical and pathologic characteristics. Clin. Cancer Res., 4: 3005-3010, 1998.[Abstract]
  39. Kurose K., Araki T., Matsunaka T., Takada Y., Emi M. Variant Manifestation of Cowden disease in Japan: hamartomatous polyposis of the digestive tract with mutation of the PTEN gene. Am. J. Hum. Genet., 64: 308-310, 1999.[Medline]
  40. Lin W. M., Forgacs E., Warshal D. P., Yeh I. T., Martin J. S., Ashfaq R., Muller C. Y. Loss of heterozygosity and mutational analysis of the PTEN/MMAC1 gene in synchronous endometrial and ovarian carcinomas. Clin. Cancer Res., 4: 2577-2583, 1998.[Abstract]
  41. Maxwell G. L., Risinger J. I., Tong B., Shaw H., Barrett J. C., Berchuck A., Futreal P. A. Mutation of the PTEN tumor suppressor gene is not a feature of ovarian cancers. Gynecol. Oncol., 70: 13-16, 1998.[Medline]
  42. Simpkins S. B., Peiffer-Schneider S., Mutch D. G., Gersell D., Goodfellow P. J. PTEN mutations in endometrial cancers with 10q LOH: additional evidence for the involvement of multiple tumor suppressors. Gynecol. Oncol., 71: 391-395, 1998.[Medline]
  43. Bockmühl U., Petersen S., Schmidt S., Wolf G., Jahnke V., Dietel M., Petersen I. Patterns of chromosomal alterations in metastasizing and nonmetastasizing primary head and neck carcinomas. Cancer Res., 57: 5213-5216, 1997.[Abstract/Free Full Text]
  44. Mollenhauer J., Wiemann S., Scheurlen W., Korn B., Hayashi Y., Wilgenbus K. K., von Deimling A., Poustka A. DMBT1, a new member of the SRCR superfamily, on chromosome 10q25.3–26.1 is deleted in malignant brain tumours. Nat. Genet., 17: 32-39, 1997.[Medline]



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