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Mutations in the k-ras and TP53 genes, as well as microsatellite instability (MIN), are frequent genetic alterations in colorectal carcinomas and represent 3 different mechanisms in the carcinogenic process. Both the incidence of colorectal cancer and the frequency of genetic alterations in such tumours have been related to different clinico-pathological variables, including age and gender of the patient and location of the tumour. A number of studies have also reported associations between different types of genetic alterations. We therefore wanted to explore the relationship between these genetic and clinico-pathological variables using multivariate analysis on material from 282 colorectal carcinomas. Three logistic regression models were constructed: 1) the presence of K-ras mutations was dependent on MIN and age and gender of patient, with an especially low frequency among younger males and in tumours with MIN (overall p = 0.0003); 2) the presence of TP53 mutations was only dependent on tumour location, with a positive association to cancers occurring distally (p = 0.002); and 3) the presence of MIN was dependent on age, gender and K-ras and TP53 mutations, as well as on tumour location. MIN was most frequent among younger male and older female patients, was rare in tumours with K-ras or TP53 mutations and was found almost exclusively in the proximal colon (overall p < 0.0001). Our data confirm that different genetic pathways to colorectal cancer dominate in the proximal and distal segments of the bowel and suggest that the K-ras- and MIN-dependent pathways are influenced by different sex-related factors.

Microdissection of tissue sections from formalin-fixed, paraffin-embedded tumor material allows separation of microscopic sites within a sample. DNA can easily be extracted, and polymerase chain reaction (PCR) technology makes it possible to perform different molecular biologic analyses on small cell populations. The presence of normal cells or tumor heterogeneity may cause false negatives in allelic imbalance (AI) studies. Microdissected well-defined cell populations from a tumor section are assumed to increase the sensitivity of AI analyses. The present study has evaluated this in colorectal liver metastases by comparing genotypes in frozen biopsies with genotypes in microdissected archival samples from the same patients. Constitutional genotypes were obtained from corresponding peripheral blood leukocytes as well as normal liver tissue. Archival samples (n = 43) from 16 patients were analyzed after microdissection with 2-5 of 10 selected microsatellite markers. Frozen biopsies from one metastasis of each patient had previously been investigated at numerous microsatellite loci. From those results we selected, for the comparable analysis of archival samples, 41 tumor genotypes at 10 loci representing 11 heterozygotes, 13 AI, 7 losses of heterozygosity (LOHs), 8 homozygotes, and 2 microsatellite unstable cases. The microdissected samples revealed AI or a complete loss of one allele (LOH) in 5 of 11 (45%) genotypes that were previously evaluated as unchanged (retained heterozygosity) in the frozen biopsies, and LOH in 8 of 13 (62%) genotypes at loci known to exhibit AI in the frozen biopsies. Microsatellite instability, LOH, and homozygosity found in the frozen samples were all confirmed by analyses of the archival material. Intertumoral genetic heterogeneity was found in samples from two patients. The same allelic intensities were seen in DNA from tumor-close liver tissue as in blood DNA from the same patient except in one sample. The present study shows a 54% increase in sensitivity of genetic alterations if pure tumor cell components are used (five "new" AIs and LOHs and eight "new" LOHs among previously scored heterozygotes [n = 11] and AI [n = 13], respectively). In total, a 93% success rate (108/115 analyses) was obtained using standard PCR conditions for the 10 selected markers. The fact that standard PCR conditions and 5-micron tumor sections are used shows how easy these analyses are to perform, and that only minor amounts of valuable archival material is used.

Microsatellite instability has been found preferentially in tumours associated with the hereditary non-polyposis-colorectal-cancer (HNPCC) syndrome. This phenotype, manifested as new alleles at microsatellite loci, and often the result of a defective mismatch-repair gene, is seen as allelic mobility shifts during electrophoretic runs. We examined possible alterations at 8 dinucleotide loci mapping to 6 different chromosomes in endometrial cancers (n = 20) and cervical cancers (n = 82). Overall instability was found in 30% of the endometrial cancers and in 6% of the cervical cancers, including 3 (15%) and 2 (2%) tumours, respectively, unstable at more than one locus. In contrast to the endometrial cancer sub-group, the affected cervical cancers were characterized by one or two new alleles at one or few loci. By DNA ploidy measurements 5 diploid endometrial cancers were microsatellite-unstable vs. one diploid of 6 unaltered cases (p = 0.015; Fisher's exact test). Our data confirm that a sub-set of diploid sporadic endometrial cancers are characterized by a mutator phenotype similar to that found in colorectal cancer. In contrast, among cervical cancers, not characterized by the HNPCC-tumour spectrum, this mutator phenotype is seen infrequently, and positive cases appear to display only minor alterations.

Forty-six benign and malignant tumours and tumour-like lesions of the thyroid were analysed for microsatellite instability (MI) at eight loci, mapping to four different chromosomes, 7 lesions (15%) displayed MI at one or more loci, including 2/13 nodular goitres, 2/15 follicular adenomas, 2/12 papillary carcinomas and 1/4 follicular carcinomas. Two benign and one malignant lesion among the seven unstable cases exhibited this phenotype at three or more loci. We found no mutations in the mismatch repair gene, hMSH2, in the seven affected cases, after screening all the exons by CDGE mutation analysis. At variance with the data on record, these results indicate that, despite being relatively infrequent, MI does occur not only in thyroid carcinomas but also in benign lesions (goitres and follicular adenomas of the thyroid).

A genome-wide instability has been found in almost all analyzed malignant tumors from patients with hereditary non-polyposis colorectal cancer (HNPCC), and in a subgroup of sporadic (non-inherited) cancers of the same type. This mutator phenotype was initially seen as novel alleles at microsatellite loci (a family of repetitive DNA sequences) and was shown to be caused by mutations in the highly conserved mismatch repair genes. Mutations have been found in each of four of these human genes: hMSH2, hMLH1, hPMS1 and hPMS2, in the germline of HNPCC patients and in their tumors, as well as in sporadic tumors. These recent discoveries provide new molecular diagnostic tools for the detection of patients at high risk of developing carcinomas of the large bowel and other HNPCC-related tumors. Ongoing international research is progressively solving many of the unanswered questions at the genotypic and phenotypic levels of this newly identified mechanism in carcinogenesis.

Several genetic alterations have been described in benign and malignant primary tumors of the colorectum, but few such associations have been made with the progression of these tumors. This study compares genetic changes found in distant metastases (n = 22) with local recurrences (n = 15) as well as with primary carcinomas (n = 12). Complete allelotypes of the tumors were obtained by analyzing 43 microsatellite loci, representing all non-acrocentric chromosome arms and mapping to the mid-portion of the arms. Allelic imbalances in the tumor DNA were evaluated by comparison with the patient's constitutional pattern in blood DNA. The allelotype profile of the distant metastases was different from those found in the local recurrences and in the primary carcinomas. More than 20% of the distant metastases exhibited allelic imbalances at loci representing 20 chromosome arms. The majority of these regions were less frequently changed in the local recurrences and in the primary tumors. The markers that most often were altered in the metastasis (>40%) represented chromosome arms 14q, 17p, 18p and 18q. Only two regions, 10p and 19p, were unaltered in all tumors analyzed. We found that the median value of fractional allelic imbalance was twice as high in the distant metastases as in the recurrent tumors. Novel alleles at microsatellite loci were observed in all 3 tumor types, but in the advanced tumors this phenotype was characterized by only a single novel allele seen at less than 10% of the analyzed loci.

The genetic changes leading to the development of malignant peripheral nerve sheath tumors (MPNSTs) are largely unknown. The few tumors that have been investigated cytogenetically had highly complex karyotypes and no consistent rearrangements, and the attempts to pinpoint consistent DNA-level changes have met with only limited success. We used comparative genomic hybridization to analyze seven MPNSTs and one dermatofibrosarcoma protuberans from eight patients with von Recklinghausen's disease (neurofibromatosis type 1), as well as three sporadic MPNSTs. Gains and losses of DNA sequences were found in all tumors, with an average of four losses (range, 0-14) and two gains (range, 0-5) per tumor. Two striking observations were made: (a) an increase in copy number of the distal part of the long arm of chromosome 17, with the smallest region of overlap 17q24-qter, was seen in five of seven MPNSTs and in the only dermatofibrosarcoma protuberans, all of which were from patients with neurofibromatosis, whereas none of the three sporadic MPNSTs had this alteration; and (b) loss of 13q, with the smallest region of overlap 13q14-q21, was found in 6 of 10 MPNSTs. The consistent involvement of these two chromosomal regions probably reflects two different pathogenetic mechanisms for MPNSTs.

Background: An isochromosome for the short arm of chromosome 12, i(12p), is the most common and characteristic cytogenetic aberration in testicular germ cell tumors. Little is known about the molecular genetic abnormalities of these neoplasms., Experimental Design: A total of 32 loci were studied in DNA from 31 primary testicular germ cell tumors and compared with corresponding normal DNA. The loci map to 17 different chromosome arms, including seven that contain known tumor suppressor genes. Southern blot analysis and PCR-based methods were used. Several microsatellite loci were included to investigate instability (seen as new alleles) at repeat loci. The TP53 tumor suppressor gene was analyzed for point mutations by constant denaturant gel electrophoresis and for expression by immunohistochemistry. Histologic sections of the tumor biopsies were evaluated with regard to components and percentage of intact tumor cells. The growth fraction, representing one component of proliferative activity of the tumor, was assessed by the Ki-67 index., Results: Changes were found at all chromosome arms investigated but at very different frequencies, 5-56% of all tumors. The most frequently affected chromosome arms, those showing loss of heterozygosity or allelic imbalance in more than 40% of the tumors, were 2q, 3p, 3q, 11p, 12p, 18q, and 22q. Complete loss of one allele was often seen at 3p and 11p loci, whereas allelic imbalances dominated on the 2p, 3q, 12p, 18q, and 22q loci tested. No mutations were detected within four known mutational hot spots of TP53, but positive immunostaining with two TP53 Ab was seen in 9 of 14 tumors. Most tumors (26 of 31) showed positive immunostaining with Ki-67. Microsatellite instability was not observed., Conclusions: High frequencies of loss of heterozygosity and allelic imbalance at several loci indicate that inactivation of several tumor suppressor genes may be of importance in developing testicular germ cell tumors. The increase in copynumber of 12p alleles seen in several tumors is likely to reflect one or more 12p isochromosomes. Our findings do not indicate that TP53 plays any major pathogenic role in this tumor type, nor was there any indication that defect repair genes, causing microsatellite instability in other cancers, participate in the progression of testicular cancer.

Previous cytogenetic studies have indicated that a subset of large bowel adenomas have distal 1p deletions. We addressed this question by examining 70 sporadic polyps (63 adenomas, 5 hyperplastic polyps, and 2 polyps of undetermined histology) from 55 patients for alterations at eight loci on the short arm of chromosome 1 and found allelic imbalance (AI) or loss of one allele (LOH) in 14 (20%). The locus most frequently changed was MSI, which maps to 1p33-35. Fluorescence in situ hybridisation with centromeric and telomeric probes for chromosome 1, performed for 11 polyps, did not yield an abnormal number of signals, in accordance with the interpretation that the observed AI and LOH were the result of interstitial deletions in 1p. Whereas allelic imbalance at five other loci (mapping to 5q, 8p, 10p, 11p and 17q) was found less frequently, and then mainly in large (> 2 cm) tumours, the 1p alterations were equally distributed among small (< 1 cm) and large polyps. They were preferentially found in left-side tumours. Instability at microsatellite loci--the mutator phenotype--is demonstrated by shifts in the electrophoretic mobility of normal alleles. The mutator phenotype was first associated with hereditary nonpolyposis colorectal cancer but is also occasionally found in sporadic colorectal carcinomas; however, it is still uncertain when in the adenoma-carcinoma sequence in this type of genomic instability arises. We therefore looked for it at 12 dinucleotide repeat loci and found that seven tumours (six adenomas and one hyperplastic polyp) from seven patients had acquired new alleles not seen in the patients' corresponding normal DNA. Our results suggest that inactivation of a putative suppressor gene distally in chromosome arm 1p is an early event in colorectal tumourigenesis. They also show that microsatellite instability can be detected in large bowel polyps, indicating that this phenomenon, too, probably plays a pathogenic role for some colorectal tumours early in the adenoma-carcinoma sequence.

Overexpression of the MDR1 product, P-glycoprotein (Pgp), has been shown to be one of the mechanisms underlying the development of multidrug resistance (MDR). Recently, one mutant p53 has been shown to stimulate the MDR1 gene promoter in vitro, whereas wild-type p53 repressed this activity. We measured Pgp and p53 expression by immunoblotting in 34 colorectal tumours, and performed mutation analyses on the p53-positive tumours to confirm the presence of mutant p53 protein. Tumour DNA indices (DIs) were also measured using flow cytometry. Pgp was detected in 44% (15/34) of the tumours and in 100% (13/13) of the normal mucosas (P = 0.0005), with highest levels of expression seen in normal mucosa, suggesting that initial drug resistance in colorectal tumours is not caused by Pgp. Highly DNA aneuploid tumours demonstrated the lowest levels of Pgp expression relative to moderately aneuploid and diploid colorectal tumours. p53 protein was detected in 53% (18/34) of the tumours, and 12 of 14 p53-positive tumours had p53 gene mutations, p53-negative tumours had approximately twice the level of Pgp expression of p53-positive tumours. Pgp expression was not associated with either p53 expression (P = 0.73) or incidence of p53 gene mutation (P = 0.70), suggesting that mutant p53 does not induce Pgp overexpression in colorectal carcinomas.

Mutations in recently identified genes on chromosomes 2 and 3 seem to be responsible for repair errors (RER+) throughout the genome. This novel genetic mechanism was first reported in hereditary non-polyposis colorectal cancer syndrome and in cancers that are characteristic of this syndrome, such as carcinomas of the right colon, stomach and endometrium. We investigated the frequency of RER+ phenotype in a series of 34 sporadic gastric carcinomas, in an attempt to see if the RER+ cases displayed any particular morphologic features and/or if they showed distinctive clinicopathologic characteristics. Twelve loci were investigated. We found 23 RER- cases (67.6%) and 11 RER+ cases (32.4%). A significant association was found between RER+ carcinomas and localization of the tumors: 9 of the 11 RER+ carcinomas (81.8%) were located at the antrum whereas all the cardiac tumors were RER-. The RER+ phenotype was also significantly related to the presence of moderate/abundant T-cell lymphoid infiltration within the tumors. The 3-year survival rate of patients with RER+ tumors was suggestively longer than that of patients with RER- tumors. No significant relationship was found between several clinicopathologic characteristics of the cases, including age, sex, staging, histologic type and ploidy, despite a trend towards an association between RER+ phenotype and advanced age of the patients and poorly differentiated, intestinal type of the carcinomas. The high frequency of microsatellite instability in sporadic gastric carcinomas supports the involvement of this genetic mechanism in gastric carcinogenesis. Gastric carcinomas with the RER+ phenotype tend to occur as poorly differentiated adenocarcinomas in the antrum of elderly patients, display abundant T-cell infiltration and carry a relatively good prognosis.

Little is known about the molecular genetic changes in malignant peripheral nerve sheath tumors (MPNST). Inactivation of the TP53 gene in 17p has been reported in a few tumors. The MPNST is one of the manifestations of neurofibromatosis 1 (NF1), suggesting that the NF1 gene in 17q might be important. We present a study of 15 neurofibromas and MPNST from nine individuals. Seven patients had NF1, and six of these developed MPNST. Genetic alterations at nine polymorphic loci on chromosome 17 were examined. Allelic imbalance was detected only in the malignant tumors from NF1 patients (4/6). Complete loss of heterozygosity of 17q loci was found in three of these tumors, all including loci within the NF1 gene. Two of the malignant tumors also showed deletions on 17p. No mutations were detected within exon 5-8 of the TP53 in any of the MPNST, and none of them were TP53 protein-positive using immunostaining with mono- and polyclonal antibodies against TP53. The numbers of chromosome 17 present in each tumor were evaluated by use of fluorescence in situ hybridization (FISH) on interphase nuclei with a centromere-specific probe. A deviation from the disomic status of chromosome 17 was observed in two of the MPNST from NF1 patients. These results support the hypothesis of inactivation of both NF1 gene alleles during development of MPNST in patients with NF1. In contrast to other reports, we did not find evidence for a homozygous mutated condition of the TP53 gene in the same tumors. Finally, FISH analysis was in accordance with the DNA analysis in the deduction of the numbers of chromosome 17 in these tumors.

One strategy for identifying genes involved in genetic predisposition to testicular germ-cell tumors (TC) is to perform association studies with polymorphic loci at or closely linked to candidate genes. Genes involved in normal fetal genital development, such as the Wilms' tumor 1 gene (WT1) located at 11p13, are among such candidates. The present study compares a TC (n = 442) and a control (n = 384) population for the allele frequencies of 2 polymorphic loci located at chromosome band 11p13. One of the polymorphisms (WT) was located within and the other (D11S325) in close proximity to the WT1 gene. No differences in allele frequencies between cancer patients and controls were found. However, the frequency of the Al allele of the WT polymorphism was significantly increased in one of the cancer subgroups most likely to carry susceptibility genes (patients with bilateral cancer) compared to controls. Furthermore, the frequency of the Al allele was increased in patients with metastatic disease. Such differences in allele frequencies were not observed for the D11S325 locus. The findings might indicate an involvement of the WT1 gene both in susceptibility to TC and in progression of the disease.

Recent reports have suggested that one or more genes may cause replication errors (RER) during colorectal tumorigenesis. Additional alleles are seen in the tumors when analyzing random microsatellite loci. We have studied seven dinucleotide repeat loci, located on seven different chromosomes, by use of polymerase chain reaction amplification and denaturing polyacrylamide gel electrophoresis. We found that 16.5% (40 of 243) colorectal cancers showed RER at one or several loci (RER+). This includes 31% (4 of 13) among cases with a strong positive family history according to previously published criteria and 17% (35 of 207) among cases with no history of familial cancer. Interestingly, no significant association was found between RER+ tumors and a general familial clustering of cancer. Microsatellite instability was significantly associated with DNA diploid status of the tumor (P < 0.001), with the location of the tumor in the proximal colon (P < 0.001), and with poorly differentiated tumor phenotype (P < 0.001). Patients with RER+ at > or = 2 loci tumors had an increased survival (P = 0.05). We further analyzed 84 breast cancers and 86 male germ cell cancers using the same seven markers. None of the tumors were RER+, indicating that this phenomenon may be specific to certain types of tumors.

Microsatellite instability implying multiple replication errors (RER+ phenotype) characterizes a proportion of colorectal carcinomas, particularly those from patients with the hereditary non-polyposis colorectal carcinoma syndrome. We studied the incidence of microsatellite instability in more than 500 sporadic tumors representing 6 different types of cancer. Apart from colorectal carcinoma [see the paper by Lothe et al. (Cancer Res., 53:5849-5852, 1993)] the RER+ phenotype was found in 18% (6 of 33) of gastric carcinomas and 22% (4 of 18) of endometrial carcinomas. In contrast, no evidence of this abnormality was detected in cancers of the lung (N = 85), breast (N = 84), and testis (N = 86). Importantly, the first three cancers, as opposed to the latter three, are characteristic of the hereditary non-polyposis colorectal carcinoma syndrome. These findings suggest that the cancers belonging to the hereditary non-polyposis colorectal carcinoma tumor spectrum may have essential pathogenetic steps in common, including a tendency to multiple replication errors.

p53 protein expression was studied by immunoblotting in 34 colorectal carcinomas and 28 of the corresponding normal mucosas, and correlated with tumor DNA ploidy as measured by flow cytometry. p53 protein was detected in 35% (12/34) of the tumors; the normal mucosas were negative. Fifty-five percent (12/22) of the tumors examined for mutations within the four hotspots (exons 5-8) of the p53 gene had point mutations. p53 expression correlated significantly with the presence of p53 gene mutations; 67% (8/12) of the tumors with mutations expressed p53, whereas only one of 10 tumors where no mutations were detected expressed the protein (p=0.01). Four tumors with p53 gene mutations did not express p53. Fifty-nine percent (20/34) of the tumors were aneuploid. p53 expression correlated significantly with aneuploidy; a total of 55% (11/20) of the aneuploid tumors were positive for p53 compared to 7% (1/14) of the diploid tumors (p=0.009). All of the 11 highly aneuploid tumors (1.31 less-than-or-equal-to DNA index (DI); less-than-or-equal-to 1.86) expressed p53, whereas all of the 9 moderately aneuploid tumors (1.11 less-than-or-equal-to DI less-than-or-equal-to 1.29) were p53-negative. Flow cytometry was also used to resolve cell cycle- and ploidy specific p53 expression in nuclei in 4 aneuploid tumors. p53 expression in these tumors was confined to the aneuploid component, whereas the diploid component was negative. p53 was seen in nuclei in all phases of the cell cycle of proliferating aneuploid cells. Neither p53 expression nor tumor DNA ploidy were correlated with Dukes' stage (p=1.00 and 0.72, respectively). The data suggest that high levels of mutant p53 may play a causative role in the generation of highly aneuploid tumors.

Deletions within the short arm of the human chromosome 11 have been found to be involved in the genesis of several tumours, including different urogenital neoplasms. We have studied 31 male germ cell tumours (19 seminomas and 12 nonseminomas), and observed loss of heterozygosity at 11p loci in 40% (12/30) of these tumours [35% (9/26) at 11p13 and 31% (8/26) at 11p15]. Our data suggest that inactivation of one or more tumour suppressor genes on 11p are involved in the genesis of testicular cancer. In addition, identification of the parental origin of the allelic losses revealed a paternal loss in six patients and a maternal loss in one case.

In a patient with neurofibromatosis (von Recklinghausen disease; NF1), normal lymphocytes, five cutaneous neurofibromas, and tumour tissue from a recurrence of a malignant schwannoma were analysed for genetic alterations. Eleven DNA markers located on chromosome 17 and nine randomly chosen markers representing chromosomes 1, 2, 3, 4, 5, 6, and 11, were analysed. High resolution Giemsa banding of lymphocytes revealed no chromosomal rearrangement. The DNA from the neurofibromas were all found to have the same restricted fragment length polymorphism pattern as the constitutional DNA from the patient. In the malignant schwannoma a complete loss of one allele was found at polymorphic loci on chromosome arm 17p. One gene copy of the TP53 gene (17p13.1) and the NF1 gene (17q11.2) was lost, as was one copy of the PGA gene (11q13). No mutations were detected in the mutational hotspots of the TP53 gene. Partial losses were detected at three loci on chromosomes 1, 2 and 6, indicating a clonal variation within the tumour since histological evaluation disclosed no normal tissue in the analysed specimen. Our data indicate that the NF1 gene may function as a tumour suppressor gene, and that, either by effect of dose reduction or complete inactivation, both the NF1 gene and the TP53 gene may be critical for the progression of a neurofibroma to a malignant schwannoma. The observations made are consistent with the concept of stepwise multigenetic changes in tumour progression.

Mutations in the TP53 gene are considered to be among the most common genetic alterations in human cancers. Both somatic and germline mutations have been found. Using polymerase chain reaction (PCR), constant denaturant gel electrophoresis (CDGE), and denaturing gradient gel electrophoresis (DGGE), we have examined 32 patients with bilateral and familial germ cell tumors (GCT) and two patients with sporadic GCT for germline mutations within the conserved regions of the gene. In addition, 15 tumors were screened for somatic mutations and analyzed for loss of heterozygosity (LOH) at the TP53 locus. Twelve tumors were analyzed for expression of TP53 via immunohistochemistry. Neither germline nor somatic TP53 mutations were detected. LOH was observed in one of five informative cases. No tumors showed increased expression of TP53 protein. These results indicate that alterations in the TP53 gene are not important for the predisposition to and development of GCT.

Cytogenetically, a marker chromosome interpreted as i(12p) is present in most testicular tumors of germ cell origin. In this study, 22 patients with testicular germ-cell tumors were investigated by Southern blot hybridization to characterize changes in chromosome 12. In comparison with normal DNA, tumor DNA of 18 patients showed increased dosages of 12p accompanied by a comparable or smaller increase or no change in the dosage of centromeric sequences of chromosome 12. A likely interpretation was that most testicular tumors had one or several isochromosomes for 12p that were formed by somatic division of the centromere and that the points of breakage and reunion in the centromeric region were different in different tumors. Allelic 12p fragments showing increased intensity were paternal in four and maternal in three of seven informative cases. Thus, there was no evidence of sex-limited parental imprinting. Furthermore, the observed patterns of allelic fragments suggested that the marker was an i(12p) formed by sister chromatids of one homolog number 12 rather than the result of interchange of genetic material between different homologues.

Studies of mutant genotypes of the retinoblastoma susceptibility gene (RB1) in different solid tumors have mainly been concentrated on the demonstration of loss of heterozygosity (LOH) at both internal and external polymorphic sites. One reason for this is the complex organization of the gene. The p105RB protein has been shown to interact with both DNA and regulatory cellular proteins and oncoproteins. The amino acids encoded by exon 21 are implicated in several of these interactions. Both point mutations and intragenic deletions involving exon 21 have previously been reported in human tumors. We have examined RB1 exon 21 from a number of human tumor types where significant LOH in or around the RB1 gene has been reported. DNA from 78 primary tumors was amplified using the polymerase chain reaction (PCR) with primers covering exon 21, followed by constant denaturant gel electrophoresis (CDGE). The 78 tumors included 11 breast carcinomas, 30 nonsmall cell lung carcinomas, 6 colon carcinomas, and 31 sarcomas. The small cell lung cancer cell line NCI-H209, previously shown to harbour a point mutation in codon 706: TGT- greater than TTT (Cys- greater than Phe), was detected using CDGE. Apart from this control mutant cell line, we did not detect any mutations in the examined region in any of the tumors.

Background: In the majority of colorectal carcinomas, both copies of the tumor suppressor gene TP53 (tumor protein 53) are known to be inactivated. In contrast to a loss of tumor suppressor function, it has been suggested that an increased copy number of the RB1 gene is involved in the progression of these tumors., Purpose: To determine genetic alterations at chromosomes 13 and 17 in colorectal tumors, we have studied several loci on these chromosomes, with special focus on the RB1 and TP53 genes at both the level of DNA sequence and the level of gene expression., Methods: Restriction fragment length polymorphism analysis was performed after alkaline Southern blotting of the DNA fragments and hybridization (in 7% sodium dodecyl sulfate and 0.5 M NaPO4) of the nylon membranes with multiprimed, radioactively labeled probes. Total RNA was extracted from tissue biopsy specimens by homogenization of the samples in guanidinium thiocyanate followed by separation in a CsCl gradient. By use of an image-processing system, x-ray film signals were measured densitometrically. Point mutations within the TP53 gene were detected by use of polymerase chain reaction (PCR) in combination with constant denaturant gel electrophoresis. Direct sequencing of PCR products revealed the exact nature of the mutations. Protein expression of TP53 was seen by immunostaining of sections from paraffin-embedded material using a mouse monoclonal antibody. The two-sided Fisher's Exact Test was used for statistical analysis., Results: An increase in allelic copy number at 13q loci was seen in 10 (32%) of 31 tumors. In the majority of the cases, this increase probably reflected a change in the diploid status of chromosome 13; in some cases, however, only part of the 13q seemed to be involved. The RB1 gene showed an elevated level of RNA compared with the beta-actin signal. Fourteen (48%) of 29 tumors showed loss of heterozygosity at loci on 17p, and base mutations within the TP53 gene were seen in 14 (42%) of 33 tumors. RNA and protein analyses of TP53 revealed an increased level of expression in the tumors compared with normal mucosa. Allelic variations seen at 13q and 17p were not associated (P = .7)., Conclusions: Our results suggest that, in addition to aneuploidy, gain of specific chromosome 13 sequences is involved in the tumorigenesis of the colon and rectum. In addition, they confirm the importance of TP53 mutations for the progression of such tumors and support the view that accumulation of events is more important than the order of events. The genetic changes observed at chromosome arms 13q and 17p seem to be independent of each other.

Patients with bilateral and/or familial testicular cancer are assumed to demonstrate germ line mutations if such mutations are instrumental in testicular cancer patients. Constitutional rearrangements have to our knowledge not been reported, while an increased level of constitutional chromosome instability has been claimed. Lymphocytes from 12 Norwegian patients with bilateral or familial testicular cancer have been studied by high resolution Giemsa banding. A possible germ line mutation would be expected to be seen in every cell. It is therefore sufficient to examine one pair of the individual chromosomes obtained, if necessary, from several cells. Based on our previous findings of loss of heterozygosity in the chromosome regions 3p and 11p in testicular tumors, these chromosome arms were paid special attention. However, no mutations were observed in these regions nor in any other chromosome. Applying a 95% confidence interval, we conclude that less than 23% of familial and/or bilateral testicular cancer patients are caused by germ line mutations large enough to be detected with high resolution banding of at least 450 G-bands per haploid genome. The presented analyses were used to standardize the resolution determination in our laboratory.

Various sublines of cells established from an osteosarcoma that developed in a patient (O.H.) with previous bilateral retinoblastoma were examined for different restriction fragment-length polymorphisms of chromosome 13q, as well as for rearrangements of the retinoblastoma gene using a cDNA probe. The independently established sublines were used to help separate primary and secondary events taking place in tumorigenesis of the osteosarcoma of this patient. Information from the present DNA analysis, taken together with data from cytogenetic and enzymatic studies on chromosome 13 in the cell lines, revealed both common and distinct genetic changes on chromosome 13q. The common changes may indicate the nature of the first and second mutational events in the development of the osteosarcoma. The first, constitutional cancer predisposing mutation seemed to be a base mutation or a small deletion/insertion, and the second event involved a deletion of a larger part of the long arm of chromosome 13. The distinct genetic changes included other deletion and duplication events of chromosome 13q. The existence of multiple sublines with different genetic constitutions provides improved possibilities for gaining insight into the nature of the genetic lesions leading to tumor formation, as these may reflect the clonal variation present in the primary tumor. We also demonstrate the difficulty of inferring from single tumor cell isolates to properties of the primary tumor.

Thirty-one males with testicular germ-cell tumors were studied by Southern hybridization using X- and Y-chromosome-specific probes as well as a pseudoautosomal probe. Densitometric analysis showed changes in the relative dosage of Y-chromosomal fragments in tumor DNA from 12 out of 31 patients (39%) as compared to normal DNA from the same patients. In 11 tumors the relative intensity ratios of Y-chromosome-specific fragments had decreased from the normal value of 1 to values between 0 and 0.77. An increase in the Y-chromosomal dosage was observed in 1 case. The entire Y chromosome was apparently involved in most patients but 2 tumors revealed regional variation. Tumor DNA of 2 patients with Y-chromosomal deficiency showed a concomitant increase in the X chromosomal dosage. The pseudoautosomal region that is shared by both sex chromosomes was involved in a total of 8 tumors (26%), 2 of which did not show any obvious dosage changes with probes detecting strictly X- or Y-chromosome-specific fragments. Autosomal alterations in the present tumor series have been described. A dosage change involving the sex chromosomes accompanied loss of heterozygosity at loci in 3p or 11p in 10 tumors out of 15 (67%). Seminomas tended to be affected more often than non-seminomas by either type of alteration. Our results indicate that changes in the sex chromosomes occur in a substantial proportion of male germ-cell tumors and, together with other defects, may constitute an important step in tumor development and/or progression.

High-resolution karyotypes were analyzed from lymphocyte cultures of 18 Norwegian polyposis patients, of whom two had classic Gardner syndrome (GS). We focused on possible rearrangements of the long arm of chromosome 5. Previously the gene for familial adenomatous polyposis (FAP) was localized to the region 5q21-22 by linkage analysis. A patient with a constitutional deletion in this region would be of great value in the course of cloning the gene. At the level of high-resolution G-banding, however, none of these patients showed any interstitial deletion or other rearrangement on chromosome 5 or any other chromosome.

Human pepsinogen (PGA) exhibits extensive polymorphism that can be detected both at the protein and the DNA level. We describe here two restriction fragment length polymorphisms, EcoRI and BglII, which provide for the detection of three of the most common PGA haplotypes (A, B, and C) in the United States population. The relationship of these polymorphisms to each PGA haplotype was determined by analysis of DNA from individuals exhibiting the corresponding protein phenotypes and by analysis of a series of human x mouse somatic cell hybrids containing the individual chromosome 11 homologous from heterozygous individuals exhibiting the AB and AC protein phenotypes. The use of the BglII polymorphism in combination with previously described EcoRI polymorphism provides a very informative marker of 11q13.

Restriction fragment length polymorphisms at 53 autosomal loci were screened for heterozygosity in 40 colorectal cancer patients. The DNA pattern in constitutional versus tumor/polyp tissue was compared. More than half of the markers tested were of the VNTR (variable number of tandem repeats) type, giving the patient panel a higher informational content, since the frequency of individuals heterozygous for a particular marker is increased. Loss of alleles was revealed in 40% of the tumors from constitutionally heterozygous patients at the chromosome 17p loci, identified by the markers YNH37 and YNZ22. Similar losses were also detected on other autosomes, but at a significantly lower frequency. Our results suggest that hemi/homozygosity of 17p alleles plays a role in the development of a major subset of colorectal carcinomas. Similar observations regarding other autosomal loci may be interpreted as random losses in these tumors, or they may indicate loci important to minor clinical subclasses of colon carcinomas.

Constitutional and tumor genotypes defined by polymorphic DNA markers were examined in 31 testicular cancer patients. Constitutional karyotypes were analyzed and clinical data presented. We analyzed 11 loci representing 8 chromosomes, including regions frequently deleted in other types of cancer. Loss of 3p or 11p sequences was detected in 8 of 28 heterozygotes (28%) and in 5 of 20 heterozygotes (25%). This gives a combined total loss of 40%. The other autosomal loci tested showed no loss or a loss of less than 10% of alleles. We suggest that this loss of heterozygosity for genetic material on chromosome 3p or on 11p is nonrandom and important in the development of a major subset of testicular neoplasms.

A cell panel from six different familial cancers, where both normal and tumor tissues were available, was examined for genotypic changes with polymorphic DNA probes. Seventeen probes were tested, representing chromosomes #1, #2, #5, #6, #13, #14, #17, and #19. One probe, p7F12 (D13S1, at 13q12-q14) revealed loss of heterozygosity in two tumors: an osteosarcoma from a patient with retinoblastoma that had been included as a control, and one polyposis tumor that had been established in nude mice from a duodenal carcinoma biopsy. Loss of heterozygosity was observed in the first passage of the mouse tumor. Chromosome analysis in later passages revealed loss of one whole chromosome #13 as the single consistent karyotypic change.

A comparative study of the Stainer and Scholte chemically defined growth medium (SS) for Bordetella pertussis, and a modification of it (MSS), was made. The Tris buffer which is reported to have adverse effects was replaced in MSS with sodium glycerophosphate. By using generation time as a measure of the growth promoting properties of the media, we found that the MSS was superior to SS for B. pertussis strain CN5612/67, which was used as a vaccine strain in Norway. Additionally by employing the 'dilution to extinction' method we showed that MSS supported B. pertussis growth from smaller inocula than the original SS medium. No significant differences in the quality of vaccines made from organisms grown in the two alternative media were observed in animal tests for potency and safety. The difference in buffers in SS and MSS had no influence on the formation of the agglutinogens, as tested by agglutination with specific factor sera. It was confirmed that liquid media gave a better total expression of the agglutinogens than solid media.

A research project initiated in 1978 comprised establishment of a national polyposis registry, a genetic linkage study using classical and DNA markers, an in vitro study of fibroblasts for transformation parameters and chromosome instability, and a comparative study of DNA-RFLPs in cancer and constitutional tissue. The linkage study (to be reported elsewhere) confirmed the recently reported close linkage between the polyposis gene locus APC and D5S71. No in vitro test for the presence of the APC gene has been confirmed or revealed, but we detected increased chromosomal instability on a statistical basis and also recorded abnormal DNA-repair. As per 1. January 1988 the prevalence of adenomatosis of colon and rectum in Norway was 1/43,500. Among patients born in the period 1931-1950 the incidence at birth of developing the disease is 1/20,000 and the mutation rate is 1/72,000 per gamete per generation. In Norway new mutants in healthy families will comprise 1/3-1/2 of all new cases in the coming two decades, or one of 36,000 births.