Identification of germline MLH1 alterations in familial prostate cancer.

Several linkage and loss of heterozygosity (LOH) analyses suggest that the region 3p21-p26, which is a chromosomal location of MLH1, could harbour a susceptibility gene for prostate cancer (PRCA). Furthermore, in a recent candidate single nucleotide polymorphism (SNP) analysis the I219V variation of the MLH1 gene was associated with PRCA. Microsatellite instability (MSI) and germ-line MLH1 mutations were originally demonstrated in hereditary non-polyposis colorectal cancer (HNPCC) but MSI and loss of MLH1 function have also been detected in PRCA. To assess the contribution of MLH1 germline mutations to the development of PRCA in Finland different approaches were used. First, the samples from 11 PRCA-colon cancer patients were screened for MLH1, MSH2 and MSH6 protein expression by immunohistochemistry (IHC). IHC revealed one patient with a putative MLH1 aberration and sequencing of this sample revealed five sequence variants including two missense variants P434L and I219V. Second, the samples from Finnish hereditary prostate cancer (HPC) families were used for the screening of MLH1 mutations which produced twelve MLH1 sequence variants including two missense mutations, I219V, as in the PRCA-colon cancer patient, and V647M. P434L and V647 were both novel, rare variants. Carrier frequencies of the I219V mutation were compared between hereditary prostate cancer (HPC) patients, unselected PRCA cases, patients with benign prostate hyperplasia and controls, but no differences between the sample groups were found. P434L was not present in this study population and V647M was a very rare variant found only in one HPC family. According to the present results, MLH1 does not have a major role in PRCA causation in Finland.

Increased risk of cancer in patients with fumarate hydratase germline mutation.

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a tumour predisposition syndrome caused by heterozygous germline mutations in the fumarate hydratase (FH) gene. The condition is characterised by predisposition to benign leiomyomas of the skin and the uterus, renal cell carcinoma (RCC), and uterine leiomyosarcoma (ULMS). To comprehensively examine the cancer risk and tumour spectrum in Finnish FH mutation positive families, genealogical and cancer data were obtained from 868 individuals. The cohort analysis of the standardised incidence ratios (SIR) was analysed from 256 individuals. FH mutation status was analysed from all available individuals (n = 98). To study tumour spectrum in FH mutation carriers, loss of the wild type allele was analysed from all available tumours (n = 22). The SIR was 6.5 for RCC and 71 for ULMS. The overall cancer risk was statistically significantly increased in the age group of 15-29 years, consistent with features of cancer predisposition families in general. FH germline mutation was found in 55% of studied individuals. Most RCC and ULMS tumours displayed biallelic inactivation of FH, as did breast and bladder cancers. In addition, several benign tumours including atypical uterine leiomyomas, kidney cysts, and adrenal gland adenomas were observed. The present study confirms with calculated risk ratios the association of early onset RCC and ULMS with FH germline mutations in Finns. Some evidence for association of breast and bladder carcinoma with HLRCC was obtained. The data enlighten the organ specific malignant potential of HLRCC.

Chromosomal changes in locally recurrent, hormone-refractory prostate carcinomas by karyotyping and comparative genomic hybridization.

The genetic mechanisms of prostate cancer recurrence during hormonal therapy are largely unknown. So far, data from conventional karyotype analysis on hormone-refractory prostate carcinomas have not been published, mainly because of the difficulties in obtaining fresh hormone-refractory prostate carcinoma samples and getting metaphases from them. Here, we have studied chromosomal changes in 12 locally recurrent, hormone-refractory prostate carcinomas using karyotyping and CGH that revealed genetic aberrations in all tumors. Loss of the Y chromosome was the most common (89%) finding, and tetraploidy or near-tetraploidy was detected in all tumors. Also non-random translocations were found in 56% of the tumors. The present study indicates that clonal chromosomal aberrations in hormone-refractory prostate carcinomas are more common than in untreated primary tumors, and also, further studies on the frequency and significance of translocations in prostate carcinoma progression during hormonal therapy are warranted.

Androgen receptor alterations in prostate cancer relapsed during a combined androgen blockade by orchiectomy and bicalutamide.

Mechanisms of prostate cancer (CaP) recurrence during a combined androgen blockade (CAB) are poorly understood. Previously, the role of androgen receptor (AR) gene mutations underlying the CAB therapy relapse has been raised. To investigate the hypothesis that AR gene aberrations are involved in CAB relapse, 11 locally recurrent CaP samples from patients treated with orchiectomy and bicalutamide were analyzed for copy number changes and DNA sequence alterations of the AR gene by fluorescence in situ hybridization and single-strand conformation polymorphism, respectively. Altogether, base changes were detected in four tumors (36%). Three of them were missense mutations (G166S, W741C, M749I) and two were silent polymorphisms. Interestingly, none of the tumors had AR amplification. These data suggest that different AR variants are developed and selected for during various types of hormonal treatments, and also, that CAB achieved by orchiectomy and bicalutamide does not act as a selective force for AR amplification.

Association of E-cadherin germ-line alterations with prostate cancer.

In our recent cancer registry-based study, the incidence of gastric carcinoma was increased up to 5-fold in male relatives of early-onset prostate cancer (PCA) patients. This association may reflect the influence of genetic factors predisposing individuals to both tumor types. Germ-line mutations of the CDH1 gene at 16q have recently been associated with familial gastric cancer. Furthermore, two genome-wide linkage studies of PCA recently reported positivity at 16q. We therefore identified families and individual patients with both gastric and PCA and investigated whether the CDH1 gene mutations were involved in cancer predisposition in these cases. Fifteen of the 180 Finnish hereditary PCA families (8.3%) had one or more gastric cancer cases. No truncating or splice site CDH1 mutations were identified by PCR single-strand conformational polymorphism in these families or in eight individual patients who had both prostate and gastric cancer. However, a novel S270A missense mutation in exon 6 of the CDH1 gene was seen in a single family with four prostate and two gastric cancers. A large-scale population-based survey indicated a higher prevalence of S270A among both familial PCA cases (3.3%; n = 120; P = 0.01) and unselected PCA patients (1.5%; n = 472; P = 0.12) as compared with blood donors serving as population controls (0.5%; n = 923). We conclude that individual rare mutations and polymorphisms in the CDH1 gene, such as S270A, may contribute to the onset of PCA and warrant further investigations in other populations. However, the CDH1 gene does not appear to explain the link between prostate and gastric cancer.

ELAC2/HPC2 involvement in hereditary and sporadic prostate cancer.

The ELAC2/HPC2 gene at 17p11 is the first candidate gene identified for human prostate cancer (PRCA) based on linkage analysis and positional cloning (S. V. Tavtigian et al. Nat. Genet., 27:172-180, 2001). A truncating mutation was found in one hereditary prostate cancer (HPC) family, whereas two missense variants, Ser217Leu and Ala541Thr, were reported to be associated with increased PRCA risk in the general population. Here, we screened for mutations of the ELAC2/HPC2 gene in 66 Finnish HPC families. Several sequence variants, including a new exonic variant (Glu622Val) were found, but none of the mutations were truncating. We then analyzed the frequency of the three found missense variants in 1365 individuals, including hereditary (n = 107) and unselected (n = 467) PRCA, benign prostatic hyperplasia (n = 223), and population controls (568 healthy male blood donors). Ser217Leu and Ala541Thr variants carried no significantly elevated risk for HPC or PRCA, although the latter variant was associated with benign prostatic hyperplasia. The previously undescribed Glu622Val variant had a 1.0% population prevalence, but a significantly higher frequency in PRCA cases (3.0% odds ratio, 2.94; 95% confidence interval, 1.05-8.23). We conclude that ELAC2/HPC2 truncating mutations are rare in HPC, but that rare variants of the ELAC2/HPC2 require additional study as risk factors for PRCA in the general population.

A missense substitution A49T in the steroid 5-alpha-reductase gene (SRD5A2) is not associated with prostate cancer in Finland.

Prostatic steroid 5-alpha-reductase gene (SRD5A2) encodes a critical enzyme involved in the conversion of testosterone to dihydrotestosterone. A germline mis-sense substitution (A49T) leads to a variant SRD5A2 protein, which has a 5-fold higher in vitro V(max)than the wild-type protein (Ross et al, 1998; Makridakis et al, 1999). The A49T variant was recently associated with 2.5 to 3.28-fold increased risk of prostate cancer (PC) in African-American and Hispanic men (Makridakis et al, 1999). Also, Jaffe et al (2000) reported an association between A49T and more aggressive disease among Caucasian patients. Here, we report that the prevalence of the A49T variant in 449 Finnish PC patients was 6.0%, not significantly different from 6.3% observed in 223 patients with benign prostatic hyperplasia or 5.8% in 588 population-based controls (odds ratio for PC 1.04, 95% C.I. 0.62-1.76, P = 0.89). There was no association between A49T and the family history of the patients nor with tumour stage or grade. Our results argue against a prominent role of the A49T variant as a genetic risk factor for prostate cancer development and progression in the Finnish population.

Primary spontaneous pneumothorax in two siblings suggests autosomal recessive inheritance.

We report on a sister and brother with recurrent spontaneous pneumothorax without underlying connective tissue disease and without other affected relatives. The occurrence of spontaneous pneumothorax in two siblings from a large Finnish family raises the possibility of autosomal recessive inheritance of this disorder in some patients.

Genetic changes in familial prostate cancer by comparative genomic hybridization.

BACKGROUND: Germline mutations in recessive cancer predisposition genes are uncovered by somatic genetic deletions during tumor development. Analysis of genetic changes in tumor tissues from patients with an inherited predisposition may therefore highlight regions of the genome containing susceptibility or modifier genes. Our aim was to characterize genetic changes in familial prostate cancer METHODS: Twenty-one primary prostate cancers from 19 Finnish prostate cancer families were analyzed for somatic genetic changes by comparative genomic hybridization (CGH). RESULTS: The average number of genetic alterations per tumor was 4.0 +/- 1.9, distributed equally among losses and gains. The most common losses were found at chromosomal regions 13q14-q22 (29%), 8p12-pter (24%), and 6q13-q16 (14%), and the most common gains at 19p (25%), 19q (14%) and 7q (14%). CONCLUSIONS: These results suggest that prostate cancers in genetically predisposed individuals arise for the most part through similar somatic genetic progression pathways as sporadic prostate cancers. This also implies that the biological properties of tumors from the two groups may not be different from one another.

Two percent of Finnish prostate cancer patients have a germ-line mutation in the hormone-binding domain of the androgen receptor gene.

Mutations of the androgen receptor (AR) gene have been reported in prostate cancer, usually from tumor tissue specimens from late-stage, androgen-independent cancer. Occasionally, germ-line mutations have been found, but a link between AR mutations and predisposition to human prostate cancer has not been firmly established. Recently, two independent studies reported the same germ-line mutation at codon 726 in exon E (CGC to CTC) in two apparently unrelated Finnish prostate cancer patients. This arginine to leucine substitution was reported to alter the transactivational specificity of the AR protein. In the present study, the R726L mutation was analyzed by allele-specific oligohybridization in DNA specimens from 418 consecutive prostate cancer patients who reported a negative family history (sporadic group) and from 106 patients with a positive family history (hereditary group). The population frequency of the R726L mutation in blood donors was 3 of 900 (0.33%). In contrast, eight (1.91%) mutations (odds ratio = 5.8; P = 0.006) were found in the sporadic group, and two (1.89%) mutations were found in the hereditary group (odds ratio = 5.8; P = 0.09). Suggestive evidence of the segregation of the mutation with prostate cancer was seen in these two families. The present study indicates that the R726L substitution in the AR may confer an up to 6-fold increased risk of prostate cancer and may contribute to cancer development in up to 2% of Finnish prostate cancer patients. These results warrant additional large-scale studies of the significance of rare mutations and polymorphisms in candidate genes along the androgen signaling pathway as risk factors for prostate cancer.

Low apoptotic activity in primary prostate carcinomas without response to hormonal therapy.

Proliferative and apoptotic activities, as well as p53 protein expression, of ten untreated primary prostate carcinomas that showed extremely poor response to hormonal therapy (primary androgen independent prostate carcinomas) were compared with the stage- and grade-matched primary tumor specimens with favorable response to hormonal therapy (androgen dependent prostate carcinomas). The mean proliferative activity measured by Ki-67 immunohistochemistry was slightly higher in the primary androgen independent prostate carcinomas (8.70+/-5.24) than in the androgen dependent prostate carcinomas (7.09+/-2.68; p=0.27). The mean apoptotic activity by in situ end-labeling technique in the primary androgen independent prostate carcinomas (0.96+/-1.03) was less than half of that in the androgen dependent prostate carcinomas (2.75+/-0.98; p=0.0001). Ten percent of the androgen dependent prostate tumors showed p53 protein expression, whereas 30% of the primary androgen independent prostate tumors were immunopositive for p53 (p=0.30). In summary, we have shown that apoptotic activity in the primary androgen independent prostate carcinomas is significantly lower than in the matched androgen dependent prostate carcinomas while the proliferative activity remains unaffected. These results suggest that primary androgen independent prostate carcinomas may have genetic properties, such as inactivation of the p53 gene, that enable them to escape apoptosis caused by androgen ablation.

Hormone-resistant prostate cancer with symptomatic pelvic tumours: patient survival and prognostic factors.

OBJECTIVES: To determine the survival and investigate the prognostic significance of immunohistochemical variables and clinical factors in patients with hormone-resistant prostate cancer (HRPC) and symptomatic pelvic tumours, in whom preliminary observations indicated that survival exceeded the median 8-10 months of patients with HRPC and painful bone metastases. PATIENTS AND METHODS: Seventy-five patients with HRPC referred for palliative pelvic radiotherapy between 1980 and 1996 were identified. For all patients at least two prostate biopsies had been obtained, one before primary hormone treatment and at least one after clinical progression despite androgen deprivation (HRPC biopsy). Bone scans at the time of referral were assessed. The medical records were reviewed for clinical variables of possible prognostic significance. Histological grade was recorded, and prostate-specific antigen (PSA), androgen receptors (ARs), Ki-67 and p53 determined immunohistochemically. In 18 HRPC specimens the degree of AR amplification was analysed. RESULTS: Positive staining for ARs was high in the HRPC biopsies, although there was no association with AR amplification. Ki-67 positivity increased after the development of HRPC. The median (range) survival was 14 (1-141) months; age < 65 years was associated with increased survival. In a multivariate analysis the following variables remained independent prognostic factors for survival from the time of the HRPC biopsy: bone metastases (0-10 vs > 10 lesions, P < 0.001), low Ki-67 score (0 vs 1-3, P = 0.006) and low p53 positivity score (0 vs 1-3, P = 0.014) in the HRPC biopsy. CONCLUSIONS: The median survival of patients with HRPC and pelvic tumours requiring palliation seems to exceed that of patients with HRPC and dominating painful bone metastases by at least 4-6 months. Simple clinical (bone metastases) and immunohistochemical variables (Ki-67, p53) enable patients with particularly long survival times to be identified, and in whom palliative treatment needs to be improved.

Apolipoprotein E genotype is not linked to locally recurrent hormone-refractory prostate cancer.

Mechanisms of prostate cancer recurrence during androgen deprivation are poorly understood. Recently, the putative role of apolipoprotein E varepsilon 4 allele in the aetiology of prostate cancer was raised. To investigate the hypothesis that varepsilon 4 allele of apolipoprotein E gene predisposes to prostate cancer and is involved in the relapse of hormonal therapy response, 38 hormone-refractory locally recurrent carcinoma samples from 38 prostate cancer patients were screened for apolipoprotein E genotype. The frequency distribution of apolipoprotein E genotypes among tumours did not differ significantly from that among controls. The allele frequency of varepsilon 4 was 19.7% and 19.3% in tumours and controls, respectively. The results suggest that clinical progression of prostate cancer during androgen withdrawal therapy is not associated with apolipoprotein E genotype. Prostate Cancer and Prostatic Diseases (2000) 3, 107-109

Androgen receptor gene alterations and chromosomal gains and losses in prostate carcinomas appearing during finasteride treatment for benign prostatic hyperplasia.

Finasteride is commonly used for the treatment of benign prostatic hyperplasia and has been suggested to prevent prostate cancer development. To gain insight to the molecular effects of finasteride on prostate cancer development, we studied six prostate cancers diagnosed during finasteride treatment for benign prostatic hyperplasia. Comparative genomic hybridization detected genetic alterations in four tumors (1-5 changes/tumor). Xq gains and 6q losses were the most common alterations. The recurrent Xq gains motivated us to study the involvement of the androgen receptor (AR) gene. One tumor with Xq gain had a 3-fold amplification of the AR gene, suggesting that tumor development in finasteride-treated patients may require increased AR copy number and expression, as has previously been shown for prostate cancers recurring during hormonal therapy. Furthermore, in another tumor, an Arg726Leu mutation of the AR gene was found. This mutation was also present in the germ-line DNA of the patient. Arg726Leu mutation has previously been reported to affect the transactivational properties of the AR. In summary, prostate cancers developing during finasteride therapy may have distinct biological properties, such as a low number of chromosomal alterations and frequent involvement of the AR gene. Further studies are needed to explore the role of germ-line AR mutations in these patients.

Androgen receptor gene amplification increases tissue PSA protein expression in hormone-refractory prostate carcinoma.

Androgen receptor (AR) gene amplification was analysed by fluorescence in situ hybridization (FISH) from 24 paraffin-embedded prostate carcinoma samples recurring locally during hormonal therapy and prostate-specific antigen (PSA) expression from 15/24 of these samples was studied by immunohistochemistry (IHC). AR gene amplification was detected in 29 per cent (7/24) of the recurrent tumours. Using modified Histoscore (MHS), PSA immunostaining in the AR gene-amplified tumours (133+/-102) was twice as high (p=0.054) as in tumours with no amplification (66+/-79) and a statistically significant (p=0.026) association between AR gene amplification and PSA positivity was found when MHS>/=20 was considered positive for PSA. AR gene copy number was positively correlated with PSA MHS in the AR gene-amplified tumours (r=0.893, p=0.012). Histological grade, Gleason's score, and tumour stage did not differ significantly between patients with and without AR gene amplification. In conclusion, these results indicate that AR gene amplification leads to up-regulation of PSA gene (and possibly other androgen-dependent genes), and that patients with AR gene amplification may have elevated serum PSA concentrations without a clear correlation with actual tumour burden.

Amplification of the androgen receptor gene is associated with P53 mutation in hormone-refractory recurrent prostate cancer.

p53 protein expression of 30 hormone-refractory locally recurrent prostate cancers was compared with their matched untreated primary tumour specimens. In addition, androgen receptor (AR) gene amplification and p53 protein immunostaining were compared. p53 positivity increased during hormonal therapy from 17 per cent of the untreated primary tumours to 40 per cent of the hormone-refractory recurrences (p = 0.078). None of the p53-positive primary tumour specimens lost p53 positivity during hormonal therapy. Hormone-refractory recurrences with AR gene amplification more frequently (p = 0.0342) showed positive p53 immunostaining than tumours without AR gene amplification, 75 and 27 per cent, respectively. In summary, this study has shown that a cell clone with P53 mutation seems to be selected for during endocrine therapy and that positive p53 immunostaining correlates with AR gene amplification. These results suggest that inactivation of P53 may lead to genetic instability in a subset of prostate carcinomas enabling them to achieve properties, such as AR gene amplification, that allow them to grow in low levels of androgens and therefore cause tumour progression.

A de novo deletion of chromosome 15(q15.2q21.2) in a dysmorphic, mentally retarded child with congenital scalp defect.

We report a rare chromosomal finding in a boy with a pronounced scalp defect, dysmorphic features and mental retardation. Initially, what seemed to be a normal karyotype by conventional karyotyping was determined to be a de novo deletion involving 15(q15.2q21.2) by high resolution banding. Consequently, prometaphase analysis is warranted in some cases when conventional karyotype analysis appears normal.

Flow cytometric analysis of tumor DNA profile related to response to treatment and survival in small-cell lung cancer.

Flow cytometric (FCM) analysis of tumor DNA ploidy and S-phase fraction (SPF) has been widely used to predict prognosis and treatment response in many malignant tumors, but rarely in small-cell lung cancer (SCLC). In the present study, tumor DNA ploidy and SPF were measured from paraffin-embedded tumor biopsy samples of 36 small-cell lung cancer patients treated with combination chemotherapy and radiotherapy. Aneuploidy was detected in 69% of the tumors. There was a statistically non-significant trend towards more aneuploidy among extensive disease (ED) patients as compared to patients with limited disease (LD): 80% versus 65%, respectively (p = 0.69). The mean SPF was 21.3% (+/-7.6) in patients with LD and 29.0% (+/-5.3) in patients with ED, the difference (7.6%) being statistically significant (p = 0.008, 95% CI for the difference 2.2-13.1). No significant differences was detected in the survival of aneuploid and diploid patients or patients with low (< or = 24.9%) and high (> 24.9%) SPF. Similarly, no significant difference was observed between aneuploid and diploid cases in relation to response to treatment or response duration. It is concluded that the difference detected in the SPF with LD and ED of SCLC may indicate the biological aggressiveness of extensive SCLC.

Molecular cytogenetics of prostate cancer.

Prostate cancer is the most common malignancy among men in many developed countries. One-fourth of prostate cancers are diagnosed at metastatic stage but there is no curative treatment for such disease and palliative androgen withdrawal therapy remains the most used one. Thus, understanding the molecular events that underlie the development and progression of prostate cancer could help to answer many clinical questions on its treatment. In this review article, I want to illustrate some of the most interesting findings (by fluorescence in situ hybridization and comparative genomic hybridization) in the molecular cytogenetics of prostate cancer.