Breast cancer after bilateral risk-reducing mastectomy.

This study aims to evaluate the incidence of breast cancer after risk-reducing mastectomy (RRM) in healthy BRCA mutation carriers. This study is a long-term follow-up of 307 BRCA mutation carriers of whom 96 chose RRM. None of the study participants had a previous history of breast or ovarian cancer nor had they undergone RRM or risk-reducing bilateral salpingo-oophorectomy (BSO) prior to the time of BRCA testing. The annual incidence of post-mastectomy breast cancer was 0.8% compared with 1.7% in the non-operated group. Implications of these findings in relation to genetic counseling and future management are discussed.

Targeted prostate cancer screening in men with mutations in BRCA1 and BRCA2 detects aggressive prostate cancer: preliminary analysis of the results of the IMPACT study.

OBJECTIVE: To evaluate the role of targeted prostate cancer screening in men with BRCA1 or BRCA2 mutations, an international study, IMPACT (Identification of Men with a genetic predisposition to ProstAte Cancer: Targeted screening in BRCA1/2 mutation carriers and controls), was established. This is the first multicentre screening study targeted at men with a known genetic predisposition to prostate cancer. A preliminary analysis of the data is reported. PATIENTS AND METHODS: Men aged 40-69 years from families with BRCA1 or BRCA2 mutations were offered annual prostate specific antigen (PSA) testing, and those with PSA > 3 ng/mL, were offered a prostate biopsy. Controls were men age-matched (± 5 years) who were negative for the familial mutation. RESULTS: In total, 300 men were recruited (205 mutation carriers; 89 BRCA1, 116 BRCA2 and 95 controls) over 33 months. At the baseline screen (year 1), 7.0% (21/300) underwent a prostate biopsy. Prostate cancer was diagnosed in ten individuals, a prevalence of 3.3%. The positive predictive value of PSA screening in this cohort was 47·6% (10/21). One prostate cancer was diagnosed at year 2. Of the 11 prostate cancers diagnosed, nine were in mutation carriers, two in controls, and eight were clinically significant. CONCLUSIONS: The present study shows that the positive predictive value of PSA screening in BRCA mutation carriers is high and that screening detects clinically significant prostate cancer. These results support the rationale for continued screening in such men.

Risk-reducing mastectomy and salpingo-oophorectomy in unaffected BRCA mutation carriers: uptake and timing.

Once female carriers of a BRCA mutation are identified they have to make decisions on risk management. The aim of this study is to outline the uptake of risk-reducing surgery in the Danish population of BRCA mutation positive women and to search for factors affecting this decision. We analysed data from 306 healthy BRCA carriers with no personal history of ovarian or breast cancer. We found a 10-year uptake of 75% for risk-reducing salpingo-oophorectomy and 50% for risk-reducing mastectomy by time to event analysis. Age and childbirth influenced this decision. The uptake rate has not changed significantly over the last decade. Risk-reducing surgeries are widely acceptable among Danish BRCA mutation positive women and the uptake of prophylactic mastectomy is higher than in most other countries.

Evaluation of a candidate breast cancer associated SNP in ERCC4 as a risk modifier in BRCA1 and BRCA2 mutation carriers. Results from the Consortium of Investigators of Modifiers of BRCA1/BRCA2 (CIMBA).

BACKGROUND: In this study we aimed to evaluate the role of a SNP in intron 1 of the ERCC4 gene (rs744154), previously reported to be associated with a reduced risk of breast cancer in the general population, as a breast cancer risk modifier in BRCA1 and BRCA2 mutation carriers. METHODS: We have genotyped rs744154 in 9408 BRCA1 and 5632 BRCA2 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) and assessed its association with breast cancer risk using a retrospective weighted cohort approach. RESULTS: We found no evidence of association with breast cancer risk for BRCA1 (per-allele HR: 0.98, 95% CI: 0.93-1.04, P = 0.5) or BRCA2 (per-allele HR: 0.97, 95% CI: 0.89-1.06, P = 0.5) mutation carriers. CONCLUSION: This SNP is not a significant modifier of breast cancer risk for mutation carriers, though weak associations cannot be ruled out.

The importance of KRAS mutations and EGF61A>G polymorphism to the effect of cetuximab and irinotecan in metastatic colorectal cancer.

BACKGROUND: The effect of anti-epidermal growth factor receptor (EGFR) antibodies (mAb) in metastatic colorectal cancer seems limited to KRAS wild-type (wt) tumours, but still a major fraction of KRASwt patients are nonresponders and supplementary selection criteria are needed. We investigated methodological aspects of KRAS testing and the predictive and prognostic value of KRAS status combined with three EGFR-related gene polymorphisms [single-nucleotide polymorphisms (SNPs)] in patients treated with cetuximab and irinotecan. PATIENTS AND METHODS: The study included 71 patients referred to third-line cetuximab-irinotecan. Blood samples were analysed for SNPs. KRAS analysis was carried out by sequencing analysis and quantitative PCR (DxS kit) in primary tumour and distant metastases. RESULTS: There was a clear correlation between KRAS status in primary tumours and metastasis. The DxS kit presented the highest sensitivity. Response was confined to KRASwt patients (40% response rate versus 0%, P < 0.1(-3)), which translated into a significant difference in PFS. The EGF61A>G polymorphism showed relation to clinical outcome. A combined biomarker analysis showed a 19% progression rate in KRASwt-EGF61 homozygote patients and 60% in the EGF61A/G patients (P = 0.006) and a significant increase in overall survival (17.1 versus 5.9 months, log-rank, P = 0.002). CONCLUSION: The combined biomarker analysis maybe an attractive approach to selection of patients for third-line treatment including anti-EGFR mAbs.

Nuclei size in relation to nuclear status and aneuploidy rate for 13 chromosomes in donated four cells embryos.

PURPOSE: The aim was to elucidate if the nuclear size and number are indicative of aberrant chromosome content in human blastomeres and embryos. METHODS: The number of nuclei and the nucleus and blastomere size were measured by a computer controlled system for multilevel analysis. Then the nuclei were enumerated for 13 chromosomes by a combination of PNA and DNA probes. RESULTS: In the mononucleated embryos there was no difference in the mean size of chromosomally normal and abnormal nuclei but a significant difference in the mean nuclei size of nuclei that had gained chromosomes compared to nuclei that had lost chromosomes. The nuclei from multinucleated blastomeres had a significant smaller mean size and the frequency of chromosomally aberrant blastomeres was significantly higher. CONCLUSION: The mean nuclear size is not a marker for the chromosome content in mononucleated embryos. However, it seems that the nuclei size can be related to multinucleation and maybe to the chromosome content.

Strategy in clinical practice for classification of unselected colorectal tumours based on mismatch repair deficiency.

OBJECTIVE: Deficiency of DNA mismatch repair (MMR) causes microsatellite instability (MSI) in a subset of colorectal cancers. Patients with these tumours have a better prognosis and may have an altered response to chemotherapy. Some of the tumours are caused by hereditary mutations (hereditary nonpolyposis colon cancer or Lynch syndrome), but most are epigenetic changes of sporadic origin. The aim of this study was to define a robust and inexpensive strategy for such classification in clinical practice. METHOD: Tumours and blood samples from 262 successive patients with colorectal adenocarcinomas were collected. Expression of the MMR proteins MLH1, MSH2, and MSH6 by immunohistochemistry (IHC) was compared with MSI DNA analysis. Methylation analysis of MLH1 and mutation analysis for BRAF V600E were compared in samples with MSI and/or lack of MLH1 expression to determine if the tumour was likely to be sporadic. RESULTS: Thirty-nine (14.9%) of the tumours showed MMR deficiency by IHC or by microsatellite analysis. Sporadic inactivation by methylation of MLH1 promoter was found in 35 patients whereby the BRAF activating V600E mutation, indicating sporadic origin, was found in 32 tumours. On the basis of molecular characteristics we found 223 patients with intact MMR, 35 patients with sporadic MMR deficiency, and four patients who were likely to have hereditary MMR deficiency. CONCLUSION: To obtain the maximal benefit for patients and clinicians, MMR testing should be supplemented with MLH1 methylation or BRAF mutation analysis to distinguish sporadic patients from likely hereditary ones. MMR deficient patients with sporadic disease can be reassured of the better prognosis and the likely hereditary cases should receive genetic counselling.

Annual surveillance by CA125 and transvaginal ultrasound for ovarian cancer in both high-risk and population risk women is ineffective.

OBJECTIVE: To assess the efficacy of annual CA125 and transvaginal ultrasound (TVU) scan as surveillance for ovarian cancer. DESIGN: Retrospective audit. SETTING: NHS Trust. POPULATION: Three hundred and forty-one asymptomatic women enrolled for ovarian cancer screening: 179 were in a high-risk group (>10% lifetime risk of developing ovarian cancer), 77 in a moderate risk group (4-10% lifetime risk of developing ovarian cancer) and 71 in a near population risk group (<4% lifetime risk). METHODS: Retrospective audit of case records, laboratory CA125 results, radiology reports, histology records and local cancer registry data. MAIN OUTCOME MEASURES: Ovarian cancers occurring in study population. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of TVU, and CA125 as a screening tool for ovarian cancer. RESULTS: Four ovarian cancers and one endometrial cancer occurred. One ovarian cancer was detected at surveillance, three occurred in women who presented symptomatically between screenings. Thirty women underwent exploratory surgery because of abnormal findings at surveillance. Two women had cancer (PPV = 6.7%); one had ovarian cancer and the other endometrial cancer. Twenty-eight women (93.3%) had no malignancy. Sensitivity, specificity, PPV and NPV for TVU in the whole cohort were 33.3, 85.8, 0.6 and 99.8%, respectively. For high-risk individuals, the figures for TVU were 33.3, 84.5, 1.1 and 99.6, respectively. Combining both modalities for the whole cohort, the sensitivity, specificity, PPV and NPV were 66.7, 82.9, 1.5 and 99.8% and 50.0, 82.8, 1.3 and 99.7%, respectively, for the high-risk group alone. CONCLUSIONS: Ovarian screening by annual TVU and CA125 is inefficient at detecting early-stage ovarian cancers.

Selecting patients with young-onset colorectal cancer for mismatch repair gene analysis.

BACKGROUND: Young patients with colorectal cancer are at increased risk of carrying a germline mutation in mismatch repair (MMR) genes. This study investigated the role of clinical criteria and immunohistochemistry for MMR proteins in selecting young patients for mutation testing. METHODS: A cohort of 56 consecutive patients with colorectal cancer aged less than 45 years were stratified into three groups based on clinical criteria: 'Amsterdam criteria', 'high risk' and 'young onset only'. Immunohistochemistry for four MMR proteins was carried out and the rate of compliance with clinical guidelines determined. RESULTS: Tumours from 11 patients (20 per cent) had abnormal MMR protein expression, of whom eight were referred for genetic assessment. Of 21 patients (38 per cent) in total referred to the genetics unit, six MMR gene mutations were identified, all associated with abnormal immunohistochemistry. CONCLUSION: MMR immunohistochemistry should be considered routine in young-onset colorectal cancer.

Evaluation of two different models to predict BRCA1 and BRCA2 mutations in a cohort of Danish hereditary breast and/or ovarian cancer families.

To meet the increasing demand for BRCA1 and BRCA2 mutation analysis, a robust system for selecting families who have a higher chance of a mutation has become important. Several models have been developed to help predict which samples are more likely to be mutation positive than others. We have undertaken a complete BRCA1 and BRCA2 mutation analysis in 267 Danish families with high-risk family history. We found deleterious mutations in 28% (76) of the families, 68% (52) of those in BRCA1 and 32% (24) in BRCA2. We compared our results with two popular manual models developed to estimate the chance of a positive result. One is the recently published Manchester model and the other is the Frank 2 model updated by Myriad Genetic Laboratories, Inc. Neither of the models would have suggested screening all mutation-positive samples. The Manchester model would have suggested screening 124 of the families in the cohort, thereby detecting 54 of 76 mutations (sensitivity 71%; specificity 63%), whereas the Frank 2/Myriad model would have found 60 of 76 mutations by screening 169 samples if a 10% likelihood was adapted (sensitivity 79%; specificity 43%). The updated Manchester model suggested screening 172 families whereby 64 mutations would have been detected (sensitivity 84%; specificity 44%). We conclude that although both models would have reduced the number of samples screened significantly, up to 28% of the mutations would not have been found by applying these models to this Danish cohort of families. This raises the question whether models designed for specific populations can be used in a wider setting.

'Indirect' BRCA1/2 testing: a useful approach in hereditary breast and ovarian cancer families without a living affected relative.

We report an approach for BRCA1/2 testing whereby genetic testing can be offered to families at high risk of hereditary breast and ovarian cancer but where no DNA from affected relatives is available. By testing two or more unaffected relatives at 50% risk of being heterozygous for a potential BRCA1/2 mutation, there is a chance of up to 99% of finding a mutation that would have been detectable in an affected individual from the same family. The overall likelihood of identifying a mutation is dependent on the family history, and therefore 'indirect' testing would be most applicable for families with a very high risk of carrying a BRCA1/2 mutation. Using this approach also requires balancing issues of testing resource limitations, family dynamics and adequate preparation of unaffected persons for a positive test, with the advantages of targeting screening and prophylactic surgery.

Sequential FISH analysis using competitive displacement of labelled peptide nucleic acid probes for eight chromosomes in human blastomeres.

BACKGROUND: The aim was to introduce a new strategy based on peptide nucleic acid (PNA) probes and competitive displacement for using fluorescence in-situ hybridization (FISH) analysis on human blastomeres. METHODS: Sequential FISH analysis with PNA probes and competitive displacement was performed using three different probe sets. The first set consisted of labelled probe only. The second and third sets included labelled as well as unlabelled probe, corresponding to the labelled probes in the previous cycles. The probes for enumeration were for chromosome 1, 13, 16, 17, 18, 21, X and Y. RESULTS: The performance of PNA probes was similar to the established DNA probes. The strategy of competitive displacement resulted in a destabilization of already bound probe before the next FISH cycle at only 50 degrees C, which allowed for up to five sequential FISH cycles without loss of signal. CONCLUSIONS: PNA probes are a good alternative to DNA probes in the present set-up, since the low temperature required both for binding and destabilization of PNA probes minimizes the loss of signal, and several FISH cycles can therefore be carried out before FISH errors occur.

Genetic analysis of males from intracytoplasmic sperm injection couples.

A total of 392 men referred for intracytoplasmic sperm injection (ICSI) participated in genetic analysis. The control group consisted of 100 normal fertile males. Chromosome and DNA analyses were performed to investigate the frequency of Y-chromosome microdeletions and CFTR mutations (the controls underwent DNA analysis only). An abnormal karyotype was found in 4.6% of all males, but the frequency among men with azoospermia was higher, at 11.7%. Y-chromosome microdeletions were found only among men with azoospermia (6.5%) and men with extreme oligospermia (2%). Compound heterozygosity for CFTR mutations was found in men with azoospermia (3.9%) and congenital bilateral absence of vas deferens (CBAVD) only. We conclude that all couples referred for ICSI should be offered chromosome analysis. DNA analysis for Y-chromosome microdeletions should be reserved for men with azoospermia or extreme oligospermia (<1 x 106 spermatozoa). Analysis for CFTR mutations should be limited to those with obstructive azoospermia or those with a family history of cystic fibrosis.

Fertility and pregnancy outcome in Danish women with Turner syndrome.

The aim of the investigation was to study fertility in Danish women diagnosed with Turner syndrome (TS), and to describe their offspring. In total, 410 women in the fertile age were registered in the Danish Cytogenetic Central Register with TS between January 1973 and December 1993. Their karyotype were as follows: 49% with 45,X, 23% with mosaicism and a structural abnormality of the second X, 19% with 45,X/46,XX mosaicism, and 9% with 46,XX and a structural abnormality of the second X. Thirty-three women, one with 45,X, 27 with mosaicism and 5 with 46,XX and a structural abnormality of the second X, gave birth to 64 children. Two women had become pregnant after in vitro fertilization, including a woman with 45,X after an egg donation. Thus, 31 women(7.6%) had achieved at least one spontaneous pregnancy, but 48% of the fertile women registered with 45,X/46,XX had 45,X in less than 10% of the analysed cells. Twenty-five of the 64 children had a chromosome analyzed. Six of the 25 examined children, including three siblings, had chromosomal aberrations. No case of Down's syndrome was present, and only two children had malformations. Fertility in women registered with TS is higher than earlier reported. However, only women with 45,X/46,XX mosaicism or 46,XX and structural abnormality of the second X, gave birth to live children after spontaneous pregnancies.

Birth of a healthy girl after ICSI with ejaculated spermatozoa from a man with non-mosaic Klinefelter's syndrome.

Klinefelter's syndrome is a major contributor to male infertility. Recent reports of births after ICSI with especially testicular spermatozoa from infertile men with this syndrome are promising. The birth of a healthy girl after ICSI treatment with ejaculated spermatozoa from a man with non-mosaic Klinefelter's syndrome is reported. The non-mosaic karyotype was confirmed by chromosome analysis of both peripheral blood leukocytes and fibroblasts from a skin biopsy. In conclusion, in a very few cases, men with apparently non-mosaic Klinefelter's syndrome have ejaculated spermatozoa that can result in a birth of a healthy child following ICSI.

Quantitative evaluation of fluorescence in situ hybridization (FISH) signals in uncultured coelomic cells.

Uncultured coelomic cells were hybridized with alpha satellite DNA probes representing chromosomes X, Y, 18, and 13/21 in order to evaluate the distribution of hybridization signals obtained by fluorescence in situ-hybridization (FISH) analysis of this cell type. Cells from 26 samples were hybridized with the X probe and the Y probe was hybridized with cells from 25 of the samples. Cells from 16 and 11 samples were hybridized with an 18 alpha satellite DNA probe and 13/21 alpha satellite probe, respectively. The evaluation demonstrated that FISH with X, Y, 18, and 13/21 alpha satellite DNA probes on uncultured coelomic cells is a reliable technique since the distribution of hybridization signals is comparable to that seen in uncultured amniotic fluid cells.

Early prenatal diagnosis: standard cytogenetic analysis of coelomic cells obtained by coelocentesis.

Coelomic fluid samples (n = 30) were obtained from normal pregnancies between 6.0 and 10.0 weeks and attempts to culture the coelomic cells were made. With one culture system, nine of ten samples were cultured and successful cytogenetic analysis was performed. There have been no previous reports of traditional cytogenetic analysis of coelomic cells and this might be the breakthrough for coelocentesis as a future method for early prenatal diagnosis.

A two-dimensional gel protein database of noncultured total normal human epidermal keratinocytes: identification of proteins strongly up-regulated in psoriatic epidermis.

A two-dimensional (2-D) gel database of proteins from noncultured total normal human epidermal keratinocytes has been established. A total of 1449 [35S]methionine labelled proteins (1112 isoelectric focusing, 337 nonequilibrium pH gradient electrophoresis) were resolved and recorded using computer assisted (PDQ-SCAN and PDQUEST software) 2-D gel electrophoresis. By matching the protein patterns of total keratinocytes and transformed human amnion cells (master database; Celis et al., Leukemia 1988, 2, 561-602) as well as by 2-D immunoblotting and microsequencing of keratinocyte proteins, it was possible to identify 72 polypeptides in the keratinocyte database. The database also includes data on polypeptides that are synthesized at a higher level by keratinocytes enriched in basal cells, and on six secreted proteins which are produced, albeit at a reduced rate, by normal keratinocytes and that are strongly up-regulated in psoriatic epidermis (Celis et al., FEBS Letters, in press).

Identification of a group of proteins that are strongly up-regulated in total epidermal keratinocytes from psoriatic skin.

Analysis using two-dimensional (2D) gel electrophoresis of the [35S]-methionine-labelled proteins synthesized by non-cultured total epidermal keratinocytes obtained from normal and psoriatic skin revealed 6 proteins that are strongly up-regulated (5 times or more) in psoriatic skin. These proteins are synthesized at albeit lower levels by keratinocytes from normal and normal-appearing (uninvolved) skin of psoriatic patients, and correspond to isoelectric focusing sample spot numbers 4311 (40.3 kDa), 4003 (12.4 kDa), 5008 (11.9 kDa), 3012 (11.6 kDa), 6016 (11.6 kDa) and 1015 (10.1 kDa) in the normal keratinocyte 2D gel protein database [Celis et al, (1990) Electrophoresis, in press]. These proteins are also detected in the labelling medium indicating that they are at least in part secreted. Given their striking regulatory behavior, these proteins may play a role in the pathogenesis of psoriasis.