Feasibility of couple-based expanded carrier screening offered by general practitioners.

Expanded carrier screening (ECS) aims to inform couples' reproductive choice, preferably before conception. As part of an implementation study in which trained general practitioners (GPs) offered a population-based ECS couple-test, we evaluated the feasibility of the test-offer and degree of participant informed choice (IC). Trained GPs from nine practices in the northern Netherlands invited 4295 female patients aged 18-40 to take part in couple-based ECS. Inclusion criteria were having a male partner, planning for children and not being pregnant. We evaluated the feasibility of the organizational aspects, GP competence and the content of the pre-test counselling. Participant satisfaction, evaluation of pre-test counselling and degree of IC were measured using a longitudinal survey. We explored GP experiences and their views on future implementation through semi-structured interviews. 130 consultations took place. All participating GPs were assessed by genetic professionals to be competent to conduct pre-test counselling. Most (63/108 (58%)) consultations took place within the planned 20 min (median 20, IQR 18-28). GPs considered couples' prior knowledge level an important determinant of consultation length. 91% of patients were (very) satisfied with the GP counselling. After pre-test counselling, 231/237(97%) participants had sufficient knowledge and 206/231(88%) had a positive attitude and proceeded with testing. Our pilot demonstrates that offering couple-based ECS through trained and motivated GPs is feasible. Future large-scale implementation requires a well-informed general public and a discussion about appropriate reimbursement for GPs and health care coverage for couples. Providing (more) test information pre-appointment may help reduce average consultation time.

Maximising the efficiency of clinical screening programmes: balancing predictive genetic testing with a right not to know.

We explored the dilemma between patients' right not to know their genetic status and the efficient use of health-care resources in the form of clinical cancer screening programmes. Currently, in the Netherlands, 50% risk carriers of heritable cancer syndromes who choose not to know their genetic status have access to the same screening programmes as proven mutation carriers. This implies an inefficient use of health-care resources, because half of this group will not carry the familial mutation. At the moment, only a small number of patients are involved; however, the expanding possibilities for genetic risk profiling means this issue must be addressed because of potentially adverse societal and financial impact. The trade-off between patients' right not to know their genetic status and efficient use of health-care resources was discussed in six focus groups with health-care professionals and patients from three Dutch university hospitals. Professionals prefer patients to undergo a predictive DNA test as a prerequisite for entering cancer screening programmes. Professionals prioritise treating sick patients or proven mutation carriers over screening untested individuals. Participation in cancer screening programmes without prior DNA testing is, however, supported by most professionals, as testing is usually delayed and relatively few patients are involved at present. Reducing the number of 50% risk carriers undergoing screening is expected to be achieved by: offering more psychosocial support, explaining the iatrogenic risks of cancer screening, increasing out-of-pocket costs, and offering a less stringent screening programme for 50% risk carriers.

Variation in mutation spectrum partly explains regional differences in the breast cancer risk of female BRCA mutation carriers in the Netherlands.

BACKGROUND: We aimed to quantify previously observed relatively high cancer risks in BRCA2 mutation carriers (BRCA2 carriers) older than 60 in the Northern Netherlands, and to analyze whether these could be explained by mutation spectrum or population background risk. METHODS: This consecutive cohort study included all known pathogenic BRCA1/2 carriers in the Northern Netherlands (N = 1,050). Carrier and general reference populations were: BRCA1/2 carriers in the rest of the Netherlands (N = 2,013) and the general population in both regions. Regional differences were assessed with HRs and ORs. HRs were adjusted for birth year and mutation spectrum. RESULTS: All BRCA1 carriers and BRCA2 carriers younger than 60 had a significantly lower breast cancer risk in the Northern Netherlands; HRs were 0.66 and 0.64, respectively. Above age 60, the breast cancer risk in BRCA2 carriers in the Northern Netherlands was higher than in the rest of the Netherlands [HR, 3.99; 95% confidence interval (CI), 1.11-14.35]. Adjustment for mutational spectrum changed the HRs for BRCA1, BRCA2 <60, and BRCA2 ≥60 years by -3%, +32%, and +11% to 0.75, 0.50, and 2.61, respectively. There was no difference in background breast cancer incidence between the two regions (OR, 1.03; 95% CI, 0.97-1.09). CONCLUSIONS: Differences in mutation spectrum only partly explain the regional differences in breast cancer risk in BRCA2 carriers, and for an even smaller part in BRCA1 carriers. IMPACT: The increased risk in BRCA2 carriers older than 60 may warrant extension of intensive breast screening beyond age 60.

Proven non-carriers in BRCA families have an earlier age of onset of breast cancer.

BACKGROUND: Risk estimates for proven non-carriers in BRCA mutation families are inconsistent for breast cancer and lacking for ovarian cancer. We aimed to assess the age-related risks for breast and ovarian cancer for proven non-carriers in these families. METHODS: A consecutive cohort study ascertained 464 proven non-carriers who had a first-degree relative with a pathogenic BRCA mutation. Kaplan-Meier analyses were used to estimate the age-related cancer risks, and we calculated standardised incidence ratios. RESULTS: In the 464 non-carriers, 17 breast cancers and two ovarian cancers were detected at a mean age of 47 years (95% confidence interval (CI) 32-61) and 49 years (95% CI 32-67), respectively. Overall, by the age of 50, the breast and ovarian cancer risks among non-carriers were 6.4% (95% CI 2.9-9.8%) and 0.4% (95% CI 0-1.3%), of which the breast cancer risk was statistically significantly higher than the risk in the general population. In particular, the number of breast cancers among non-carriers in BRCA1 families was higher than expected for the general population (standardised incidence ratio (SIR) 2.0, 95% CI 1.1-3.3). In the BRCA1 cohort, the mean number of breast cancer cases was higher in families in which non-carriers were diagnosed before the age of 50 (p=0.04). CONCLUSION: The age at diagnosis of breast cancer in non-carriers in BRCA mutation families is younger than expected, yielding an increased risk in the fifth decade. This effect is most evident in BRCA1 families. If our results are confirmed by others, this could affect the advice given on breast cancer screening to proven non-carriers between the age of 40 and 50 in such families.

Breast Cancer Incidence After Risk-Reducing Salpingo-Oophorectomy in BRCA1 and BRCA2 Mutation Carriers.

Premenopausal risk-reducing salpingo-oophorectomy (RRSO) in BRCA1/2 mutation carriers effectively reduces ovarian cancer risk, but also reduces breast cancer risk. Breast cancer risk reductions up to 50% have been reported for both BRCA1 and BRCA2 mutation carriers, but recent prospective studies were not able to reproduce this finding for BRCA1 mutation carriers. Breast cancer incidence after RRSO was assessed in a consecutive series of 104 BRCA1 and 58 BRCA2 mutation carriers. On the basis of data from our own centre, and assuming a 50% risk reduction through RRSO at premenopausal age, we expected to find 8 breast cancers (range 6-10) in this population for the reported screening period (532 women-years). In 162 carriers with a median age of 41 years at RRSO, 13 incident breast cancers were diagnosed. In BRCA1 mutation carriers, 12 incident breast cancers were found compared with 5 (range 3-6) expected and in BRCA2 mutation carriers 1 breast cancer was found compared with 3 (range 2-5) expected. Breast cancer incidence after premenopausal RRSO is still high, especially in BRCA1 mutation carriers. Previously reported breast cancer risk reductions up to 50% were not confirmed. As a consequence, continued intensive screening for breast cancer is warranted in BRCA1 and BRCA2 mutation carriers after RRSO.

Penetrance of breast cancer, ovarian cancer and contralateral breast cancer in BRCA1 and BRCA2 families: high cancer incidence at older age.

Accurate estimations of lifetime risks of breast and ovarian cancer are crucial for counselling women from BRCA1/2 families. We therefore determined breast and ovarian cancer penetrance in BRCA1/2 mutation families in the northern Netherlands and compared them with the incidence of cancers in the general population in this region. We identified 1188 female mutation carriers and first-degree female relatives in 185 families with a pathogenic BRCA1 or BRCA2 mutation. The occurrence of breast cancer, contralateral breast cancer and ovarian cancer was recorded. The cumulative incidence of breast cancer by age 70 was 71.4% (95% CI 67.2-82.4%) in BRCA1 and 87.5% (82.4-92.6%) in BRCA2 mutation carriers. For ovarian cancer at age 70, it was 58.9% (53.5-64.3%) in BRCA1 and 34.5% (25.0-44.0%) in BRCA2 mutation carriers. For breast cancer we saw a rise of 24.2% in the cumulative incidence in the seventh decade for BRCA2 mutation carriers versus 6.3% for BRCA1. For ovarian cancer the rise in the seventh decade was 17.3% for BRCA1 mutation carriers and 15.1% for BRCA2. The 10-year risk for contralateral breast cancer was 34.2% (29.4-39.0%) in BRCA1 families and 29.2% (22.9-35.5%) in BRCA2. We show that the incidence of breast and ovarian cancer in BRCA2 mutation carriers and of ovarian cancer in BRCA1 mutation carriers is still high after 60 years. This may justify intensive breast screening as well as oophorectomy even after age 60. The risk of contralateral breast cancer rises approximately 3% per year, which may affect preventive choices.

Selective expression of cholesterol metabolism genes in normal CD34+CD38- cells with a heterogeneous expression pattern in AML cells.

OBJECTIVE: To identify adenosine triphosphate-binding-cassette (ABC) transporters that are selectively expressed in normal and/or leukemic CD34+CD38- stem cells. METHODS: Microarray experiments on murine stem cells identified 13 ABC transporters with a high expression level. Corresponding human transporters were analyzed in normal CD34+CD38- and CD34+CD38+ bone marrow cells by quantitative reverse transcriptase polymerase chain reaction. RESULTS: Five ABC transporters, including ABCG1, were differentially expressed with a higher expression in CD34+CD38- cells. Besides ABCG1, known to be involved in cholesterol metabolism, expression of another major cholesterol transporter (ABCA1), some cholesterol metabolism genes (3-hydroxy-3-methyl-glutaryl-CoA reductase, low-density lipoprotein receptor), and the transcription factor controlling ABCA1 and ABCG1 expression, liver-X-receptor-alpha (LXR-alpha), were assessed. All these genes were predominantly expressed in the more primitive subpopulation, indicating a high rate of cholesterol metabolism and transport. Conversely in acute myeloid leukemia (AML), a heterogeneous expression pattern was found consisting of a considerably higher expression of particularly LXR-alpha in CD34+ cells and a reverse expression pattern in a subset of AML CD34+CD38+ cells. CONCLUSION: These data suggest an active cholesterol metabolism and efflux in normal CD34+CD38- cells, although a subgroup of AMLs potentially demonstrate a hyperactive cholesterol metabolism.

Breast cancer resistance protein (BCRP) in acute leukemia.

Multidrug resistance, cross-resistance to structurally and functionally unrelated drugs, is an important cause of treatment failure in acute leukemia. Multidrug resistance can result from the overexpression of ATP-dependent efflux pumps, such as P-glycoprotein and members of the multidrug resistance associated protein (MRP) family. Recently a novel transporter has been identified, which is called breast cancer resistance protein (BCRP), ABCG2 or mitoxantrone resistance protein. BCRP confers resistance to chemotherapeutic agents, such as mitoxantrone, doxorubicin and daunorubicin. This review describes BCRP detection techniques and the normal physiology of BCRP. The role of BCRP in the physiology of hematopoietic stem cells is addressed as well as the involvement of BCRP in multidrug resistance in acute leukemia. In AML and ALL, several studies showed that BCRP is expressed and functionally active at low, but variable levels. However, further studies are warranted to investigate its effect on clinical outcome, and explore whether patients could benefit from the combination of BCRP inhibitors and chemotherapy.

The role of breast cancer resistance protein in acute lymphoblastic leukemia.

PURPOSE: Overexpression of the transporter ABCG2, also known as breast cancer resistance protein and mitoxantrone resistance protein, can confer resistance to a variety of cytostatic drugs, such as mitoxantrone, topotecan, doxorubicin, and daunorubicin. This study analyzes the ABCG2 expression and activity in 46 human de novo acute lymphoblastic leukemia B- and T-lineage (ALL) samples. EXPERIMENTAL DESIGN: ABCG2 expression was measured flow cytometrically with the BXP-34 monoclonal antibody. ABCG2 functional activity was determined flow cytometrically by measuring mitoxantrone accumulation in combination with the ABCG2 inhibitor fumitremorgin C (FTC). To determine a possible effect of the transporters P-glycoprotein and multidrug resistance-associated protein (MRP1 and MRP2) on mitoxantrone accumulation, the accumulation was investigated in the presence of the P-glycoprotein inhibitor PSC 833 and MRP inhibitor MK-571. The ABCG2 gene was sequenced to investigate the amino acid at position 482. RESULTS: In B-lineage ALL (n = 23), the median BXP-34:IgG1 ratio was higher, namely 2.4 (range, 1.7-3.7), than in T-lineage ALL (n = 23; 1.9; range, 1.2-6.6; P = 0.003). The addition of FTC to mitoxantrone treatment caused a median increase in mitoxantrone accumulation of 21% (range, 0-140%) in B-lineage ALL. In T-lineage ALL, this FTC effect was less pronounced (5%; range, 0-256%; P = 0.013). The influence of FTC on mitoxantrone accumulation correlated with ABCG2 protein expression (r = 0.52; P < 0.001; n = 43). The increase in mitoxantrone accumulation, when FTC was added to cells treated with both PSC 833 and MK-571, correlated with the ABCG2 expression in B-lineage ALL but not in T-lineage ALL. Sequencing the ABCG2 gene revealed no ABCG2 mutation at position 482 in patients who accumulated more rhodamine after FTC. CONCLUSIONS: This study shows that ABCG2 is expressed higher and functionally more active in B-lineage than in T-lineage ALL.

High functional P-glycoprotein activity is more often present in T-cell acute lymphoblastic leukaemic cells in adults than in children.

There is a distinct difference in prognosis between childhood versus adult acute lymphoblastic leukaemia (ALL). To define whether multidrug resistance (MDR) genes might contribute to this distinction, the expression and functional activity of P-glycoprotein (P-gp) and MDR associated proteins (MRP) were determined with RT-PCR (MDR-1, MRP1, MRP2, MRP3) and flow cytometry (P-gp and MRP). Patient samples were obtained from 36 children and 35 adults with de novo ALL. Of these patients, 38 showed a T-lineage and 33 showed a B-lineage immunophenotype. In the samples, large variability in P-gp activity (0.8-4.9) and MRP activity (1.1-13.9) was observed. Most T-ALL patients with high P-gp activity were adults (89%). The mRNA expression of MDR-1 correlated weakly with P-gp activity. In contrast, MRP activity did not correlate with the mRNA expression of MRP1, MRP2 and MRP3. In T-ALL, a worse overall survival and event-free survival was observed with increasing P-gp activity. P-gp activity had no prognostic impact in B-lineage ALL. In addition, high MRP activity did not influence treatment outcome in either T- or B-lineage ALL. Multivariate Cox regression analysis, showed P-gp activity to be the only unfavourable prognostic factor for overall survival in T-ALL. In conclusion, this study demonstrates the prognostic relevance of P-gp activity in T-ALL. Since the majority of the patients with high P-gp activity were adults, P-gp might contribute to the poor prognosis of adult T-ALL.

The role of drug efflux pumps in acute myeloid leukemia.

A major problem in the treatment of patients with acute myeloid leukemia (AML) is the occurrence of resistance to structurally and functionally unrelated chemotherapeutic agents, called multidrug resistance (MDR). One of the known MDR mechanisms is the overexpression of adenosine triphosphate (ATP)-dependent efflux pumps. Permeability-glycoprotein (P-gp), the best characterized of the human drug efflux pumps, has been shown to be associated with poor treatment outcome in AML patients. Besides P-gp, in addition the multidrug resistance protein 1 (MRP1) appeared to contribute to the observed resistance in AML. Alternative transporter proteins, such as the MRP1 homologues MRP2, MRP3, MRP5 and MRP6, and the breast cancer resistance protein (BCRP), have been shown to be expressed at variable levels in AML patient cells. The latter proteins have been described to confer resistance to chemotherapeutic agents, such as daunorubicin, mitoxantrone, etoposide and 6-mercaptopurine, which are generally used in the treatment of AML patients; however, theyhave not yet proven to play a role in drug resistance in AML. The present review gives an overview of the current knowledge concerning these drug transporters, with a focus on the role of the transporter proteins in AML.

Expression and activity of breast cancer resistance protein (BCRP) in de novo and relapsed acute myeloid leukemia.

Overexpression of the breast cancer resistance protein (BCRP) efflux pump in human cancer cell lines results in resistance to a variety of cytostatic agents. The aim of this study was to analyze BCRP protein expression and activity in acute myeloid leukemia (AML) samples and to determine whether it is up-regulated due to clonal selection at relapse/refractory disease. BCRP protein expression was measured flow cytometrically with the monoclonal antibodies BXP-34 and BXP-21 in 20 paired samples of de novo and relapsed/refractory AML. BXP-34/immunoglobulin G1 ratios were observed of 1.6 +/- 0.5 (mean +/- SD, range 0.8-2.7) and BXP-21/immunoglobulin G2a ratios of 4.9 +/- 3.0 (range 1.1-14.5) in the patient samples versus 9.8 +/- 6.8 and 6.5 +/- 2.4, respectively, in the MCF-7 cell line. BCRP activity was determined flow cytometrically by measuring mitoxantrone accumulation in absence and presence of the inhibitor fumitremorgin C. Mitoxantrone accumulation, expressed as mean fluorescence intensity (MFI), varied between 44 and 761 MFI (227 +/- 146 MFI) and correlated inversely with BCRP expression (r = -0.58, P <.001). Addition of fumitremorgin C showed a small increase in mitoxantrone accumulation (11 +/- 29 MFI, n = 40) apart from the effect of PSC833 and MK-571. No consistent up-regulation of BCRP expression or activity was observed at relapse/refractory disease; some cases showed an increase and other cases a decrease at relapse. Relatively high BCRP expression correlated with immature immunophenotype, as determined by expression of the surface marker CD34 (r = 0.54, P =.001). In conclusion, this study shows that BCRP protein is expressed at low but variable levels in AML, especially in immature CD34(+) cells. BCRP was not consistently up-regulated in relapsed/refractory AML.