Performance of BRCA1/2 mutation prediction models in male breast cancer patients.

To establish whether existing mutation prediction models can identify which male breast cancer (MBC) patients should be offered BRCA1 and BRCA2 diagnostic DNA screening, we compared the performance of BOADICEA (Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm), BRCAPRO (BRCA probability) and the Myriad prevalence table ("Myriad"). These models were evaluated using the family data of 307 Dutch MBC probands tested for BRCA1/2, 58 (19%) of whom were carriers. We compared the numbers of observed vs predicted carriers and assessed the Area Under the Receiver Operating Characteristic (ROC) Curve (AUC) for each model. BOADICEA predicted the total number of BRCA1/2 mutation carriers quite accurately (observed/predicted ratio: 0.94). When a cut-off of 10% and 20% prior probability was used, BRCAPRO showed a non-significant better performance (observed/predicted ratio BOADICEA: 0.81, 95% confidence interval [CI]: [0.60-1.09] and 0.79, 95% CI: [0.57-1.09], vs. BRCAPRO: 1.02, 95% CI: [0.75-1.38] and 0.94, 95% CI: [0.68-1.31], respectively). Myriad underestimated the number of carriers in up to 69% of the cases. BRCAPRO showed a non-significant, higher AUC than BOADICEA (0.798 vs 0.776). Myriad showed a significantly lower AUC (0.671). BRCAPRO and BOADICEA can efficiently identify MBC patients as BRCA1/2 mutation carriers. Besides their general applicability, these tools will be of particular value in countries with limited healthcare resources.

Proliferation of precursor B-lineage acute lymphoblastic leukaemia by activating the CD40 antigen.

No reliable culture system exists for B-lineage acute lymphoblastic leukaemia (ALL). Recently we found that many different mature B-cell malignancies proliferate upon stimulation via the CD40 antigen, and this led us to investigate whether a similar CD40 activation on ALL cells could also induce proliferation. First, we measured CD40 expression in 21 ALL cases; all were CD40+, although mostly weak. Next, we triggered the CD40 antigen by anti-CD40 antibodies and by a CD40 ligand-expressing cell line. In addition, we measured the influence of IL-3, IL-4 and IL-7 with and without these stimuli. In 8/10 cases proliferation, measured by 3H-thymidine incorporation, could be induced after CD40 crosslinking, especially in the presence of IL-3. Stimulation via the CD40 ligand was more successful than using crosslinked anti-CD40 antibodies. IL-4 inhibited the spontaneous proliferation found in three cases, but stimulated proliferation after CD40 crosslinking. IL-7 did not contribute to proliferation. Morphology, immunophenotyping and surface marker analysis, combined with DNA flow cytometry confirmed that the proliferation found could be ascribed to the ALL cells. In conclusion, B-lineage ALL cases are CD40+, and many can be cultured using CD40 stimulation and IL-3.

Proliferation of B cell malignancies in all stages of differentiation upon stimulation in the 'CD40 system'.

Stimulation of the CD40 antigen on normal B cells by crosslinking of anti-CD40 mAbs via their Fc receptor using a Fc gamma RII(CD32)-transfected mouse fibroblast cell line ('CD40 system') results in activation and proliferation. Not only normal B cells, but also malignant B cells fitting in the low-grade malignancy category such as chronic lymphocytic leukemia (CLL), hairy cell leukemia and follicular lymphoma could be induced to proliferation upon CD40 stimulation. Here, the 'CD40 system' has also been used to culture intermediate and high grade malignancies. Proliferation was measured by 3H-thymidine incorporation and cell counting after culture. Time curves showed that at day 7 most cultures were optimal. By flow cytometry, morphology and assessment of light chain restriction the monoclonal nature of the cultured B cells was proven. We confirmed that B cell malignancies with a more slowly evolving course, such as CLL (n=11), PLL (n=5), and low-grade NHL (immunocytoma and follicular cb/cc n=9), could successfully be cultured in the 'CD40 system'. In contrast, four out of seven cases of mantle cell lymphoma did not proliferate. Cases of precursor B lineage ALL (n=7), high grade NHL (n=3) and multiple myeloma (n=10) showed a heterogenous growth pattern. We conclude that the 'CD40 system', although not always successful, is a useful tool to culture a whole variety of B cell malignancies.

Transfer of specific immunity from donor to recipient of an allogeneic bone marrow graft: evidence for donor origin of the antibody producing cells.

A rapid recovery of specific humoral immunity in the recipient of an allogeneic bone marrow transplantation (BMT) can be observed after immunization of the donor before graft sampling. This has been attributed to transfer of specific immunity from donor to recipient. However, to maintain the concept of transfer the origin of the antibody producing cells in the recipient after BMT must be demonstrated. To this end, donor-recipient pairs with differences in Gm-allotypes were selected and immunized before BMT with the neo-antigen Helix pomatia haemocyanin (HPH) according to three immunization protocols. Additionally, the recipients were immunized at day 42 after BMT. Serum samples were weekly obtained from the recipients in the first 100 d after BMT. The origin of HPH-specific antibody producing cells was assessed by two approaches: (1) determination of the Gm-allotypes of anti-HPH antibodies within a distinct IgG subclass, (2) analysis of anti-HPH antibody spectrotypes by isoelectric focusing combined with immunoblotting. The results obtained with these two approaches show concordance in most instances and led to the conclusion that the antibody producing cells are of donor origin.

Transfer of specific immunity from donor to recipient of an allogeneic bone marrow graft: effect of conditioning on the specific immune response of the graft recipient.

Transfer of specific immunity was investigated in a group of 28 paediatric and adult leukaemia patients during the first 100 d after allogeneic bone marrow transplantation (BMT). These patients and/or their donors were immunized 7-13 d before transplantation with the recall antigen tetanus toxoid (TT) and the neo-antigen Helix pomatia haemocyanin (HPH). The recipients were booster immunized with both antigens at day 42 after transplantation. Transfer of a primary IgM and IgG response to HPH was successful in most paediatric and adult patients, but transfer of a secondary response to TT was established in only a few paediatric recipients. After booster immunization at day 42 most paediatric recipients responded with a rise in serum antibody titre to HPH as opposed to only two of 18 adult recipients. This incapability of the adult recipients to mount a secondary immune response may be related to their conditioning regimen which included Campath-IG in vivo. The results from this study indicate that transfer of immunity against recall- and neo-antigens is possible. However, the establishment of long-term memory may be affected by the regimen used to condition the graft recipient.