Pitfalls in molecular analysis for mismatch repair deficiency in a family with biallelic pms2 germline mutations.

Heterozygous germline mutations in the mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2 cause Lynch syndrome. Biallelic mutations in the MMR genes are associated with a childhood cancer syndrome [constitutional mismatch repair deficiency (CMMR-D)]. This is predominantly characterized by hematological malignancies and tumors of the bowel and brain, often associated with signs of neurofibromatosis type 1 (NF1). Diagnostic strategies for selection of patients for MMR gene analysis include analysis of microsatellite instability (MSI) and immunohistochemical (IHC) analysis of MMR proteins in tumor tissue. We report the clinical characterization and molecular analyses of tumor specimens from a family with biallelic PMS2 germline mutations. This illustrates the pitfalls of present molecular screening strategies. Tumor tissues of five family members were analyzed for MSI and IHC. MSI was observed in only one of the analyzed tissues. However, IHC analysis of brain tumor tissue of the index patient and his sister showed absence of PMS2 expression, and germline mutation analyses showed biallelic mutations in PMS2: p.Ser46IIe and p.Pro246fs. The same heterozygous mutations were confirmed in the father and mother, respectively. These data support the conclusion that in case of a clinical phenotype of CMMR-D, it is advisable to routinely combine MSI analysis with IHC analysis for the expression of MMR proteins. With inconclusive or conflicting results, germline mutation analysis of the MMR genes should be considered after thorough counselling of the patients and/or their relatives.

Immunotherapeutic strategies in neuroblastoma: antitumoral activity of deglycosylated Ricin A conjugated anti-GD2 antibodies and anti-CD3xanti-GD2 bispecific antibodies.

BACKGROUND: The antigen GD2 is selectively expressed on the surface of neuroblastoma cells, and is detected by the monoclonal antibody BW704. In this study, we describe the antitumoral capacity of the immunotoxin BW704dgA (BW704 conjugated to deglycosylated ricin A), and of anti-CD3xanti-GD2 bispecific antibodies that are capable of redirecting cytotoxic T cells towards neuroblastoma cells. We further investigate the in vivo activity of BW704dgA immunotoxins in a human neuroblastoma model in SCID mice. PROCEDURE: BW704dgA immunotoxins were injected i.p. as a single close (48 microg/mouse) on day 4 or divided into three doses on day 4, 5, and 6 after i.v. inoculation of the human neuroblastoma cell line IMR5-75. RESULTS: The mean survival time (MST) of BW704dgA treated animals was significantly increased (MST 49 days) compared to the control animals treated with irrelevant immunotoxin, unconjugated BW704, or control buffer (MST 33 to 39 days, P < 0.0001), without differences in the application schedules. Anti-CD3xanti-NP antibodies and NP-conjugated GD2-antibodies (BW704-NP) were used in a cytotoxicity assay with cytotoxic T-cells as effectors, and tracer labeled neuroblastoma cell line IMR5 as target cells. Anti-CD3xanti-NP antibodies, together with BW704-NP, showed increased cytotoxic activity compared to the incubation with CD3xanti-NP antibodies alone or with unconjugated anti-GD2. Additionally, a dose-dependent effect of NP-conjugated anti-GD2-antibodies upon the lysis of the target cells could be demonstrated. In this report, we describe two immunotherapeutic approaches using GD2 binding BW704 antibodies, modified as immunotoxin and a bispecific antibody, for the targeting and elimination of neuroblastoma cells. CONCLUSIONS: We envisage a combined immunotherapeutic regimen consisting of BW704dgA mediated stem cell purging, followed by a systemic treatment with anti-CD3xanti-GD2 bispecific antibodies in neuroblastoma.