Genotype-phenotype study of familial haemophagocytic lymphohistiocytosis due to perforin mutations.

BACKGROUND: PRF1 gene mutations are associated with familial haemophagocytic lymphohistiocytosis type 2 (FHL2). Genotype-phenotype analysis, previously hampered by limited numbers of patients, was for the first time performed by data pooling from five large centres worldwide. PATIENTS AND METHODS: Members of the Histiocyte Society were asked to report cases of FHL2 on specific forms. Data were pooled in a common database and analysed. RESULTS: The 124 patients had 63 different mutations (including 15 novel mutations): 11 nonsense, 10 frameshift, 38 missense and 4 in-frame deletions. Some mutations were found more commonly: 1122 G-->A (W374X), associated with Turkish origin, in 32 patients; 50delT (L17fsX22) associated with African/African American origin, in 21 patients; and 1090-91delCT (L364fsX), in 7 Japanese patients. Flow cytometry showed that perforin expression was absent in 40, reduced in 6 and normal in 4 patients. Patients presented at a median age of 3 months (quartiles: 2, 3 and 13 months), always with fever, splenomegaly and thrombocytopenia. NK activity was absent in 36 (51%), 5% in 4 (6%), "reduced" in 2 (3%) (not reported, n = 54). Nonsense mutations were significantly associated with younger age at onset (p<0.001) and absent natural killer activity (p = 0.008). CONCLUSION: PRF1 mutations are spread over the functional domains. Specific mutations are strongly associated with Turkish, African American and Japanese ethnic groups. Later onset and residual cytotoxic function are observed in patients with at least one missense mutation.

Novel Munc13-4 mutations in children and young adult patients with haemophagocytic lymphohistiocytosis.

Familial haemophagocytic lymphohistiocytosis (FHL) is a genetically heterogeneous disorder characterised by constitutive defects in cellular cytotoxicity resulting in fever, hepatosplenomegaly and cytopenia, and the outcome is fatal unless treated by chemoimmunotherapy followed by haematopoietic stem-cell transplantation. Since 1999, mutations in the perforin gene giving rise to this disease have been identified; however, these account only for 40% of cases. Lack of a genetic marker hampers the diagnosis, suitability for transplantation, selection of familial donors, identification of carriers, genetic counselling and prenatal diagnosis. Mutations in the Munc13-4 gene have recently been described in patients with FHL. We sequenced the Munc13-4 gene in all patients with haemophagocytic lymphohistiocytosis not due to PRF1 mutations. In 15 of the 30 families studied, 12 novel and 4 known Munc13-4 mutations were found, spread throughout the gene. Among novel mutations, 2650C-->T introduced a stop codon; 441del A, 532del C, 3082del C and 3226ins G caused a frameshift, and seven were mis sense mutations. Median age of diagnosis was 4 months, but six patients developed the disease after 5 years of age and one as a young adult of 18 years. Involvement of central nervous system was present in 9 of 15 patients, activity of natural killer cells was markedly reduced or absent in 13 of 13 tested patients. Chemo-immunotherapy was effective in all patients. Munc13-4 mutations were found in 15 of 30 patients with FHL without PRF1 mutations. Because these patients may develop the disease during adolescence or even later, haematologists should include FHL2 and FHL3 in the differential diagnosis of young adults with fever, cytopenia, splenomegaly and hypercytokinaemia.

bcr-abl rearrangement in adult T-lineage acute lymphoblastic leukemia.

The bcr-abl rearrangement in T-lineage ALL has been rarely described. In the last three years we studied all new patients with ALL at diagnosis by cytogenetic and molecular analysis. Three out of eleven T-lineage ALL patients presented the rearrangement and only one was Philadelphia positive.

Successful engraftment of allogeneic PBSC after conditioning with busulfan alone.

A 44-year-old woman with AML, while receiving a conditioning treatment with BU-CY for an allogeneic sibling transplant, developed septic shock with pulmonary embolism and heart failure. Conditioning was stopped at the end of the busulfan course and cyclophosphamide omitted. After antibiotics, dopamine and steroids the patient was allografted, using the donor's G-CSF-primed PBSC. She recovered her peripheral blood counts promptly and developed an acute GVHD grade II that responded to steroids. The DNA microsatellite analysis showed full donor engraftment up to a year from transplantation. This case suggests that the use of PBSC may facilitate engraftment in the absence of an effective immunosuppression during conditioning.

Transplantation of unmanipulated allogeneic PBSC: preliminary report on 24 patients.

To explore the feasibility and potential advantages of PBSC in allogeneic transplantation, we grafted 24 patients (age 16-57, median 37) with different hematologic diseases (ALL = 10, AML = 5, MM = 4, NHL = 2, CML = 1, MDS = 1, AA = 1), 23 HLA-identical to their siblings and 1 partially matched. Cells were collected from donors by apheresis after G-CSF 10 to 16 mg/kg/day for 4 to 5 days, and stored at 4 degrees C until infusion. The patients were conditioned with chemotherapy regimens including busulfan and cyclophosphamide in the majority of cases and received GVHD prophylaxis with CSA-MTX in all but two. The graft consisted of PBSC alone, with a median of 143.5 (range 18.1-358.9) x 10(4)/kg CFU-GM, 9.0 (range 3.3-18.0) x 10(6)/kg CD34+ cells and 2.8 (range 1.2 to 8.6) x 10(8)/kg CD3+ and cells. An ANC >0.0.5 x 10(9)/L was recovered on (median) day 13 (range 11-17), and a platelet count >50 x 10(9)/L on (median) day 13 (range 12-55) post graft. There was no correlation between CD34+ cells or CFU-GM number in the inoculum and time to hematologic reconstitution. Acute GVHD (grade II-IV) occurred in 10 out of 22 (45%), chronic GVHD in 10 out of 18 evaluable (55%) patients. We found no relationship between occurrence of acute or chronic GVHD and number of CD3+ cells in the graft. Four patients relapsed and 7 died after transplantation. Fifteen patients are currently alive and disease-free 67 to 710 (median 286) days from the graft. Allogeneic transplantation with unmanipulated PBSC ensures a fast and stable engraftment. Acute GVHD incidence and severity seems comparable to that of bone marrow transplantation, but there may be an increase in chronic GVHD, mainly of the extensive form.

Steroid-growth factor interaction in human prostate cancer. 2. Effects of transforming growth factors on androgen metabolism of prostate cancer cells.

The ability of human prostate cancer cells to metabolize androgens was assessed through administration of physiological concentration (0.5-10 nM) of tritiated testosterone (T) as precursor and one-step analysis of both T degradation and products' formation by reverse-phase HPLC and on-line radioactive detection after either 24 h or 72 h incubation. Overall, different prostate cancer cells degraded T quite differently, favoring alternatively reductive or oxidative metabolic pathways. In particular, both LNCaP and DU145 cells retained high levels of unconverted T, with a limited production of androstenedione and its 17-keto derivatives and relatively high amounts of dihydrotestosterone (DHT) and 3 alpha-androstanediol (3 alpha-diol). In contrast, PC3 cells quickly degraded T and exhibited high formation rates of androstenedione and 17-keto metabolites, while neither dihydrotestosterone nor 3 alpha-diol were detected after short or longer incubation times. The effects of both TGF alpha (50 ng/mL) and TGF beta 1 (5 ng/mL) on rates and direction of T metabolism were also explored. In LNCaP cells TGF alpha induced a significant (P < 0.04) decrease of the reductive metabolism of T with a corresponding enhancement of the oxidative pathway (P < 0.002), while TGF beta 1 did not significantly affect T metabolism. On the other hand, both reductive and oxidative pathways were only partially influenced by either growth factor in DU145 and PC3 cells, although TGF alpha significantly raised 5 alpha-androstanedione formation and reduced androsterone production in DU145 cells. All the above evidence was confirmed at both 24 h and 72 h or using increasing doses of TGF alpha and TGF beta 1, a peak activity of 50 ng/mL and 5 ng/mL, respectively, being generally encountered. Overall, our data suggest that TGFs may have a role in the growth regulation of hormone-responsive prostate tumor cells through changes of the intracellular contents of biologically active androgen metabolites.

Oxidative and reductive pathways of estrogens in hormone responsive and non-responsive human breast cancer cells in vitro.

In order to measure the formation and degradation rates of estradiol by human breast cancer cells, after assessing the biochemical basis of hormone responsiveness and growth response to estrogens, we considered both responsive, estrogen receptor (ER) positive, and non-responsive, ER-negative, breast cancer cell lines, i.e. MCF7, ZR75-1 and MDA-MB231. To this end, we employed a novel "intact cell" approach which allows us, after 24 h incubation, to analyze several enzyme activities in sequence, concurrently with the monitoring of labeled precursor degradation. Our investigations led to the following evidence: (a) the reductive activity of the 17 beta-hydroxysteroid oxoreductase (17 beta-HSOR) appears to be higher than the oxidative only in responsive, ER-rich MCF7 and ZR75-1 cells, as also previously observed by others; (b) this activity is, on the contrary, much lower in MDA-MB231 cells and other unresponsive, ER-poor breast cancer cell lines; (c) conversely, the oxidative activity shows an opposite pattern, being limited in MCF7 and ZR75-1 cells and much higher in MDA-MB231 cells. Overall, a 17 beta-HSOR reductive pathway prevails in both MCF7 and ZR75-1 cells, whilst the oxidative pathway is prevalent in MDA-MB231 cells, leading to a large formation of estrone that is no further metabolized, at least in the experimental conditions used. Our results may provide a likely explanation of previous data on the different estrogen content of breast tumor tissues.

Different conversion metabolic rates of testosterone are associated to hormone-sensitive status and -response of human prostate cancer cells.

The main goal of the present work was to compare the ability of human prostate cancer (PCa) cells to metabolize testosterone (T) in living conditions. To this end we studied three different human PCa cell lines (LNCaP, DU145 and PC3) having different hormone-sensitive status and capability of response to androgens. We used an original approach which allows the evaluation of conversion metabolic rates in growing cells after administration of labeled steroid precursor (presently T), at physiological concentrations (1-10 nM). Analysis of both precursor degradation and formation of several products was carried out using reverse phase-high performance liquid chromatography (RP-HPLC) and "on line" radioactive detection. Comparison of the three human PCa cells revealed that their metabolic aptitude differed in many respects: (i) rates of precursor degradation, (ii) different products' formation, and (iii) extent of conjugate production. In detail, PC3 cells quickly degraded T and exhibited high formation rates of androstenedione (A-4-ene-Ad); both DU145 and LNCaP cells mostly retained high levels of unconverted T, with a limited production of A-4-ene-Ad and its 17-keto derivatives (if any). Either LNCaP or DU145 cells generated a relatively high amount of dihydrotestosterone (DHT). In contrast, neither DHT nor its main metabolites were detected in PC3 cells at both short and longer incubation times. As expected, T degradation and A-4-ene-Ad production were highly correlated (r = 0.97; P < 0.03); similarly, A-4-ene-Ad and DHT formation showed a negative, significant correlation. Negligible production of conjugates was noted in both PC3 and DU145 cells, whilst it was remarkable in LNCaP cells (ranging from 43 to 57%). Overall, our data indicate that human PCa cells degrade T quite differently, favoring alternatively reductive or oxidative patterns of androgen metabolism.