Allogeneic stem cell transplantation after fludarabine, melphalan and thymoglobulin followed by early withdrawal of prophylactic immunosuppression in patients with acute lymphoblastic leukemia - update of single center study.

Presented are updated results of allogeneic hematopoietic stem cell transplantations (HSCTs) in 25 adult patients with acute lymphoblastic leukemia (ALL) in complete remission (CR) after a reduced intensity conditioning (RIC) combining fludarabine (150 mg/m2) and melphalan (140 mg/m2) with thymoglobulin (4.5 mg/kg or recently 4.0 mg/kg) followed by early initiation of reduction and withdrawal of prophylactic posttransplant immunosuppression. The median post-transplant follow-up was 32 (range, 4-87) months. Stable engraftment of donor's hematopoiesis was achieved in all patients. Acute graft versus host disease (GVHD) as well as the chronic one were equally observed in four cases (16%). Five patients (20%) relapsed with ALL in the median of 9 (range, 3-15) months after HSCT. During the above post-transplant follow-up, 4 recipients (16%) died. Disease progression and posttransplant complications were the cause of death in three (12%) and one (4%) of them, respectively. The probabilities of 2-year event-free (EFS) and overall survival (OS) were 70.3% (95% CI 51.9-88.7%) and 86.1% (95% CI 71.6-100%), respectively. Presented study confirmed our previously reported promising results and this approach may be considered as an alternative to traditional HSCTs performed in high-risk patients with ALL.

The quality of life following allogeneic hematopoietic stem cell transplantation - a multicenter retrospective study.

UNLABELLED: Although allogeneic haematopoietic stem cell transplantation (allo-HSCT) offers a unique curative potential, it may be connected with high treatment-related morbidity and mortality. Besides many organ complications, allo-HSCT may significantly affect quality of life (QOL). PATIENTS AND METHODS: Between January 2011 and December 2012, five hundred and ninety patients (pts) from 6 transplant centers in the Czech Republic filled in the questionnaire for the quantitative measurement of QOL using Functional Assessment of Cancer Therapy-General (FACT-G) version 4. Study cohort characteristics were as follows: 325 males, 340 pts received myeloablative conditioning, 383 pts received PBPC, representation of diagnoses; acute leukemia (n=270), bone marrow failure (n=36), chronic myeloid leukemia (n=74), myelodysplastic/myeloproliferative syndrom (n=110), lymphoproliferative disease (n=93). The median age at allo-HSCT was 43 years (range: 1.7 - 71.0), the median time from allo-HSCT to questionnaire completing was 3.8 years (range: - 0.2 - 21.6). The earliest allo-HSCT was performed in November 1989, the last in September 2012. In this retrospective study, we investigated the impact of various factors on the QOL after allo-HSCT: age, gender, diagnosis, type of conditioning, time from diagnosis to allo-HSCT, disease stage, graft type, donor type, time from allo-HSCT to questionnaire completing, GVHD, relapse. Only data from patients who were more than 3 months after allo-HSCT were used for the multivariate analysis. The overall results of the total FACT-G score (median=85.0; range: 29-108) as well as the results of each specific dimension - PWB (median=23.0; range: 5-28), SWB (median=24.0; range: 7-28), EWB (median= 19.0; range: 4-24), FWB (mean=21.0; range: 2-28) showed a value in the highest quartile of the possible evaluation. In multivariate analysis, an inferior QOL score was reported for patients with aGVHD (p=0.002), cGVHD (p<0.001), QOL decreased with increasing age (p=0.048) and increased with time elapsed since allo-HSCT (p<0.001).Allogeneic HSCT represents an important intervention into the overall integrity of the organism. In particular, the development of GVHD can cause very serious organ, but also mental problems which can significantly reduce the QOL. The QOL is steadily increasing with increasing interval from allo-HSCT but improvement and disappearance of these complications may take many years, and sometimes these effects may probably persist permanently.

A novel HLA-B allele, HLA-B*35:279, identified by sequencing-based typing in a Czech patient.

The identification of a novel HLA-B*35:279 allele in a Czech patient is described. This allele is identical to the B*35:03:01 variant except the G/A nucleotide exchange at position 652 of the HLA-B gene that corresponds to the amino acid substitution from valine to isoleucine in alpha 3 domain of the HLA-B antigen.

Prognostic factors to predict outcome of reduced intensity allogeneic haematopoietic cell transplantation for chronic lymphocytic leukemia.

Despite advances in immunochemotherapy CLL remains an incurable disease.. Allogeneic haematopoietic cell transplantation (HCT) has proven curative potential with ability to overcome adverse prognostic factors, however due to its toxicity it is generally perceived as the last option. We performed retrospective study to explore the outcomes and possible determinants of survival in the unselected consecutive cohort of 68 CLL patients (median age 59 years) receiving reduced intensity HCT as a part of salvage therapy in 2 Czech centers. The median interval from diagnosis to HCT was 69 months with median 3 of prior regimens, all patients were refractory to purine analogues. 49% of patients were transplanted with advanced (i.e. refractory or progressive disease or CR/PR>3), 38% had high risk cytogenetics. With median follow-up of 35 months the 3-year Kaplan-Meier survival probability for OS and PFS were 39% and 26%, respectively. Altogether 18 patients (26%) have relapsed or progressed. During the follow-up 41 patients died, 32 (78%) of transplant related factors (NRM), the others of relapse or disease progression.Univariate analysis failed to identify any clinical and pre- or post-transplant variables having clear prognostic significance for OS or PFS. The marginal OS advantage favoring HCT performed recently was detected (3-year OS: 31% for HCT until 2006 and 47% thereafter, p=0.0923). In multivariable hazards model only the female donors were associated with shorter OS (HR 2.278, p=0.016) whereas transplanted T-cell> 2.75x108/kg predicted inferior PFS(HR 1.957, p=0.035). No prognostic impact of donor type, age of donor and recipient, HLA mismatch, disease status pre-HCT, number of previous therapy lines, interval from dg. to HCT and number of transplanted hematopoietic cells was found. Our findings support the conclusion that alloHCT is able to overcome well known negative cytogenetic prognostic factors and that preferring male to female donors could be beneficial.

Allogeneic stem cell transplantation after fludarabine, melphalan and thymoglobulin followed by early withdrawal of prophylactic immunosuppression could be an effective approach to patients with acute lymphoblastic leukemia.

Presented are results of allogeneic hematopoietic stem cell transplantations (HSCTs) in 13 patients with high-risk acute lymphoblastic leukemia (ALL) in the first complete remission after a reduced intensity conditioning combining fludarabine (150 mg/m2) and melphalan (140 mg/m2) with thymoglobulin (4.5 mg/kg). The immunosuppressive effect of T-cell depletion reducing the risk of graft-versus-host disease (GVHD) and non-relapse mortality was compensated by early initiation of reduction and withdrawal of prophylactic immunosuppression aimed at maintaining effective immunological antileukemic control. The median post-transplant follow-up was 23 (range, 10-65) months. Stable engraftment of donor's hematopoiesis was achieved in all patients. Acute GVHD was observed in two cases (15.4%); the chronic form was not noted. Two patients (15.4%) relapsed with ALL at 3 and 16 months after transplantation. During the above post-transplant follow-up, all 13 recipients were alive, with a probability of 2-year disease-free survival of 76.9% (95% CI 51-100%). Although the results were obtained with a small pilot study group it may be assumed that, given the prognostic risk of most patients and the nearly 2-year median post-transplant follow-up, the approach may be considered as an alternative to HSCTs after traditional myeloablative or reduced conditioning regimens with standard GVHD prophylaxis.

Acute promyelocytic leukemia successfully treated also in elderly patients with significant comorbidities: a 20-year single-center experienc.

UNLABELLED: Acute promyelocytic leukemia is a unique entity among acute leukemias. Introduction of all-trans retinoic acid and, subsequently, arsenic trioxide in its treatment has markedly improved treatment outcomes for this once frequently fatal disease. Improved outcomes have also been observed in elderly patients, including those in whom standard intensive therapy is contraindicated because of comorbidities.In our center, a total of 60 APL patients were treated in 1993-2013, of whom 9 were aged 60 or more years. Although most of them had significant comorbidities at the time of diagnosis, eight achieved complete remission. At the time of the analysis, six patients were alive and in long-term remission; two patients died of causes other than APL. The median follow-up was 59 months.Included is case report of a patient with a high comorbidity score whose treatment was markedly reduced and individualized.Our experience shows that, in APL patients a curative approach is generally tolerated and should always be attempted regardless of age and comorbidities. KEYWORDS: APL - elderly patients - comorbidity.

Somatic mutation in acute myelogenous leukemia cells imitate novel germline HLA-A allele: a case report.

A somatic mutation of the human leukocyte antigen (HLA)-A gene revealed in tumour cells of acute myelogenous leukemia (AML) is described. A patient with AML and her siblings were routinely typed for HLA in order to find a suitable donor for haematopoietic stem cell transplantation. Sequencing-based typing of the initial patient's sample characterized by high proportion of blasts revealed unknown G/A exchange at position 781 of the HLA-A gene (exon 4) associated with HLA-A*02:01 allele. Importantly, this G781A variant was completely absent in the patient's remission sample obtained after the clearance of blasts. Our results are a reminder that HLA mutations in tumour cells may interfere with routine HLA typing and should always be considered, namely, in patients with haematological malignancies.

The outcome of allogeneic HSCT in older AML patients is determined by disease biology and not by the donor type: an analysis of 96 allografted AML patients ≥ 50 years from the Czech acute leukaemia clinical register (alert).

Older patients with AML have poor prognosis after chemotherapy and allo-SCT was historically limited to the young patients. In the multicentre retrospective study we analyzed 96 consecutive AML patients ≥ 50 years allografted with related (n=59) or unrelated (n=37) donor. The 2- year OS and DFS rates were 45 % and 42 % for the whole group. The corresponding figures for related patients were 48% and 42% whereas for unrelated 42% and 42%, respectively (OS p=0,721, DFS p= 0,896). The cumulative incidences of relapse (28% of all patients) and NRM mortality (26%) were low with no significant differences among related and unrelated cohorts. Multivariate analysis revealed the only major independent variables associated with an inferior OS were unfavourable cytogenetics (RR 3.36; CI 1.66-6.83; p=0.001) and advanced disease status (RR 2.30; CI 1.21-4.37; p=0.011). Unfavourable cytogenetics (RR 3.00; CI 1.50-5.99; p=0.002) and advanced disease at SCT (RR 2.27; CI 1.22-4.22; p=0.009) were also the only independent variables associated with inferior DFS. In conclusion, our analysis indicates that outcomes of allografted AML patients aged ≥ 50 years are determined by cytogenetic risk category and disease status at transplantation and not by the type of donor.

[Treatment of patients with relapsed/refractory Hodgkin lymphoma]. / Lécba pacientu s relabovaným/refraktérním Hodgkinovým lymfomem.

BACKGROUNDS: This retrospective study evaluated treatment outcomes in patients undergoing autologous stem cell transplantation (ASCT) for relapsed/refractory Hodgkin lymphoma (HL). PATIENTS AND METHODS: Overall, 194 HL patients treated with ASCT between 2000 and 2009 were analyzed. Survival was calculated using Kaplan-Meier method and differences in survival between subgroups with log-rank test. RESULTS: Best responses observed after ASCT: 124 complete and 35 partial remissions, 2 patients with stable disease and 33 relapses/progressions. During a median follow-up of 44 months, seventy patients after ASCT progressed/relapsed. Thirty-seven patients received salvage chemotherapy only with or without radiotherapy, 25 underwent allogeneic stem cell transplantation (SCT), 4 the second ASCT and 4 refused treatment. 5-year overall survival after ASCT was 71% and progression-free survival 54%. Median survival of the 70 patients relapsing after ASCT was 16.9 months. Median survival in patients after allogeneic SCT was 31.8 months and 12.4 months in patients treated with other modalities (p = 0.21). Overall mortality was 26.3% (51/194 patients): 13.4% progressions/relapses of HL and 12.9% non-relapse mortality. CONCLUSION: Efficacy of ASCT was confirmed in 54% progression-free survivors. Median survival after ASCT failure is relatively short. There is a slightly longer overall survival after allogeneic SCT, although not statistically significant when compared to other approaches.

FcγRIIIA receptor genotype does not influence an outcome in patients with follicular lymphoma treated with risk-adapted immunochemotherapy.

Antibody (rituximab) dependent cellular cytotoxicity is a key mechanism in killing CD20+ lymphoma cells. FcγRIIIA-158 V/F gene polymorphism results in expression of 3 variants of the FcγRIIIA receptor (FcγRIIIA) on cytotoxic lymphocytes with different receptor affinity. We studied 102 patients with newly diagnosed FL to assess whether the FcγRIIIA genotype influences outcome in patients treated with risk-adapted immunochemotherapy. The median age was 52 years (31-84); 90% of the patients had advanced (III/IV) clinical stages. The Follicular Lymphoma International Prognostic Index (FLIPI) scores were as follows: low 18.9%, intermediate 33.7% and high 47.4%. The front-line treatment was stratified according to the commonly used risk factors (FLIPI, beta-2-microglobuline and serum-Tyrosine-Kinase levels, bulky disease) into 3 treatment groups: (1) patients with FLIPI 0-1 treated with (R)-CHOP (51%), (2) patients under 60 (65) years of age with intermediate-risk disease (FLIPI 2) indicated for an intensive protocol (ProMACE-CytaBOM or sequential chemotherapy) (21%), and (3) patients under 60 (65) years with high-risk disease (FLIPI ≥3) treated with intensive chemotherapy plus autologous stem cell transplantation (28%). Rituximab was added to front-line chemotherapy in 59% of the patients. Generally, complete remission (CR) or unconfirmed CR was achieved in 85% of the patients, 11% had partial remission and 4% stable disease. Molecular CR (CRm) was achieved in 67.4% of 86 evaluable patients. Overall survival (OS) at 5 years reached 84% (95% CI 0.74-0.93); event-free survival (EFS) at 5 years was 58% (95% CI 0.45-0.71). The frequencies of FcγRIIIA-158 gene polymorphisms V/V, V/F and F/F were 8%, 50% and 42%, respectively. The FLIPI score distribution was not different in F/F patients as compared to V/F+V/V carriers (chi-square, P=0.7). The treatment modalities (treatment arm or rituximab administration) had the same distribution in V/V+V/F vs F/F patients (chi-square, P=0.16 and P=0.62, respectively). The CRm rates were similar in both subgroups of V/V+V/F vs F/F patients (chi-square, P=0.92). Survival curves for OS and EFS were not significantly different when comparing the subgroups of V/V+V/F vs F/F patients (P=0.28 and P=0.57, respectively). We found no difference in the quality of treatment response or survival after front-line immunochemotherapy between FcγRIIIA subgroups. FcγRIIIA polymorphism have no influence on the outcome of patients treated with risk-adapted chemotherapy with or without rituximab.

[Toxoplasmosis after immunosuppressive therapy--our experience]. / Toxoplazmová infekcia u pacientov po transplantácii krvotvorných buniek--skúsenosti jedného pracoviska

Toxoplasmosis is a parasitic disease associated with high mortality in immunocompromised patients. It may lead to life-threatening conditions, usually neuroinfections, pneumonia or disseminated disease. It may be potentially dangerous, especially for patients with prolonged lymphopenia or those treated with immunosuppressive drugs. In our centre, we have observed 3 cases of toxoplasmosis in patients after allogeneic haematopoietic stem cell transplantation (HSCT) (2.6% of 116 allo-HSCT patients since 2000) and one case after autologous HSCT (0.3% of 395 auto-HSCT patients since 1997). Toxoplasmosis is manifested by neurological symptoms including hemiparesis and paraparesis, cerebral salt-wasting syndrome (hyponatraemia and hypoosmolality), psychoorganic syndrome and signs of respiratory infection. The diagnosis was made by combining clinical signs and results of PCR and CT examinations. The patients were treated with high-dose pyrimethamine, clindamycin, co-trimoxazole and folic acid. Three of the four patients have survived with no signs of the disease. One patient died prior to treatment. The increasing use of highly immunosuppressive chemotherapy and conditioning regimens (including rituximab, fludarabine and anti-thymocyte globulin) is associated with a significant risk of toxoplasmosis. Variable manifestations, non-specific results of MRI or CT examinations and possibility of PCR negativity are the main obstacles to successful diagnosis.

Association of IL6 and CCL2 gene polymorphisms with the outcome of allogeneic haematopoietic stem cell transplantation.

Various polymorphisms of non-HLA genes have recently been investigated as candidate risk factors in allogeneic haematopoietic SCT (aHSCT). Our study aimed at exploring possible associations of IL6 and CCL2 single nucleotide polymorphisms (SNP) with aHSCT outcome. A total of 166 HLA-identical aHSCT pairs recruited in were genotyped for IL6 -174 G/C, IL6 -597 G/A, CCL2 -2518 A/G and CCL2 -2076 A/T SNPs by PCR with sequence-specific primers (PCR-SSP). The association between IL6 -174 GG genotype and increased risk of acute GVHD was found in whole study group (P=0.03) and in the subgroup of related aHSCT (P=0.01), association between IL6 -597 GG genotype and the occurrence of acute GVHD was detected only in the related aHSCT pairs (P=0.02). Furthermore, reduction in OS was revealed among recipients possessing IL6 -174(*)G allele in the group of related aHSCT pairs (P=0.04). Presence of CCL2 -2076 TT genotype was associated with decrease of OS (P=0.04) and increase of TRM (P=0.02) in patients transplanted by related donor. These results, in the context of previous findings, suggest that IL6 gene polymorphisms may be associated with aHSCT outcome, particularly in patients transplanted from a related donor.

[Selective depletion of alloreactive T cells and study of anti-tumor activity of specific T cell clones in patients with leukemia]. / Selektivní deplece aloreaktivních T lymfocytu a studium protinádorové aktivity specifických klonu T lymfocytu u pacientu s leuikémií.

BACKGROUND: Graft-versus-host disease (GVHD) is a severe complication of allogeneic transplantation of hematopoietic stem cells. Donor T cells play a major role in GVHD leading to the host tissue damage, mainly the skin, liver, and gastrointestinal tract. A selective depletion using an anti-CD25 immunotoxin can eliminate harmful alloreactive T cells while preserving other donor T cells with antileukemic and antiinfectious reactivity. PATIENTS AND METHODS: We performed 15 mixed lymphocyte reactions with clinical specimens from 12 patients with various types of leukemia (7x AML, 3x ALL, 1x CML, 1x CLL) and PBMC from 15 healthy volunteers from Transfusive station FN Brno Bohunice. RESULTS: In our experiments we have demonstrated, that antileukemic (GVL) effect of donor, especially CD4+ T cells was well preserved (7.46%), while unfavourable alloreactive (GVH) reaction of donor T cells was completely removed. The graft-versus-host (GVH) reactivation of donor cells was negligible ever after repeated stimulation with irradiated patient's PBMC. CONCLUSION: We have shown that anti-CD25 immunotoxin (IT), RFT5-SMPT-dgA, launched against alpha chain for human interleukin 2 (IL-2), led to long-term selective depletion of alloreactive donor T cell clones while their antileukemic activity was well preserved. Base on our results the clinical phase I/II study was designed. This study was initiated in year 2007 in three clinical centers in Czech Republic.

Association of IL-6 gene polymorphism with the outcome of allogeneic haematopoietic stem cell transplantation in Czech patients.

Interleukin-6 (IL-6) is an important pro-inflammatory mediator implicated in immune-mediated complications of allogeneic haematopoietic stem cell transplantation (aHSCT). In accord with previous reports, this preliminary study on 56 donor-recipient pairs revealed IL-6-174 single nucleotide polymorphisms as a risk factor for the development of acute graft-versus-host disease and decreased survival after aHSCT.

High-dose therapy and autologous stem cell transplantation in patients with diffuse large B-cell lymphoma in first complete or partial remission.

Improved survival has been observed in poor-risk diffuse large B-cell lymphoma (DLBCL) patients treated with high-dose therapy (HDT) followed by autologous stem cell transplantation (ASCT) in first complete remission. Retrospective studies have suggested that HDT with ASCT can improve survival also in partial responders but some doubts about the advantage of intensive therapy in such patients still remain. We evaluated retrospectively the results of HDT and ASCT in 55 patients with confirmed DLBCL treated between May 1999 and July 2006. Thirty-six patients (65%) showed partial remission (PR) and 19 patients (35%) reached complete remission (CR) after induction treatment with (44%) or without (56%) concomitant rituximab (R) immunotherapy. After HDT and ASCT, 69% of patients fulfilled the criteria of CR, 22% had unconfirmed CR (CRu), 7% remained in PR and 1 patient (2%) relapsed. Twenty patients in PR after the induction treatment reached CR after ASCT, 12 other PR patients achieved CRu. The 5-year event-free survival (EFS) of the 55 transplanted patients was 76% (95% confidence interval /CI/, 63% to 89%) and the 5-year overall survival (OS) was 85% (95% CI, 73% to 97%). The EFS and OS rates differed significantly only between patients younger than 40 years and older groups (p=0.022 and p=0.046, respectively). On univariate analysis of prognostic factors, EFS and OS were not affected by any of the following: age, sex, stage, subtype of DLBCL, initial lactate dehydrogenase, beta-2-microglobulin and serum thymidine kinase levels, International Prognostic Index (IPI) and age-adjusted IPI scores, induction treatment with or without rituximab and type of primary therapeutic response (CR vs PR). These results show that first-line HDT and ASCT for adults up to the age of 65 years with poor-risk DLBCL is a feasible and effective treatment option even in the era of R-chemotherapy in CR as well as for patients in PR.

[Chemotherapy of BOVAPEC in the primary treatment of Hodgkin's lymphoma intermediate stages]. / Chemoterapie BOVAPEC v primární lécbe stredne pokrocilých stadií Hodgkinova lymfomu.

DESIGN: Chemotherapy of BOVAPEC is the modification of temporary intensified Stanford V protocol, an effective primary treatment of advanced Hodgkin's lymphoma (HL) in spite of limited toxicity. Nitrogen mustard was substituted by less myelotoxic cyclophosphamide and the protocol has been used in the treatment of patients with an intermediate stage of HL. METHODS: The primary treatment with BOVAPEC was started in 62 patients. Complete chemotherapy schedule was administered to 60 patients (97%) and the median of its overall duration was 13 (12-18) weeks. 31 patients (50 %) underwent adjuvant "involved field" radiotherapy (RT). The median of posttherapeutic follow-up was 37 (range 8-85) months. RESULTS: During the treatment, a neutropenia of grade 3 and 4 was observed in 14 patients (23%) but without the development of any serious infectious complications. The manifestation of early non-hematological toxicity did not overcome grade 2.58 patients (94%) achieved the complete remission of HL. A relapse was observed in 11 cases (19%) and estimated five years disease-free survival (DFS) is 72%. The combination of BOVAPEC and RT in primary treatment was associated with higher probability of five years DFS but actually without statistical significance (88% vs. 58%; p = 0.08). CONCLUSION: The BOVAPEC regimen with its acceptable toxicity may represent effective primary therapeutic approach to the patients with the intermediate stage of HL. Adjuvant RT is essential in all patients diagnosed with nodal bulk and/or residual lymphadenomegaly.

A single amino acid exchange shifts the serological reactivity of the novel HLA-B*4442 allele product from HLA-B44 to HLA-B21.

A novel HLA-B (human leukocyte antigen-B) allele, HLA-B*4442, was identified both in a Czech patient with leukaemia and in his mother. The presence of a novel allele was initially suspected because conflicting results were obtained by serological and DNA typing techniques. The HLA typing using the polymerase chain reaction-sequence-specific primers (PCR-SSP) at the two-digit level indicated an allele belonging to the HLA-B*44 group, whereas serological typing indicated HLA-B21. Typing with PCR-sequence-specific oligonucleotides (PCR-SSO) resulted in a unique reaction pattern that could not be assigned to a known allele, PCR-SSP typing at the four-digit level did not match any known B*44 allele, either. The sequencing-based typing of the HLA-B locus then revealed the novel B*4442 allele that is identical with B*4405 except a single C-->G nucleotide exchange at position 572. This exchange results in an amino acid substitution from serine to tryptophan at position 167 of the expressed HLA-B protein. The B21 serological reactivity of the novel B*4442 allele product was confirmed by employing an additional serological panel of typing sera. Our findings support previous reports claiming that serine at the position 167 in the alpha-2 domain of the HLA-B protein is a major determinant of the HLA-B44(12) serological epitope.

A novel HLA-B*420502 allele identified by PCR-SSO/SSP routine typing and confirmed by Sequencing-based typing.

A novel human leukocyte antigen-B (HLA-B) allele, B*420502, was identified in a patient with leukemia (Caucasoid, Czech ancestry) and his mother during intrafamily search for the hematopoietic stem cell donor. The novel allele was initially detected by HLA typing at low resolution using both sequence specific primers and sequence specific oligonucleotides techniques that resulted in unique reaction patterns. The alleles of the HLA-B locus were separated by the haplotype-specific extraction technique. Sequencing of those alleles revealed a novel allele, B*420502, that is identical with B*420501 except a T-->G exchange (synonymous mutation) at position 618.

High incidence of unbalanced chromosomal changes in mantle cell lymphoma detected by comparative genomic hybridization.

Comparative genomic hybridization (CGH) was carried out in 30 mantle cell lymphoma (MCL) patients at the time of diagnosis. CGH results were supported by conventional cytogenetics (CC), FISH, molecular genetic PCR methods and 2 patients were examined by array CGH. Using all cytogenetic, molecular cytogenetic and PCR methods, chromosomal changes were detected in 28 (93%) patients. Using CGH, unbalanced chromosomal changes were detected in 24 (80%) cases. The most frequent aberrations were losses of 1p (8 cases), 8p (10 cases), 9q (6 cases), 11q (11 cases), 13q (10 cases) and 17p (9 cases), and gains of chromosome 3 and 3q (12 cases) and 8q (7 cases). Total number of 60 gains and 116 losses were detected. The primary chromosomal change t(11;14) was detected using FISH and/or PCR in 20 (66.6%) patients, and in 9 of them, the breakpoint was determined using PCR in the major translocation cluster (MTC). The evaluation of the frequencies of CGH changes in groups of patients with and without t(11;14) revealed the differences only in losses 6q and 9q, which were only found in patient with t(11;14). An important result was obtained using array CGH method. In a patient without the primary t(11;14), the gain of CCND1 gene was found. Our results show high heterogeneity of the additional chromosomal changes in MCL cases, which involved specific chromosomal subregions. We did not confirm the importance of subdividing of MCL cases with and without t(11;14). Also, statistical significance in survival rates between both subgroups was not confirmed.