Distinguishing myelodysplastic syndromes (MDS) from idiopathic cytopenia of undetermined significance (ICUS): HUMARA unravels clonality in a subgroup of patients.

BACKGROUND: Patients not fulfilling minimal criteria for myelodysplastic syndromes (MDS) but presenting with persisting cytopenia(s) not attributable to a haematological or non-haematological disease are defined as 'idiopathic cytopenia of undetermined significance' (ICUS). DESIGN AND METHODS: We retrospectively analysed 67 of 3504 patients from our MDS Registry fulfilling the criteria for ICUS. Furthermore, we used the human androgen receptor gene-based assay (HUMARA) to look for clonality. RESULTS: Of all 67 patients, 66% had unilineage, 18% bilineage and 12% trilineage cytopenias. The majority of patients (67%) presented with anaemia. Median overall survival was 44 months (range: 1-199 months). In the entire group, eight patients (12%) developed acute myeloid leukaemia (AML). Of the 23 patients eligible for HUMARA, 17 had non-clonal X-chromosome inactivation patterns, while 6 patients showed clonal patterns. Two of these six patients developed AML indicating that a clonal stem cell disorder was the reason for the anteceding cytopenia, while there was no AML observed among the 17 patients with non-clonal patterns (P = 0.013). CONCLUSIONS: Since some of the ICUS patients had a clonal bone marrow disease when presenting with cytopenia(s) and 8 of 67 patients with ICUS later developed AML, we recommend to follow these patients thoroughly. As demonstrated here, HUMARA can facilitate the discrimination between ICUS and a 'manifest' MDS.

The ratio between CD4+ and CD8+ cells in the peripheral blood of patients with hematological malignancies is not altered by thalidomide.

Thalidomide is thought to have anti-angiogenic and immunomodulatory properties, including suppression of tumor necrosis factor-alpha, effects on interleukins and interferons, down-regulation of some cell adhesion molecules, and changes in the proportion of lymphocyte subsets. It is unclear whether the clinical response to thalidomide in patients with multiple myeloma (MM), idiopathic myelofibrosis (IM), and myelodysplastic syndromes (MDS) is related to its ability to inhibit angiogenesis or its immunomodulatory effects. We examined the effect of thalidomide on T-lymphocyte subsets in 18 patients with MDS, 6 patients with MM, 4 patients with IM, and 3 patients with angioimmunoblastic lymphoma (AILD). These patients had either a relapse or progressive disease following cytotoxic chemotherapy including high-dose chemotherapy with autologous stem cell support. Thalidomide was first administered at 100 mg/day p.o. and increased to 400 mg/day. T-lymphocyte subsets (CD4+, CD8+) were measured by fluorescence-activated cell sorter (FACS) before and during treatment with thalidomide. Twenty-six of 31 patients responded to thalidomide, most of them achieving partial remission. The median concentration of CD4+ cells was 443/microl, the median of CD8+ cells was 359/microl (CD3 992/microl). In our cohort, no significant changes in absolute numbers or proportions of CD3+ (P = 0.12), CD4+ (P = 0.668), or CD8+ (P = 0.143) cells were observed following the treatment with thalidomide. Although the CD4/CD8 ratio declined from 1.6 to 1.0 during 3 months of thalidomide treatment, this had no statistical significance (P = 0.1). Our findings show that an effect of thalidomide on the T lymphocytes studied is unlikely to be of major importance for the clinical effects.

Transplantation of allogeneic CD34+-selected cells followed by early T-cell add-backs: favorable results in acute and chronic myeloid leukemia.

BACKGROUND: The aim of this study was to investigate preservation of anti-leukemic activity and protection from opportunistic infections after transplantation of allogeneic + cells in patients with hematologic malignancies and bad prognosis. Methods Thirty-three patients [median age 42 years, range 23-55 years, diagnosis AML/myelodysplastic syndrome (MDS) 14, ALL nine, CML seven and multiple myeloma (MM) three] received myeloablative conditioning followed by infusion of selected CD34+ cells from matched unrelated donors (31) or HLA-identical siblings (two). Early donor lymphocyte infusions (DLI; 0.5 and 1.0 x 10(6) CD3+ cells/kg) were given while patients were on immunosuppressive therapy. RESULTS: Ninety-seven per cent of patients engrafted and 24 of 29 patients surviving more than 30 days received at least one pre-emptive DLI. Three patients (10%) developed acute (a)GvHD (two grade I-II, one grade III-IV) spontaneously, and 16 patients (67%) developed aGvHD after DLI (12 grade I-II, four grade III-IV). Eight of 24 evaluable patients developed chronic (c)GvHD (33%, six limited, two extensive). After a median follow-up of 590 days (range 138-1610 days) 18 patients were alive (55%), 16 in complete remission (CR), one in hematologic and one in molecular relapse. Seven patients died after relapse (21%) and eight died from transplantation-related causes (24%). Patients with myeloid malignancies had a significantly better survival than patients with ALL or MM (74%+/-10 vs. 30%+/-13, P<0.05). DISCUSSION: Early pre-emptive low-dose DLI following transplantation of selected CD34+ cells from unrelated donors after myeloablative conditioning is feasible and effective without undue toxicity, especially in patients with myeloid malignancies.

Clinical, morphological, cytogenetic, and prognostic features of patients with myelodysplastic syndromes and del(5q) including band q31.

We analyzed data of 76 consecutive patients with myelodysplastic syndrome (MDS) and isolated del(5q) (n=66) or del(5q) plus one additional chromosomal abnormality (n=10) included in our MDS database over the last 26 years. The median age of our patient population was 66.8 years. The male to female ratio was 1:1.7. In all, 14 patients (18%) had advanced MDS with an increased medullary blast count. A total of 17 patients (22%) had significant dysplasia in the nonmegakaryocytic cell lines. Nearly half of the study population showed erythroid hypoplasia in the bone marrow. The projected median survival of patients with isolated del(5q) is 146 months for a median follow-up of 67 months. Patients with an increased medullary blast count and those with an additional chromosomal abnormality have a significantly shorter overall survival (24 and 45 months, respectively) than patients with isolated del(5q). We did not find survival differences for different cytogenetic breakpoints, nor did the amount of dysplasia have an impact on survival in our population. In total, 29 patients have died. Deaths occurred primarily due to transformation into acute leukemia, infection, or cardiac failure. Our data support the current definition of a separate entity of MDS with del(5q) that has been suggested by the World Health Organization.

The emergence of a C/EBPalpha mutation in the clonal evolution of MDS towards secondary AML.

Recently, mutations in the transcription factor CCAAT/ enhancer binding protein alpha (C/EBPalpha) have been described in acute myeloid leukemia (AML). We performed a mutational analysis of the C/EBPalpha gene in the myelodysplastic syndromes and AML with antecedent MDS. No mutations were found in patients with refractory anemia (0/27), refractory anemia with ringed sideroblasts (0/7), refractory anemia with excess of blasts (RAEB 0/16) or chronic myelomonocytic leukemia (CMML 0/5). One out of 13 patients with RAEB-T/AML secondary to MDS showed a mutation in the C/EBPalpha gene. In this patient a 4 bp insertion disrupted codon 69 in one allele. This novel +1 frame shift is predicted to result in a truncated protein of 107 amino acids. However, the dominant protein translated was the C/EBPalpha isoform p30, which was previously shown to inhibit the DNA-binding and transactivation properties of C/EBPalpha p42. Interestingly this mutation could not be detected at diagnosis in the initial RAEB and RAEB-T stage. The mutation appeared at relapse after chemotherapy for RAEB-T. We conclude that the C/EBPalpha mutation was not essential for the initial blast accumulation. The emergence of a bast clone carrying a C/EBPalpha mutation at relapse indicates that this mutation may confer a growth advantage in a myeloid cell with an established differentiation block.

Favourable response to antithymocyte or antilymphocyte globulin in low-risk myelodysplastic syndrome patients with a 'non-clonal' pattern of X-chromosome inactivation in bone marrow cells.

OBJECTIVE: Antithymocyte and antilymphocyte globulin (ATG/ALG) have a therapeutic effect in about 30% of patients with myelodysplastic syndromes (MDS). We were interested to know whether responding patients achieve clonal or polyclonal remissions. PATIENTS: Ten women with low-risk MDS received either ALG or ATG. Before treatment and 3, 6, and 12 months later, X-chromosome inactivation patterns of peripheral blood T lymphocytes were compared with those of peripheral blood granulocytes or bone marrow cells, using the human androgen receptor gene assay and the phosphoglycerate kinase-1 assay. RESULTS: Six women did not respond to therapy. Prior to treatment, four of them had a monoclonal, one had an oligoclonal, and one had a skewed X-chromosome inactivation pattern (XCIP). Four patients responded to ATG/ALG. Three of them were informative in our X-inactivation assays, and showed a non-clonal XCIP which did not change significantly after treatment with ATG/ALG. CONCLUSION: A non-clonal XCIP in the bone marrow was associated with a response to ATG/ALG. Non-clonal XCIPs do not necessarily imply that there is no pathological clone. By definition, they just indicate that there is no evidence of a clone contributing more than 50% of cells in a sample. Non-clonal XCIPs may therefore be attributable to incomplete clonal expansion. This, in turn, might be explained by a vigorous immune attack against the MDS clone, which simultaneously causes collateral damage in the remaining normal haemopoiesis. In such patients, ATG/ALG may improve normal haemopoiesis by relieving the immunological pressure on the innocent bystanders.

Testicular infiltration in acute myeloid leukemia with complex karyotype including t(8;21).

Testicular infiltration is a well-known complication in acute lymphoblastic leukemia. In acute myeloid leukemia (AML), it has rarely been described and preferably occurred in cases with myelomonocytic or monoblastic differentiation. We report on a patient with AML with complex karyotype including translocation t(8;21) who presented with testicular infiltration at the time of his third bone marrow relapse. Cytological analysis of the specimen showed infiltration with blasts displaying the typical morphology of AML with translocation t(8;21) and comparable to those detected in the bone marrow. Fine needle aspiration cytology might suffice in these cases and should be performed if testicular involvement is suspected.

Angioimmunoblastic lymphadenopathy (AILD) may respond to thalidomide treatment: two case reports.

The histological morphology of AILD-type T-cell lymphoma shows proliferation of small, arborizing high endothelial venules. Patients typically have generalized lymphadenopathy, fever, weight loss, skin rash, polyclonal hypergammaglobulinemia and autoimmune phenomena, and are susceptible to infections. The clinical course is usually aggressive. About 30% of patients have an initial remission on steroids alone, but most require some form of cytotoxic chemotherapy. Median survival ranges from 15 to 24 months, and it appears that the disease cannot be cured. We have used anti-angiogenetic therapy with thalidomide in two AILD patients and observed remarkable responses.

Thalidomide for the treatment of patients with myelodysplastic syndromes.

We examined the efficacy of thalidomide in 34 patients with myelodysplastic syndromes (MDS): five RAEB-T, four RAEB, three CMML, six RARS, and 16 RA. Patients belonged to the following cytogenetic groups: 15 complex abnormal karyotypes, 12 normal karyotypes, four cases with 5q- as sole anomaly and three single aberrations. The median thalidomide dose was 400 mg/day (25/34 patients). Four patients discontinued the study after less than 5 weeks, because of fatigue (three) or skin rash (one). One patient died of heart failure after 4 weeks. In the remaining 29 patients (median follow-up: 13 months), treatment responses were classified according to the IWG criteria. Six patients (four RA, two CMML) showed progressive disease (five with transformation into AML) and four patients showed stable disease. Hematological improvement (HI) was observed in 19 patients. Nine of the responders (three RA, one RARS, two RAEB, three RAEB-T) achieved partial remission with granulocytes > or = 1500/microl, Hb > 11 g/dl and platelets > or =100,000/microl. Four patients (one RARS, one CMML, one RAEB, one RAEB-T) had a major response, with platelet and RBC transfusion independence. Six patients (five RA, one RARS) showed minor responses (three HI-E, two HI-E+HI-P, one HI-E+HI-N). Hematological improvement occurred after a median of 2 months of thalidomide treatment. Two patients (RAEB-T) relapsed after a partial remission lasting 8 and 16 months, respectively. In summary, a therapeutic benefit was achieved in 19 of 34 study patients (56%).

Acute basophilic leukemia.

Acute basophilic leukemia has recently been included into a revised classification of acute leukemias proposed by the WHO panel. Due to the rarity of the disease, consistent diagnostic criteria are lacking. We report on two cases of acute basophilic leukemia that occurred in our department during the last 10 yr. We focus on their clinical, morphological and cytogenetic presentation. Both patients were >60 yr of age, and presented in good clinical condition with alterations to their full blood count. None had cutaneous symptoms such as erythema or urticaria. Cytogenetic analyses in the first patient showed a normal karyotype, while the second displayed a translocation t(2;6); (q23?4;p22?3), as well as a del (12)(p11). Earlier observations have linked bone marrow basophilia either to a deletion of the short arm of chromosome 12 (p11-13), to translocations involving the long arm of chomosome 6 at 6q23 or to the translocation t(6,9); (p23;q34). However, other translocations involving chromosome 6p23 have not been described before. Treatment of our patients consisted of supportive treatment in the one with normal karyotype and aggressive chemotherapy in the other patient. Both patients died within one year after diagnosis due to progressive or recurrent leukemia.

Treatment of severe steroid refractory acute graft-versus-host disease with infliximab, a chimeric human/mouse antiTNFalpha antibody.

Acute graft-versus-host disease (aGVHD) is a serious complication of allogeneic peripheral blood stem cell transplantation (PBSCT). Patients with severe aGVHD not responding to treatment with steroids have a poor prognosis. We treated four patients with severe aGVHD refractory to steroids with infliximab, a chimeric human/mouse antiTNFalpha antibody. Patients (CML 2, MM 1, AML 1) developed grade III-IV GVHD at a median of 34 days (range 15-76) after myeloablative PBSCT (two), donor lymphocyte infusion for relapsed CML (one) or non-myeloablative PBSCT (one), respectively. All patients had severe intestinal involvement in addition to skin and/or liver disease and had received treatment with high-dose steroids (four) for a median of 11 days (range 5-17) in addition to CsA (four) and MMF (three). Infliximab (10 mg/kg) was given once a week until clinical improvement. In three of four patients a complete resolution of diarrhea and significant improvement of skin and liver disease were observed. Two patients received one, one patient two and one patient three infliximab infusions. At present two patients are alive >200 days after therapy, one with limited cGVHD. Two patients died, one of progressive malignant disease without GVHD and one of refractory GVHD. Infliximab is apparently an active drug for the treatment of aGVHD.

Validation of the WHO proposals for a new classification of primary myelodysplastic syndromes: a retrospective analysis of 1600 patients.

In 1982, the French-American-British (FAB) cooperative group proposed a classification of myelodysplastic syndromes (MDS) based on morphological features in blood and bone marrow, namely on medullary and peripheral blast count, Auer rods, ring sideroblasts and the number of monocytes in the peripheral blood. This classification has been used for numerous studies regarding morphology, prognosis and treatment of MDS. Some details of this morphological classification remained unclear, and some patients were unclassifiable. A working group of the World Health Organization (WHO) recently proposed a new classification of MDS, based on a significant modification of the original FAB proposals. CMML and RAEB-T were removed from the MDS classification and RAEB was split into two groups with medullary blast counts below and above 10%. In addition, a group of patients with less than 5% medullary blasts but evidence of multilineage dysplasia was defined. MDS patients with 5q- as the sole chromosomal anomaly were also considered a separate group. The aim of the present study was to validate the new classification with respect to prognostic importance, and to correlate it with cytogenetic and hematological features in a large series of patients (n=1600) with a long-term follow up. We were able to confirm a significant difference in prognosis between RAEB I and RAEB II, as well as a difference between refractory anemia and multilineage dysplasia. Furthermore, patients with 5q- anomaly had a much better prognosis than other WHO subtypes, but this was only true for patients with a medullary blast count below 5%. In summary, the WHO classification appears to define morphological subgroups that are more homogeneous with respect to prognosis than the FAB subtypes.

["Field cancerization"--an additional phenomenon in development of colon tumors? K-ras codon 12 mutations in normal colonic mucosa of patients with colorectal neoplasms]. / "Field cancerization"--ein zusätzliches Phänomen in der Entwicklung von Colontumoren? K-ras-Codon-12-Mutationen in normaler Colonmucosa von Patienten mit colorectalen Neoplasien.

INTRODUCTION: The c-K-ras2-gene (K-ras) encodes the p21ras protein, which participates in the transduction of mitogenic signals from the cell surface to the nucleus. About 40-50% of colorectal cancers bear a point mutation of the K-ras codon 12 within exon I, leading to activation of the K-ras oncogene. METHODS: Using a polymerase chain reaction (PCR)-based two-step restriction fragment length polymorphism (2sRFLP)-technique, we assessed the frequency of point mutations in the first or second base of K-ras codon 12 in patients suffering from colorectal carcinoma, adenoma, and their normal colon mucosa, respectively. RESULTS: Similar to other investigators, we found mutations in 7 out of 17 (41%) colorectal carcinomas and 1 out of 4 (25%) adenomas. Using this very sensitive 2sRFLP technique (detection level = 0.2% of mutated cells), we were able to find K-ras codon 12 mutations in normal mucosa in 4 out of 18 (22.2%) patients with colorectal neoplasia. CONCLUSIONS: This result can be explained by the phenomenon of "field cancerization" or by a local micrometastasis of K-ras-positive tumor cells.

Quality assurance in RT-PCR-based BCR/ABL diagnostics--results of an interlaboratory test and a standardization approach.

Here we describe the results of an interlaboratory test for RT-PCR-based BCR/ABL analysis. The test was organized in two parts. The number of participating laboratories in the first and second part was 27 and 20, respectively. In the first part samples containing various concentrations of plasmids with the ela2, b2a2 or b3a2 BCR/ABL transcripts were analyzed by PCR. In the second part of the test, cell samples containing various concentrations of BCR/ABL-positive cells were analyzed by RT-PCR. Overall PCR sensitivity was sufficient in approximately 90% of the tests, but a significant number of false positive results were obtained. There were significant differences in sensitivity in the cell-based analysis between the various participants. The results are discussed, and proposals are made regarding the choice of primers, controls, conditions for RNA extraction and reverse transcription.

Long-term remission after intensive chemotherapy in advanced myelodysplastic syndromes is generally associated with restoration of polyclonal haemopoiesis.

The clonality of peripheral blood cells was assessed in eight female patients with myelodysplastic syndrome (MDS) by means of the human androgen receptor gene-based assay (HUMARA). The patients were in complete remission for a median follow-up time of 83 months after intensive chemotherapy. X-chromosome inactivation patterns (XCIPs) indicated polyclonal haemopoiesis in five patients. Two patients had skewed lyonization (i.e. unbalanced XCIPs in both granulocytes and T cells) and one patient presented monoclonal granulocytes together with polyclonal T cells. We conclude that long-term remission in MDS following intensive chemotherapy is usually associated with polyclonal haemopoiesis.

Two types of acquired idiopathic sideroblastic anaemia (AISA): a time-tested distinction.

In 1982, acquired idiopathic sideroblastic anaemia (AISA) was included by the French-American-British (FAB) Co-operative Group in their classification of myelodysplastic syndromes (MDS). However, the malignant potentiality of AISA has always been a matter of debate. In different series, median survival and rates of transformation into acute myeloid leukaemia (AML) varied extensively. On cytomorphological grounds, AISA can be divided into pure (dyserythropoietic) sideroblastic anaemia (PSA), in which dysplasia is confined to erythropoietic cells, and a true myelodysplastic form (RARS), which is characterized by additional dysplastic features of granulopoiesis and/or megakaryopoiesis. In a previous study, based on retrospective analysis of 94 patients with AISA, we found that both types of sideroblastic anaemia differed considerably in terms of survival and risk of AML transformation. Almost identical results have now been obtained through a prospective study of 232 new patients with AISA. The difference in survival between PSA and RARS remained significant over the whole period of follow-up (survival after 3 years being 77% vs. 56%; P = 0.003), and the incidence of AML did not increase with time in the PSA group, even in the long term. This prospective study strongly supported our conclusion that cytomorphological distinction between PSA and RARS provides valuable prognostic information.

Acute promyelocytic leukemia and pregnancy.

In acute promyelocytic leukemia (APL), the use of all-trans-retinoic acid (ATRA) as a differentiating agent induces complete remission in a high percentage of patients. In pregnancy, however, this drug bears the risk of severe teratogenicity to the child. We report the case of a 23-yr-old woman at 21 weeks' gestation suffering from APL. She was treated with ATRA (45 mg/m2) for 40 d and two courses of standard chemotherapy. The mother achieved complete remission within 22 d of treatment. Fetal development was normal, and a healthy premature girl was born in the 35th week of pregnancy. In a review of the literature we have identified 14 cases of APL in pregnancy treated with ATRA alone or in combination with chemotherapy. ATRA has been used as early as in the 3rd week of gestation and in no case have malformations or other teratogenic effects occurred. Side-effects, however, ranged from fetal cardiac arrhythmias to induction of labour. Although known to exhibit severe teratogenic effects during the first trimester of pregnancy, ATRA seems to be reasonably safe during the second and third trimesters in the treatment of APL. However, careful obstetric follow-up is mandatory regarding fetal cardiac complications.

Granulocyte colony-stimulating factor (G-CSF) mediated mobilization of leukemic cells in Philadelphia chromosome positive acute lymphoblastic leukemia expressing myeloid antigens (my+Ph+ALL).

The therapeutic benefit of G-CSF in the treatment of acute lymphoblastic leukemia has been well established. G-CSF has been used to shorten neutropenia induced by conventional dose cytotoxic chemotherapy and allogeneic bone marrow transplantation. Recently autologous peripheral blood progenitor cell transplantation has been explored to treat high-risk ALL. Several in vitro studies suggest that subpopulations of lymphoblasts express G-CSF receptors. Furthermore, enhanced growth of Ph+ ALL cells expressing myeloid antigens stimulated by G-CSF has been demonstrated in vitro. However, the clinical relevance of these findings has been questioned. We report a patient with my+Ph+ALL in whom the administration of G-CSF after high-dose Cytarabin and Mitoxantrone led to a significant mobilization of leukemic cells and contamination of the stem cell harvest during cytologic marrow remission.