Cytogenetic studies in bone marrow transplant recipients

Chromosome studies were performed in 24 patients who underwent allogeneic bone marrow transplantation (BMT) for severe aplastic anaemia (8), chronic myeloid leukemia (5 in chronic, 2 in accelerated phase and 1 in lymphoid blast crisis), acute myeloid leukemia (6), acute lymphoblastic leukemia in relapse (1) and Hodgkin's disease (1). Donor-cell type engraftment was demonstrated in 21 patients: in all 17 sex-mismatched transplants and - as demonstrated by reconstitution with Ph-negative cell populations - in 4 CML patients with a sex-matched donor. Recipient-type mitoses were seen in the bone marrow of 5 cases (1 SAA, 3 CML, 1 AML) after transplantation. They were only observed on one occasion in patients with SAA (4 of 25 on day 33) and AML (44 of 50 on day 14). Despite the continued demonstration of some Ph-positive mitoses in 3 patients with CML up to day 28, 323 and 451 after BMT, respectively, all surviving CML patients are still in complete haematological and clinical remission. So far the significance of these cytogenetically abnormal persisting host cells remains unknown.     

Monosomy 7 in donor cell-derived leukemia after bone marrow transplantation for severe aplastic anemia: Report of a new case and review of the literature

Monosomy 7 arises as a recurrent chromosome aberration in donor cell leukemia after hematopoietic stem cell transplantation. We report a new case of donor cell leukemia with monosomy 7 following HLA-identical allogenic bone marrow transplantation for severe aplastic anemia (SAA). The male patient received a bone marrow graft from his sister, and monosomy 7 was detected only in the XX donor cells, 34 months after transplantation. The patient’s bone marrow microenvironment may have played a role in the leukemic transformation of the donor hematopoietic cells.     

Growth and growth hormone in children after bone marrow transplantation

Growth and growth hormone (GH) were investigated every year in 24 children after allogeneic bone marrow transplantation (BMT) for severe aplastic anemia (SAA) or leukemia. Conditioning included total body irradiation (TBI) in all cases of leukemia. The young leukemic children grew poorly. At 4 years after BMT, the mean standard deviation score for attained height had decreased from 0 to -1.73. GH deficiency was diagnosed with provocation tests. Three years after BMT, 10/18 children had a subnormal response. Ten children were further investigated with 24-hour GH profiles. Children with SAA had normal growth and GH levels. TBI seemed to be the major factor responsible for impaired growth.     

Acute leukemia in adults: assessment of remission induction with combination chemotherapy by clinical and cell-culture criteria.

Remission induction was assessed by clinical and cell-culture criteria for 65 patients with acute myelogenous leukemia (AML), 11 patients with chronic myelogenous leukemia (CML) in blast crisis and 19 patients with acute lymphoblastic leukemia (ALL). Cyclophosphamide, cytosine arabinoside and vincristine (CAV) therapy resulted in complete remission in 23 of 50 previously untreated patients with AML and in 3 of the 11 patients with CML. Fourteen patients with ALL responded to vincristine-prednisone induction therapy and two to induction therapy with CAV. The median duration of survival of the responding patients was 2.2 years, compared with 4 months for the patients who did not respond to treatment. Granulopoietic colony formation, assessed by assay of colony-forming units dependent on colony-stimulating activity in culture (CFU-C), was abnormal in 37 of 42 bone marrow aspirates from patients with AML before treatement. CFU-C concentration increased when leukocyte-conditioned medium (LCM) was added to the cultures; 13 cultures had normal or elevated CFU-C concentration with LCM. Marrow cells of patients with ALL or CML in blast crisis demonstrated a similar pattern. Serial studies of marrow CFU-C concentration of 31 patients with AML demonstrated a change to a normal pattern with successful remission induction. Results of this study suggest that administration of purified LCM to leukemic patients might increase granulocyte production from potential but unstimulated granulopoietic precursors. This therapy would lessen the probability of death from infection during remission induction.     

Anti-thoracic duct lymphocyte globulin stimulates human megakaryocytopoiesis in vitro

Anti-thoracic duct lymphocyte globulin (ALG) therapy is effective in patients with aplastic anemia. We examined the effect of ALG on human megakaryocyte progenitor cells (colony-forming unit-megakaryocyte, CFU-Meg) in vitro. Normal human bone marrow mononuclear cells (MNC) were cultured in plasma clots with varying concentrations of ALG or non-immunized horse IgG. After 12 days of culture, significant megakaryocyte colony formation was observed in cultures containing ALG but not in cultures containing non-immunized horse IgG. The peak stimulatory effect seemed to occur with 10-25 micrograms/ml of ALG. When marrow MNC, depleted of adherent and T cells, were cultured in plasma clots with ALG, its stimulatory effect on megakaryocytopoiesis decreased markedly. Finally, it was demonstrated that ALG stimulated marrow MNC to produce a factor stimulatory for CFU-Meg. The in vitro megakaryocytopoietic stimulatory effect of ALG may be related to its clinical efficacy in some patients with aplastic anemia.     

Bone marrow transplantation for leukemia and aplastic anemia: management of ABO incompatibility.

Between February 1971 and October 1980, 34 patients with leukemia or aplastic anemia received bone marrow transplants from HLA-identical siblings whose lymphocytes did not react in a mixed leukocyte culture. The donors of 10 patients were ABO-incompatible, and for five pairs the ABO incompatibility was major. Plasma exchanges followed by a red blood cell exchange transfusion reduced the anti-A titres to 1:4 or less in these patients. The ABO incompatibility had no adverse effect on the results of marrow transplantation. Twenty-two patients, including 16 of the 20 who received their transplant after Jan. 1, 1980, are still living. Seven of the 15 patients with acute leukemia have survived 89 to 466 days, and 4 of the 6 with chronic myelogenous leukemia (CML) have survived 117 to 545 days. Of the 13 patients with aplastic anemia, 11 are alive up to 8 years after transplantation. Marrow transplantation, when possible, is the treatment of choice for young patients with acute leukemia in remission and for patients with aplastic anemia. Marrow transplantation may also prove to be effective in patients with CML.     

Identification of B-cell translocation gene 1 as a biomarker for monitoring the remission of acute myeloid leukemia

Acute myeloid leukemia (AML) is a biologically heterogeneous disease of the hematopoietic system characterized by a clonal accumulation of immature blast cells in bone marrow. We used a proteomic approach based on two-dimensional electrophoresis and mass spectrometry to search for biomarkers related to the complete remission (CR) state of AML patients. We detected one AML-related protein, which was identified as the B-cell translocation gene 1 (BTG1) protein that belongs to anti-proliferative protein family. In the CR state of AML-M2 and M3 patients (by French-American-British subtype classification), the BTG1 protein was upregulated in bone marrow mononuclear cells. It was also expressed robustly in normal bone marrow mononuclear cells. In addition, the BTG1 levels in AML-M2 patients in a non-remission state after therapy did not increase as they did before therapy. Overexpression of BTG1 mRNA was also observed in the CR state of all-trans-retinoic acid (ATRA)-treated AML-M3 patients and ATRA-treated HL-60 cells. Taken together, these results suggest that BTG1 may play a role in the differentiation process of myeloid cells and can therefore be used as a potential treatment-related biomarker for monitoring the remission status of AML-M2 and M3 patients.     

Bone marrow necrosis intravitally recognized in four cases of blastic leukaemia.

Bone marrow necrosis (BMN) is a rare intravitally recognized finding in acute leukaemia with an uncertain clinical significance. The clinical events in 4 patients with AML, ALL, AMoL and blastic transformation of CGL in whom bone marrow cytology and histology revealed BMN are reviewed. One patient with BMN at clinical presentation of AML entered complete, long lasting remission with marrow restoration after the standard DAT therapy. In the three remaining patients survival after BMN diagnosis was 6, 11, and 14 weeks. Clinical, haematological, histological and marrow scanning findings and their significance for early diagnosis and means to asses the extent and evaluation of BMN will be discussed. In contrast to the most earlier reports, BMN does not appear to confer a poor prognosis in all patients with blastic leukaemia.     

Effect of recombinant human alpha interferon (INF) and antilymphocyte globulin (ALG) on normal and aplastic anaemia haematopoietic progenitor cell growth.

The effect of recombinant alpha interferon (INF) and of antilymphocyte globulin (ALG) to the colony stimulating factor (CSF) production was examined with in vitro culture of the bone marrow of healthy and of aplastic anaemia (AA) persons. In healthy persons the supernatant of lymphocytes preincubated with PHA and ALG was found to show a stimulating effect to clonogenic properties of marrow progenitors, the mentioned effect being not in proportion to the concentration value. Similar properties are shown by interferon in these persons. In patients with aplastic anaemia, a considerable stimulating ALG effect to the granulocytic formation of colonies and a lesser stimulating effect of interferon were shown.     

The Clinical and Immune Characteristics of Patients with Hepatitis-Associated Aplastic Anemia in China

Hepatitis-associated aplastic anemia (HAAA) is a variant of severe aplastic anemia (SAA) in which bone marrow failure follows an acute attack of hepatitis. Its pathogenesis is poorly understood. We investigated the prevalence of HAAA among cases of newly diagnosed SAA presenting to our hospital between January 1998 and February 2013, and analyzed the clinical and immune characteristics of HAAA and non-hepatitis-associated SAA (non-HASAA) patients. The prevalence of HAAA among cases of SAA was 3.8% (36/949), and the majority of patients (33/36) were seronegative for a known hepatitis virus. Compared with non-HASAA patients, HAAA patients had a larger proportion of CD8+ T cells, a lower ratio of CD4+/CD8+ T cells, and a smaller proportion of CD4+CD25+ regulatory T cells. There was no significant difference in peripheral blood count, bone marrow cellularity, or the number of blood transfusions received between HAAA and non-HASAA patients. HAAA patients had a higher early infection rate and more infection-related mortality in the first 2 years after diagnosis than non-HASAA patients, and their 2-year survival rate was lower. The results demonstrate that HAAA patients have a more severe T cell imbalance and a poorer prognosis than non-HASAA patients.     

An abnormal CD34+ myeloid/CD34+ lymphoid ratio at the end of chemotherapy predicts relapse in patients with acute myeloid leukemia.

During conventional follow-up of patients with acute myeloid leukemia (AML), the emergence of cytopenias is considered to be a sign of impending relapse, and it represents an example of how leukemic hematopoiesis affects normal hemopoietic differentiation. In the present study, we have explored the possible value of the analysis of the distribution of CD34+ myeloid and CD34+ lymphoid progenitor cells in follow-up complete remission bone marrow samples from de novo AML patients as a prognostic parameter for predicting relapse. A total of 213 bone marrow samples from 36 AML patients in morphological complete remission, obtained at the end of induction, consolidation, and intensification therapy and every six months thereafter were analyzed. The normal CD34+ myeloid/CD34+ lymphoid ratio ranged between 2.4 and 8.9. In contrast, in most AML cases an abnormally high ratio (> or =10) was observed at the end of induction and consolidation therapy: 96% and 75% of cases, respectively. On the other hand, at the end of intensification, 70% of the patients displayed a normal CD34+ ratio. Patients with a myeloid/lymphoid CD34+ ratio higher than 10 at the end of intensification showed a significantly lower overall survival (median survival of 19 months versus median not reached, P = 0.05), as well as a lower disease-free survival (median of 7 months versus 30 months, P = 0.0001). Regarding sequential studies, 67% of the relapses were preceded by the re-appearance of an abnormal CD34 ratio, whereas relapse was not predicted in four patient with leukemia classified as M3 undergoing maintenance therapy. From the remaining 18 patients who are still in continuous complete remission, all except 3 cases (17%) displayed a normal CD34 myeloid/lymphoid ratio. In summary, the present study shows that the persistence at the end of chemotherapy of an abnormally high (> or =10) ratio between CD34+ myeloid and CD34+ lymphoid progenitors in the bone marrow of AML patients is associated with high risk of relapse and a shorter overall survival.     

Complete remission in a patient with acute myelogenous leukemia treated with leukocyte α-interferon and cimetidine

Summary A 76-year-old woman with acute myelogenous leukemia with approximately 65% myeloblasts on bone marrow examination was treated daily with a combination of 4 megaU of leukocyte interferon IM and 1,000 mg cimetidine PO. During therapy there was a gradual decrease of bone marrow myeloblasts down to 9% and a normalization of peripheral white blood cells. The treatment was discontinued after 6 weeks because of increasing fatigue and anorexia. The general condition improved greatly during the following weeks and the patient entered complete remission, which has continued for 6 months so far. In the course of therapy there was a gradual appearance of antibodies showing a selective binding capacity to autochthonous leukemic cells with no tendency to increased binding to remission cells. The aim of this report is to stimulate a further evaluation of this form of therapy in additional AML patients whenever this might be justified as an alternative to conventional chemotherapy.     

Poor prognosis of acute lymphoblastic leukemia with translocation (1;19) in childhood: potential interest of allogeneic bone marrow transplantation

We report a new case of childhood acute lymphoblastic leukemia with translocation (1;19). At the time of diagnosis, the only adverse prognosis factor was the existence of this translocation. Under conventional chemotherapy, the girl experienced early marrow relapse (duration of first remission was 2 months). She received allogeneic bone marrow transplantation during the second remission and is alive in continuous complete remission 20 months after transplant. Several earlier reports have suggested that children with the (1;19) have a poor prognosis; If this poor response to conventional therapy is confirmed, an allogeneic bone marrow transplantation should be considered during the first remission.     

Day-6 bone marrow aspirate for the prediction of response to remission induction therapy for acute myelogenous leukaemia

Seventy-two adults were treated for acute myelogenous leukaemia (AML). Forty-two had previously untreated AML and 30 had AML after a preleukaemic phase, refractory AML or relapsed AML. The previously untreated patients received a 7-day course of cytosine arabinoside (100 or 200 mg/m2 daily), daunorubicin and vincristine while the remaining patients received a 7-day course of cytosine-arabinoside (1 g/m2 q 12h for 6 days) and amsacrine (on day 7). The percentage of malignant cells and the reduction in the percentage of malignant cells were determined by means of bone marrow aspirates taken on day 6 of the chemotherapy course and at the time of diagnosis. Both variables correlated significantly with the ultimate treatment outcome; the reduction in the percentage of malignant cells correlated even more significantly than the absolute percentage malignant cells in the day-6 bone marrow. By means of multiple regression analysis it became possible to calculate the probability of achieving complete remission for the individual patient; this is given by the equation: probability = 1.9-0.009X (% malignant cell reduction). In addition, the mean percentage of malignant cells in the day-6 bone marrow was significantly higher for patients who failed to achieve than those who entered complete remission. Eighty-six per cent of the patients with less than 20% malignant cells on day 6 entered remission, while 75% of the patients with more than 21% malignant cells failed to achieve complete remission (p less than 0.001). Although all of these calculations support the predictive value of the day-6 bone marrow aspirate, the 95% confidence intervals are too large to allow reliable and safe predictions; therefore more patients must be studied to demonstrate the reliability of this test.     

Follow-up study on bone marrow reticulin fibrosis in AML

Planimetric analysis of argyrophilic (reticulin) fibers of the bone marrow was performed on 17 patients (7 males, 10 females, median age 52 years) with acute myeloid leukemia (AML). Trephine biopsies were investigated at presentation and during the course of chemotherapy. Density of reticulin was determined by examining the total marrow area as well as the hematopoietic tissue. In about half of the patients with FAB-subtypes M4, M5 an increased amount of fibers was recognizable on admission in comparison with age-matched controls. Two to five bone marrow biopsies (total 81) of each patient were obtained at intervals ranging mostly between three and seven weeks after cytotoxic treatment. The following results could be demonstrated: in several patients with a normal or elevated fiber count at onset, partial and complete remission was characterized by an increase in reticulin in the regenerative hematopoietic tissue. On the other hand, a relapse was most frequently heralded by a reduction of the formerly increased amount of fibers. Because of the inverse relationship between the density of argyrophilic fibers and the amount of fat cells, these findings were observable only by considering the hematopoietic tissue and not the total marrow area, as has been done in previous studies. For this reason a significant decrease in reticulin fibers in the areas of regenerative hematopoiesis may be suggestive of an impending relapse of AML.     

Distribution of the cytoskeletal protein,Nestin, in acute leukemia

Abstract

Nestin is a neuroepithelial stem cell marker that is expressed in some types of tumor cells. Recent reports suggest that Nestin may be closely related to malignant cell proliferation and migration. Acute leukemia (AL) is characterized by a lack of differentiation, which results in uncontrolled proliferation in the bone marrow and accumulation of immature cells. The expression and function of Nestin in AL is unclear. We investigated Nestin immunohistochemical patterns of 87 patients that included 47 cases of acute myeloid leukemia (AML) and 40 cases of acute lymphoblastic leukemia (ALL), and 20 patients in complete remission (CR) from AML or ALL. We also investigated the clinico-pathological features of 87 cases of AL and their CR and overall survival (OS). Nestin was expressed in leukemic blasts and mature granulocytic cells in most cases (39/47) of AML. Conversely, Nestin was expressed in mature granulocytic cells in fewer cases (6/40) of ALL, but not in blasts. Nestin expression appeared in leukemic blasts of AML, but not ALL. Nestin expression in AML blast cells was not associated with CR or OS. We provide evidence that Nestin is expressed in AL and might be a useful immunohistochemical marker for identifying AML and ALL.     

Flow cytometric determination of atypical antigen expression in acute leukemia for the study of minimal residual disease.

The aim of this study was to investigate to which extent acute leukemias could be monitored for residual disease by using atypical antigen combinations as leukemia-related markers. Atypical antigenic features were determined by double color flow cytometry and included coexpression of lymphoid and myeloid related antigens, unphysiological coexpression of immature and mature antigens, and lack of an antigen that is normally expressed during maturation. Atypical immunophenotypes were detected in 35 of 68 patients with AML (51.5%) and 15 of 24 patients with ALL (62.5%). When 12 patients with leukemia-associated markers were again analyzed at relapse, the relevant antigen combinations were retained in 11 of them. The sensitivity of this two color flow cytometric assay as determined in dilution experiments was 1 in 10(3) to 10(4) cells. Follow-up studies of bone marrow samples revealed that, after induction chemotherapy cells with leukemia-associated markers were detectable in several patients at a frequency of 0.5 to 4%, but only patients in whom the cells with atypical antigens never disappeared suffered from relapse. In contrast, patients who became negative for the atypical cells remained in complete remission (median remission duration after the first negative bone marrow assessment by flow cytometry 52 weeks, range 20-102). We conclude that atypical antigen combinations, which are present in a meaningful number of acute leukemias, are a valuable means of monitoring acute leukemia patients during follow-up. This flow cytometric approach can complement other strategies to get a more accurate definition of remission in acute leukemia.     

流式细胞术动态监测微小残留病灶在急性髓系白血病中的意义*

目的:探讨急性髓系白血病(AML)在治疗过程中应用多参数流式细胞术(MFC)动态微小残留病灶(MRD)的意义。方法:选择2015年1月至2017年2月在我院血液科收治的60例AML患者,在诱导治疗第8天,第21天,每次巩固治疗前1天,结束化疗随访过程中,检测骨髓形态学变化和应用MFC监测患者骨髓MRD,并动态随访。定义未发现异常表型细胞(MRD10~(-4))为阴性,其余为阳性。结果:平均随访时间为11个月(3到16个月),早期MRD(诱导化疗第8天)阴性的患者占58.33%,MRD阳性的患者占41.67%。MRD阴性的患者在诱导治疗接受第21天100%达到CR,MRD阳性的患者80%达到CR。第一次巩固治疗结束后,MRD阴性的患者预后明显较MRD阳性的患者好。结论:动态监测MRD对预测AML患者对治疗的反应和预测复发有重要意义。     

Detection of minimal residual disease (MRD) after bone marrow transplantation (BMT) by multi-parameter flow cytometry (MPFC)

Multi-parameter flow cytometry (MPFC) was used to detect minimal residual disease (MRD) following bone marrow transplantation (BMT) in 21 patients. Bone marrow (BM) was analyzed pre-transplant and 3–4 months post-BMT while the patients were in clinical and morphological remission. MRD was detected by identifying cells with aberrant antigen expression and/or leukemia-associated phenotype (LAP) using MPFC. Prior to BMT, 8 out of 21 patients exhibited normal antigen expression based on normal BM samples while 13 BM aspirates had abnormal MPFC. Pre-BMT MPFC was abnormal in all 10 patients who were not in complete remission (CR) (>5% blasts in BM) as well as 3 patients acute lymphoblastic leukemia (ALL) who were in CR. In BM from ALL patients, an abnormal uniform B cell population was observed however antigen expression patterns varied greatly between patients. BM from acute myeloblastic leukemia (AML) patients showed an abnormal distribution of CD34+ cells. In addition, a correlation was observed between pre-BMT cytogenetics and MPFC. Only 2 out of 8 (25%) patients with normal MPFC pre-autologous bone marrow transplantation (ABMT) relapsed (AML), while 6 out of 13 (46%) patients with abnormal pre-BMT MPFC relapsed including 2 out of 3 patients who were transplanted in clinical CR. Pre-BMT MPFC may thus be an effective tool for detection of MRD by detection of a pre-transplant MPFC abnormality.     

Systemic mastocytosis associated with t(8;21)(q22;q22) acute myeloid leukemia

Although KIT mutations are present in 20–25% of cases of t(8;21)(q22;q22) acute myeloid leukemia (AML), concurrent development of systemic mastocytosis (SM) is exceedingly rare. We examined the clinicopathologic features of SM associated with t(8;21)(q22;q22) AML in ten patients (six from our institutions and four from published literature) with t(8;21) AML and SM. In the majority of these cases, a definitive diagnosis of SM was made after chemotherapy, when the mast cell infiltrates were prominent. Deletion 9q was an additional cytogenetic abnormality in four cases. Four of the ten patients failed to achieve remission after standard chemotherapy and seven of the ten patients have died of AML. In the two patients who achieved durable remission after allogeneic hematopoietic stem cell transplant, recipient-derived neoplastic bone marrow mast cells persisted despite leukemic remission. SM associated with t(8;21) AML carries a dismal prognosis; therefore, detection of concurrent SM at diagnosis of t(8;21) AML has important prognostic implications.