Sequential Bone Marrow Research Articles

Monitoring Informed Consent in an Oncology Study Posing Serious Risk to Subjects

States, reviewed an oncology protocol presenting serious risk to research participants. Given the degree of risk to subjects posed by the study and the potential vulnerability of the study population, the IRB decided that direct monitoring of the informed consent process was required to ensure that subjects were adequately informed and that the decision to participate was voluntary. In this paper, we describe the consent monitoring process devised by the IRB and the results obtained. The study in question was a phase II regimen of dose intensification therapy for women with advanced (stage IIIB, IV, or inflammatory carcinoma) breast cancer ineligible for hormonal therapy (estrogen-receptor negative tumor or failure of two hormonal therapies).' The protocol combined a variety of aggressive (and investigational) treatments for advanced cancer. The induction regimen consisted of treatment with a very high dose of an anthracycline, an experimental cardio-protective drug, methotrexate, and 5-flourouracil. After 6 cycles of induction chemotherapy, patients received 2 sequential autologous bone marrow transplants (ABMT). If after the second ABMT no evidence of tumor was seen in biopsy samples, patients received irradiation to any (previous) areas of bulky disease and to the cranium. Finally, patients received tamoxifen for up to 5 years after completion of radiation therapy. The entire regimen (excluding the tamoxifen) took 10 months to complete, of which an estimated 110 days were spent in hospital. Intensive supportive measures were required, including growth factors, peripheral stem cell transfusion, parenteral nutrition, antibiotics, and, if necessary, admission to the intensive care unit. The anticipated treatment mortality rate was 10 to 15%.

Prognostic influence of blood transfusion on minimal residual disease in resected gastric cancer patients: Heiss MM, Allgayer H, Gruetzner KU, Tarabichi A, Babic R, Mempel W, Jauch KW, Schildberg FW. Anticancer Res 17:2657–2662, 1997

The effect of blood transfusion on prognosis of resected cancer patients has been debated controversially. Therefore, we raised the hypothesis that transfusion-associated immunomodulation affects minimal residual disease after curative tumour resection, an unknown and uncontrolled phenomenon in all former studies which might significantly influence long-term prognosis.104 patients of a prospective study with curatively resected gastric cancer were stratified according to the immunocytochemical detection of disseminated tumour cells in bone marrow and the prognostic impact of allogeneic blood transfusion was tested. Multiple sequential bone marrow aspirations during follow-up were performed in 74 patients to investigate the blood transfusion effect on long-term development of this systemic disease component.Whereas in patients with tumour cell detection in bone marrow a significant association of blood transfusion and survival was seen (P = 0.048; relative risk 2.91; 95% CI 1.51-5.61), this was not found in patients without disseminated tumour cells (P = 0.129). Quantitative development of tumour cells in bone marrow during follow-up demonstrated a significant quantitative increase of tumour cells in transfused patients only (P = 0.028).These findings might explain the contradictory results of recent studies and suggest that the prognostic effect of transfusion is mediated through an impact on minimal residual disease in resected cancer patients.

Effect of IFN-alpha on normal human hematopoiesis: an immunohistochemical and morphometric study on trephine biopsy specimens.

To elucidate the effects of interferon-alpha (IFN-alpha) on normal human bone marrow in vivo, an immunomorphometric study was performed using trephine biopsy specimens without hematopoietic pathology. Samples were derived from patients with mycosis fungoides but no marrow involvement, who were undergoing low-dose IFN-alpha treatment. Parameters included density of reticulin (argyrophilic) fibers, CD61+ megakaryocytes, PGM1+ macrophages, the GSA-I lectin-expressing (activated) macrophage subpopulation, proliferative activity (PCNA staining), and apoptosis. Following IFN-alpha therapy (3 x 3 x 10(6) U/week between 6 and 21 months), morphometric evaluation of sequential bone marrow examinations revealed a significant increase in the number of megakaryocytes and the amount of reticulin fibers. Additionally, there was an overall decrease in PCNA+ cells, accompanied by a reduction in the incidence of apoptotic bodies. On the other hand, total number of macrophages and their activated subfraction remained unchanged. Opposed to in vitro findings, a fibrogenetic capacity of IFN-alpha associated with megakaryocyte growth was detectable. Moreover, contrasting with effects of IFN-alpha treatment in chronic myelogenous leukemia, the incidence of apoptosis was significantly reduced. This feature was assumed to contribute to a maintenance of steady-state hematopoiesis expressed by a nonaltered bone marrow cellularity in our specimens.

Achievement of a complete cytogenetic response with hydroxyurea in a patient with chronic myelogenous leukemia

Hydroxyurea rarely produces a complete cytogenetic remission in patients with chronic myelogenous leukemia (CML). In this report, we decribe one case of the CML patient who achieved complete cytogenetic remission (no Ph chromosome in 20–25 metaphase cells) by treatment with hydroxyurea alone. By the fluorescent in situ hybridization (FISH) methodology using bcr/abl specific translocation probe, sequential bone marrow specimens from the patient showed the characteristic 9;22 translocation at a higher rate (9–10%) than the normal control range (2.49–4.88%) at the time of complete cytogenetic remission. Thus, it is suggested that FISH is a more sensitive method to detect the bcr/abl fusion gene than conventional cytogenetic analysis for the detection of minimal residual disease in CML.

Sequential molecular monitoring of chimerism in chronic myeloid leukemia patients receiving donor lymphocyte transfusion for relapse after bone marrow transplantation

Recent observations of chimerism in patients relapsed following an allotransplant suggest the persistence of immunotolerance, thus offering a biologic rationale for the use of donor lymphocyte transfusion (DLT). In this study, we have analyzed by PCR amplification of several VNTR regions, sequential bone marrow and peripheral blood DNA samples in four patients who received DLT for CML relapse after bone marrow transplantation. Prior to DLT, all patients showed mixed chimerism in peripheral blood cells while two had mixed chimerism and two no chimerism in the BM. None of these four patients showed evidence of chimerism at the cytogenetic level (all had 100% +ve metaphases). After DLT, a complete hematologic and molecular remission (ie disappearance of the BCR/ABL fusion transcript) was obtained in the two patients who had bone marrow mixed chimerism prior to DLT. The two patients without evidence of marrow chimerism prior to DLT converted to a pattern of mixed chimerism after DLT, but both developed a severe bone marrow aplasia occurring at day 56 and 36, respectively. With regard to the sequential analysis of bone marrow chimerism after DLT we observed that: (1) the disappearance of BCR/ABL +ve cells paralleled the conversion to a pattern of full donor chimerism; and (2) the time interval to achieve CR was inversely correlated with the percentage of donor DNA in bone marrow. In conclusion, we have shown here that the assessment of bone marrow pre-DLT chimerism by PCR analysis might predict the response in patients with favorable characteristics, and also might identify patients at high risk of developing severe myelosuppression.

Management of relapsed and refractory Hodgkin's disease

Hodgkin's disease remains highly curable after relapse or even after an ineffective initial therapy. The likelihood of attaining a lasting second remission is particularly high for patients who failed to respond to a primary program of radiation therapy (RT) alone. The preferred treatment for these patients is with a doxorubicin-containing chemotherapy and additional RT (if feasible). The prospect for a “second cure” after failure to respond to initial chemotherapy is smaller and for most cases a program of high-dose therapy followed by stem-cell transplantation is required. Advances in using this modality over the last 15 years have resulted inn a diseasefree survival of approximately 40% with a follow-up that extends up to 10 years. Most important, a recent dramatic decrease in treatment-related mortality of highdose therapy as well as a reduction in cost have been documented. Analysis of prognostic factors allows better selection of patients and a sounder choice of salvage options. Although it appears that further dose escalation of chemotherapy is limited by nonhematologic toxicity, the benefit from incorporation of RT into high-dose programs has only recently been recognized. Because of the availability of hematopoietic growth factors and easier mobilization and collection of peripheral blood stem cells, these have become the preferred source for hematopoietic support. The use of peripheral blood stem cells and growth factors was shown to correlate with more rapid recovery of granulocytes and platelets and a shorter hospitalization period. Still, many patients remain refractory to salvage despite intensive therapy. These patients can be identified by their response to re-induction chemotherapy before transplant and should be considered for alternative approaches such as allogeneic bone marrow transplantation, sequential bone marrow transplant, and other experimental programs. This article reviews standard-dose salvage as well as current development in high-dose therapy with particular attention to the role of RT.

Idiopathic primary osteo-myelofibrosis: a clinico-pathological study on 208 patients with special emphasis on evolution of disease features, differentiation from essential thrombocythemia and variables of prognostic impact.

A retrospective clinico-pathological study was performed on 208 consecutively recruited patients (94 males, 114 females, median age 67 years) with idiopathic (primary) osteo-/ myelofibrosis (IMF). According to bone marrow histology (cellularity) as well as extent (semiquantitative grading) and quality (reticulin/collagen) of myelofibrosis, stages of the disease process were determined. At closure of this study (observation time 65 months) 133 patients were dead and 75 alive and median survival was 56 months. The wide spectrum of clinical signs and symptoms and laboratory data on admission was reflected by a corresponding variety of histological features. Significant differences of hematological values could be calculated between patients with or without early reticulin fibrosis (fiber scores 0 and 1) and advanced fibro-osteosclerotic stages (fiber scores 2 and 3). Evolution of disease features was elicited by longitudinal follow-up studies and sequential bone marrow biopsies. Morphometric assessment of fiber density in patients without preceding chemotherapy revealed an unpredictable and varying progression of myelofibrosis associated with alterations of certain laboratory parameters (hemoglobin level, spleensize, thrombocytosis). Differentiation from essential (primary) thrombocythemia (ET) was required in 25 patients who fulfilled the postulated diagnostic criteria. In fact, this group was consistent with hypercellular, early stages of IMF without relevant reticulin fibrosis and an excessively raised platelet count (> or = 1000 x 10(9)/1). Discrimination was only feasible by regarding histology carefully, particularly abnormalities of megakaryopoiesis and follow-up data. Parameters of predictive value indicating a significant loss in life expectancy in comparison with a sex- and age-adjusted normal population included: age (> or = 60 years), hemoglobin levels (< or = 10 g/dl), thrombocyte count (< or = 600 x 10(9)/1) and the presence of myeloblasts and promyelocytes. Statistical analysis disclosed that in the so-called early stages of IMF without relevant myelofibrosis, findings indicative for extramedullary hemopoiesis or generalization of the disease process exerted an unfavourable influence on survival.

The impact of megakaryocyte proliferation of the evolution of myelofibrosis. Histological follow-up study in 186 patients with chronic myeloid leukaemia.

A histological study on sequential bone marrow biopsies in patients with chronic myeloid leukaemia (CML) was performed. We wished to answer the question as to whether a different content of megakaryopoiesis in the bone marrow of CML patients has a prognostic significance for the development of myelofibrosis during the course of disease. In addition, the significance of possible changes in the quantity of megakaryopoiesis in this process was assessed. In 186 patients who had no fibre increase at first diagnosis, the rate of subsequent myelofibrosis varied from 19% for the common or granulocytic subtype (CML.CT) to 40% for patients with features of megakaryocytic increase (CML.MI). No significant differences were found either in the rapidity of progression to fibrosis or in the final rate of osteomyelosclerosis. Whereas in CML.MI most patients (75%) showed an increase of fibres only, this was accompanied by an additional increase of megakaryocytes in CML.CT, changing the histological pattern from CML.CT to .MI or .MP, respectively. The data therefore revealed a correlation between fibre increase and subtyping of CML as suggested by the Hannover classification of chronic myeloproliferative diseases. Subtypes of CML with megakaryocytic increase could be shown to present a "pre-myelofibrotic" stage of disease and may therefore be conceived as a particular pathway of acceleration.

Bone marrow and blood findings after marrow transplantation and rhGM-CSF therapy.

Sequential bone marrow biopsy specimens and peripheral blood findings were evaluated from patients treated with recombinant human granulocyte-macrophage colony-stimulating factor after autologous bone marrow transplantation for recurrent non-Hodgkin's lymphoma. Parallel increases were found in white blood cell count and marrow cellularity and in absolute neutrophil count, monocyte count, and marrow myeloid:erythroid ratio. Platelet count recovery and the reappearance of megakaryocytes occurred later than granulocyte/monocyte recovery. Elevated peripheral eosinophil counts and eosinophilic hyperplasia in the marrow were noted during recombinant human granulocyte-macrophage colony-stimulating factor administration, as was the appearance of distinctive, prominently granulated and/or vacuolated neutrophils and neutrophilic precursors in the blood and bone marrow. No detrimental effects of recombinant human granulocyte-macrophage colony-stimulating factor administration in the marrow or peripheral blood were observed.

Effect of subcutaneously administered human recombinant erythropoietin on erythropoiesis in patients with myelodysplasia

In a phase II study, 12 patients with a myelodysplastic syndrome (MDS) and anaemia (nine transfusion-dependent) were treated with recombinant human erythropoietin (rHuEpo) to assess the therapeutic effect on erythropoiesis and on transfusion requirement. Patients with a low risk of developing acute leukaemia were included, i.e. refractory anaemia (RA), RA with ringed sideroblasts (RARS) and RA with excess blasts (RAEB), providing the percentage of myeloblasts in the bone marrow did not exceed 10%. Recombinant HuEpo treatment was initiated at a dose of 50 units/kg body weight and administered subcutaneously three times weekly. At 3-week intervals the dose was increased with 50 units/kg per injection, until after 15 weeks a maximum dose of 250 units/kg three times weekly was reached. All patients completed the study. Recombinant HuEpo was well tolerated and no serious side effects were seen. There was no evidence of the emergence of a new malignant clone in response to rHuEpo as shown by sequential karyotyping. In none of the patients was an increase in haemoglobin level or a diminished red blood cell transfusion requirement seen. In four out of 10 evaluable sequential bone marrow smears, an increase in erythropoiesis was seen, suggesting stimulation of ineffective red cell production. One of these patients also showed a rise in reticulocyte count. The number of erythroid progenitor cells (BFU-E and CFU-E) in blood and bone marrow was not affected by rHuEpo treatment. Also no change in the number of myeloid progenitor cells (CFU-GM) in blood and bone marrow was noted. In conclusion, subcutaneous treatment with rHuEpo at dosages up to 250 units/kg body weight (three times weekly) fails to increase the haemoglobin level or to diminish the transfusion requirement in patients with MDS and anaemia. It is unclear whether higher doses of rHuEpo are effective or whether patients with less severe anaemia who are transfusion independent, have a higher likelihood of response.

Prompt engraftment using autologous peripheral blood stem cells for double autologous bone marrow rescue

A patient with Hodgkin's Disease, stage IIIA, was treated for recurrent disease with autologous peripheral blood stem cells after ablative chemotherapy. Bone marrow rescue occurred promptly and his disease was significantly reduced. Ablative chemotherapy was administered again and a second bone marrow rescue with peripheral blood stem cells was successful. Sequential peripheral blood stem cell bone marrow rescue, following high dose chemotherapy, may provide an alternative approach in selected patients.

Prognostic implication of sequential bone marrow cultures in the myelodysplastic syndromes

Twenty-five patients with newly-diagnosed myelodysplastic syndromes were studied by the clonal culture method at least three times during the clinical course. Clinical outcomes of the patients were classified into: a stable disease (ten patients); subsequent leukemic transformation (eight patients) and nonleukemic death (seven patients). The growth of the marrow granulocyte-macrophage progenitors (CFU-GM) at the time of diagnosis was significantly related to the survival. In addition, sequential changes in the CFU-GM growth patterns correlated with the different clinical outcome of myelodysplastic syndromes patients.

Follow-up Studies with Sequential Bone Marrow Biopsies in Chronic Myeloid Leukaemia and So-called Primary (Idiopathic) Osteo-myelofibrosis: Evolution of Histopathological Lesions and Clinical Course in 40 Patients

A clinicopathological follow-up study was performed on 17 patients with chronic myeloid leukaemia (CML) and 23 cases with so-called primary (idiopathic) osteo-/myelofibrosis (OMF) concentrating on a comparison between clinical data and multiple sequential biopsies of the bone marrow. Histological classification of bone marrow lesions was done according to the subtypes proposed by Georgii et al. At clinical diagnosis initial trephine biopsies in CML showed in only 6/17 cases a pronounced granulocytic proliferation or CGL. In 9/23 patients with OMF a so-called hyperplastic or early hypercellular stage was encountered with a mixed megakaryocytic-granulocytic pattern without or with minimal reticulin fibres (CMGM/EMS). The histopathology of this early stage OMF as well as the later evolving advanced fibrosclerotic lesions (AMS/OMS) were by morphological aspects alone not distinguishable from cases with CML showing prominent fibrosclerotic alterations. At presentation 5/17 patients with CML displayed already some degree of reticulin fibre formation (EMS). Following serial trephine biopsies in CML with an increased megakaryocyte proliferation (CMGM), a remarkable tendency for myelofibrosis was present. The dynamics of this fibrosclerotic transformation seem to be variable in CML and OMF likewise. However, they are related to abnormal megakaryopoiesis as well as to duration respectively progress of disease, paralleled by corresponding haematological parameters. This longitudinal case control study emphasizes that histopathology of the bone marrow taken at clinical diagnosis may reflect different stages of chronic myeloproliferative diseases and therefore should be always accompanied by relevant clinical and cytogenetic findings to enable a correct diagnosis.

Myelofibrosis with myeloid metaplasia: pathophysiologic implications of the correlation between bone marrow changes and progression of splenomegaly

We undertook a study of 35 cases of myelofibrosis with myeloid metaplasia to assess the histopathologic findings in the bone marrow of patients with this disorder, to ascertain if changes in morphology occurred with time, and to attempt to correlate marrow findings with splenic size. We reviewed 71 bone marrow biopsies and studied 13 splenectomy specimens. Sequential bone marrow biopsies were obtained in 21 cases over intervals ranging from two to ten years (mean, 4 1/2 years). We noted a patchy nature and variable degree of stromal proliferation in most marrow biopsies, and were unable to demonstrate a correlation between the extent of medullary fibrosis and duration of disease, splenic weight, or degree of splenic myeloid metaplasia. We were unable to document a progression of medullary fibrosis as a cause for increasing splenomegaly. However, the alteration in the marrow stroma in this disorder is responsible for the presence of distended marrow sinusoids with intravascular hematopoiesis, a phenomenon we observed in all cases. We believe that this morphological feature, not emphasized by previous investigators, is of significance in understanding the pathophysiology of myeloid metaplasia.

Myelofibrosis With Myeloid Metaplasia: Pathophysiologic Implications of the Correlation Between Bone Marrow Changes and Progression of Splenomegaly

Abstract We undertook a study of 35 cases of myelofibrosis with myeloid metaplasia to assess the histopathologic findings in the bone marrow of patients with this disorder, to ascertain if changes in morphology occurred with time, and to attempt to correlate marrow findings with splenic size. We reviewed 71 bone marrow biopsies and studied 13 splenectomy specimens. Sequential bone marrow biopsies were obtained in 21 cases over intervals ranging from two to ten years (mean, 4 1/2 years). We noted a patchy nature and variable degree of stromal proliferation in most marrow biopsies, and were unable to demonstrate a correlation between the extent of medullary fibrosis and duration of disease, splenic weight, or degree of splenic myeloid metaplasia. We were unable to document a progression of medullary fibrosis as a cause for increasing splenomegaly. However, the alteration in the marrow stroma in this disorder is responsible for the presence of distended marrow sinusoids with intravascular hematopoiesis, a phenomenon we observed in all cases. We believe that this morphological feature, not emphasized by previous investigators, is of significance in understanding the pathophysiology of myeloid metaplasia.

Sequential bone marrow biopsy in chronic lymphocytic leukaemia.

Sequential bone marrow biopsy in chronic lymphocytic leukaemia.

Cell Kinetic Responses in Childhood Acute Nonlymphocytic Leukemia During High-Dose Therapy With Cytosine Arabinoside

Sequential bone marrow aspirates obtained from 10 children with relapsed acute nonlymphocytic leukemia (ANLL) after a high dose of cytosine arabinoside (Ara-C; 1000 mg/sq m) were analyzed by flow cytophotometry. The drug causing elimination of proliferating cells followed by a synchronous wave of cell recruitment. Among individual patients, considerable variation was observed in the degree of recruitment as well as in the time of appearance of the recruitment maximum (range 17-36 hr). However, both parameters appeared inversely correlated with the proliferative status in the bone marrow before treatment. In 6 other patients, cell kinetic responses were studied during treatment with repeated Ara-C injections scheduled individually according to the expected optima of recruitment. Waves of recruitment could be observed during 4-5 consecutive injections. The results suggest that in childhood ANLL, characteristic and individual cytokinetic responses to treatment with high-dose Ara-C can be monitored during therapy. These observations may allow the development of individual treatment schedules.

Cell kinetic responses in childhood acute nonlymphocytic leukemia during high-dose therapy with cytosine arabinoside

Sequential bone marrow aspirates obtained from 10 children with relapsed acute nonlymphocytic leukemia (ANLL) after a high dose of cytosine arabinoside (Ara-C; 1000 mg/sq m) were analyzed by flow cytophotometry. The drug causing elimination of proliferating cells followed by a synchronous wave of cell recruitment. Among individual patients, considerable variation was observed in the degree of recruitment as well as in the time of appearance of the recruitment maximum (range 17–36 hr). However, both parameters appeared inversely correlated with the proliferative status in the bone marrow before treatment. In 6 other patients, cell kinetic responses were studied during treatment with repeated Ara-C injections scheduled individually according to the expected optima of recruitment. Waves of recruitment could be observed during 4–5 consecutive injections. The results suggest that in childhood ANLL, characteristic and individual cytokinetic responses to treatment with high-dose Ara-C can be monitored during therapy. These observations may allow the development of individual treatment schedules.

Assessment of marrow trephine in relation to staging in chronic lymphocytic leukaemia

The prognostic value of the Rai clinical staging system and of a series of bone marrow features was examined in 167 untreated patients with CLL. The patients fell into three prognostic groups: those with limited disease, Rai stages 0 and I, median survival 107 months; those with intermediate disease, Rai stage II, median survival 44 months; and those with extensive disease, Rai stages III and IV, median survival 27 months. Two types of infiltration patterns, diffuse and nodular, were found in the bone marrow, and these were associated with significantly different median survival times: 34 and 115 months respectively.It is suggested that diffuse and nodular patterns in the bone marrow relate to the rapidity of progress of the disease rather than to different stages in CLL.The data suggest that a simplified clinical staging system together with an histological classification reflects both the extent as well as the rate of progression of CLL. The course of CLL may be monitored by sequential bone marrow biopsies to assess changes in the proliferation pattern, in the proliferative cell system and in the tumour mass, which give early warning of shifts in the rate of progression and therefore in the prognosis of the disease.

Progression of bone marrow abnormalities in malignant hemopoietic dysplasia. Evaluation of cytological changes with serial bone marrow samples.

Progression of bone marrow abnormalities in 12 patients with malignant (fatal) hemopoietic dysplasia (HD) was evaluated retrospectively in sequential bone marrow samples, using a simple morphological scoring technique. Special attention was paid to alterations in cytological abnormalities in the myeloid series. Patients who developed acute myeloid leukemia showed increasing scores during the clinical course and a rapid final increase in particularly myeloid nucleolar abnormalities. Patients who did not develop overt myeloid leukemia but died from bleeding and/or infection had low and stable scoring and showed no progression of cytological abnormalities. Repeated evaluation, especially of cytological changes in the bone marrow, and morphological scoring might be useful as predictive tools to estimate the risk of a leukemic transformation in some patients with HD.