Proposal of a morphologic bone marrow response score for imatinib mesylate treatment in chronic myelogenous leukemia

Cytogenetic and molecular analyses are essential disease-monitoring parameters in chronic myelogenous leukemia (CML) treated with imatinib. However, a bone marrow morphologic response has not been defined. We reviewed bone marrow histology and cytology of 39 imatinib-treated patients with CML over 49 weeks and introduced a morphologic response score. A significant positive correlation with a complete cytogenetic response was shown for absence of dry tap (P = .04) and abnormal megakaryocytes (P < 0.001), normalization of cellularity (P = .001) and reduction of fibrosis (P = .01), myelopoiesis:erythropoiesis index (P = .001), blast (P = .001) and basophil count (P < 0.001). The morphologic score integrating these parameters showed an early and late correlation with cytogenetic response. In conclusion, morphologic criteria for complete cytogenetic response in patients with CML treated with imatinib can be defined. Persistent high-level morphologic abnormalities herald early on a high likelihood to fail treatment and call for more intense or alternative therapy.     

In vitro inhibitory effects of imatinib mesylate on stromal cells and hematopoietic progenitors from bone marrow

Imatinib mesylate (IM) is used to treat chronic myeloid leukemia (CML) because it selectively inhibits tyrosine kinase, which is a hallmark of CML oncogenesis. Recent studies have shown that IM inhibits the growth of several non-malignant hematopoietic and fibroblast cells from bone marrow (BM). The aim of the present study was to evaluate the effects of IM on stromal and hematopoietic progenitor cells, specifically in the colony-forming units of granulocyte/macrophage (CFU-GM), using BM cultures from 108 1.5- to 2-month-old healthy Swiss mice. The results showed that low concentrations of IM (1.25 µM) reduced the growth of CFU-GM in clonogenic assays. In culture assays with stromal cells, fibroblast proliferation and α-SMA expression by immunocytochemistry analysis were also reduced in a concentration-dependent manner, with a survival rate of approximately 50% with a dose of 2.5 µM. Cell viability and morphology were analyzed using MTT and staining with acrydine orange/ethidium bromide. Most cells were found to be viable after treatment with 5 µM IM, although there was gradual growth inhibition of fibroblastic cells while the number of round cells (macrophage-like cells) increased. At higher concentrations (15 µM), the majority of cells were apoptotic and cell growth ceased completely. Oil red staining revealed the presence of adipocytes only in untreated cells (control). Cell cycle analysis of stromal cells by flow cytometry showed a blockade at the G₀/G₁ phases in groups treated with 5-15 µM. These results suggest that IM differentially inhibits the survival of different types of BM cells since toxic effects were achieved.     

Transformation of chronic myelogenous leukemia: clinical, morphologic, and cytogenetic features

The morphologic, cytogenetic, and clinical features of 58 patients with transformation of Philadelphia chromosome (Ph1) positive chronic myelogenous leukemia (CML) were evaluated. The patients were divided into two groups on the basis of blood and marrow findings: blast crisis and subacute transformation. The evolution of the leukemic process in 41 patients was classified as blast crisis based on one of three criteria: 30% or more blasts in blood and/or marrow smears, intramedullary focus of blast transformation in a marrow trephine biopsy, or blast transformation in an extramedullary site. The 17 patients with subacute transformation of CML had a deteriorating clinical and hematologic picture but did not manifest any of the criteria for blast crisis. The blood and marrow findings in this group of patients were characterized by several qualitative and quantitative changes, including anemia, thrombocytopenia, decreasing leukocyte count, increasing basophilia, myelofibrosis, dysplastic alterations in hematopoietic cells, and increased blasts which, however, never exceeded 25%. Chromosome abnormalities in addition to the Ph1 were found in 65% of the patients with blast crisis and 86% of the patients with subacute transformation. The 41 patients with blast crisis had a median survival of nine weeks; the 17 with subacute transformation had a median survival of 26 weeks. The shortest median survival for patients with blast crisis, four weeks, occurred in the patients with myeloid blast crisis with chromosome abnormalities in addition to the Ph. The longest median survival, 52 plus weeks, occurred in patients with lymphoid blast crisis with only the Ph1 at transformation.     

The predictive value of bone marrow morphologic characteristics and immunostaining in primary (AL) amyloidosis.

The authors previously demonstrated that bone marrow plasmacytosis in primary (AL) amyloidosis may be monoclonal or polyclonal. However, the clinical implications of the degree of plasmacytosis and its clonality have not been studied. The authors evaluated 62 patients with AL amyloidosis, 40 of whom had monoclonal medullary plasma cells. There was complete concordance between the light chain class of the plasma cells in the monoclonal cases and that of the circulating paraprotein in the 22 cases associated with a paraprotein. The remaining 22 patients had polyclonal plasma cells, although a paraprotein was detected in 6. The degree of plasmacytosis was significantly higher among patients with monoclonal plasma cells and correlated inversely with length of survival. The authors' findings indicate that the quantitation of bone marrow plasma cells in AL amyloidosis by immunoperoxidase studies may predict the clinical course.     

Evaluation of CML model cell lines and imatinib mesylate response: determinants of signaling profiles

Our understanding of the mechanisms by which BCR-ABL drives CML is based, in part, on the use of model cell lines such as the K562 cell line. However, the BCR-ABL translocation may occur via a number of different junction points. In addition, CML is a disease of hematopoietic stem cells and, as a result, can give rise to multiple lineages of tumor cells. In this study, we examined the cellular signaling profiles following imatinib mesylate treatment of eight model CML and ALL cell lines that encompass three BCR-ABL junction points and multiple lineages. We used phosphorylation-specific antibodies and flow cytometry to determine the kinase and pathway activation states with each of the cell lines before and after imatinib mesylate exposure. The comparisons of signaling response profiles, junction points and lineages indicate that cell line lineage rather than BCR-ABL junction point may determine cellular response to imatinib mesylate. The large amount of variation observed among the cell lines suggests that further analysis is required to understand the complex signaling profiles present in CML patients.     

Effects of chemotherapy (busulfan-hydroxyurea) and interferon-alfa on bone marrow morphologic features in chronic myelogenous leukemia. Histochemical and morphometric study on sequential trephine biopsy specimens with special emphasis on dynamic features

We performed a retrospective clinicopathologic study on sequential biopsy specimens from 90 patients with Philadelphia chromosome-positive chronic myelogenous leukemia to study therapy-specific effects of busulfan (28 patients), hydroxyurea (32 patients), and interferon-alfa (IFN-alfa; 30 patients). Bone marrow specimens were evaluated by morphometry after silver impregnation and staining with monoclonal antibodies to identify reticulin fibers, nucleated erythroid precursors, megakaryocytes, and macrophages. To compute dynamics of histopathology implicating corresponding changes in time, relevant indices were calculated. Quantification of megakaryocytopoiesis and its precursor cell population showed a significant increase in the IFN-alfa and busulfan groups compared with the hydroxyurea group. These changes were associated with a development of myelofibrosis during therapy. Although a significant increase in fiber density was detectable in the busulfan group, the progression index proved to be twice as high after IFN-alfa therapy. In contrast, a considerable number of patients displayed a regression of myelofibrosis after hydroxyurea treatment. The general association of the megakaryocyte lineage with myelofibrosis was in line with experimental findings. The mature macrophage population and its activated subfraction revealed a marked proliferation (IFN-alfa group) during treatment. Growth and activation of macrophages may be compatible with their putative function during erythrocytopoietic regeneration and with stimulation of their phagocytic properties.     

Analysis of bone marrow by flow cytometry: morphologic and immunologic aspects

Bone marrow aspirates from 28 healthy donors (18 adults, 10 children) were analysed by flow cytometry (FACS analyser) after purification of low density bone marrow cells (Ld BMC) on a density gradient (d = 1,077) and labeling with 23 anti-hematopoietic cells monoclonal antibodies. Based on physical properties the Ld BMC could be divided into four different populations called E, My, Mo et L including 13 +/- 8%, 33 +/- 15%, 12 +/- 5% and 42 +/- 14% of these cells respectively. The phenotypic analysis of these different populations allowed us to identify in E, erythrocytes (glycophorin A+, Rhesus D+ but negative for early erythroid differentiation markers like the transferrin receptor and/or the FA6-152 antigen); in My, the myeloid lineage (VIM2+, HLADR-); in Mo, the monocytic lineage (CD14+) and some myeloblasts (CD14-, VIM2+, HLADR+) and finally in L, an heterogeneous population including: 1) leucocyte cells, in which 27.3 +/- 9.0% are T cells labelled to the same extent by CD2, CD3, CD5 and CD6, 13.2 +/- 5.9% are B cells assessed by CD19 and CD20, 8.3 +/- 5.7% are Pré-B (CD10+), less than 5% are "natural killer" cells (CD16+ or Leu7+) and finally less than 6% are myelomonocytes (CD14+ or VIM12); 2) the erythroid lineage (Rhesus D+ = 43.7 +/- 12.9%, transferrin receptor and FA6-152+ 36.7 +/- 9.6%); 3) undifferentiated cells or progenitor cells (CD34+ = 6.5 +/- 3.5%); 4) cells unlabelled with any antibodies (approximatively 6%). We have not observed difference between adults and children bone marrow in regard to physical properties properties and all but also immunological markers. Indeed, a significant (p less than 0.02) higher proportion of B cells (CD19 and CD10) was observed in children. These data get from a large number of bone marrow could be used to quantify the imbalance of some bone marrow disorders.     

Rare KIT (CD117) expression in multiple myeloma abrogates the usefulness of imatinib mesylate treatment

Background Imatinib mesylate blocks the tyrosine kinase activity of KIT (CD117) and is an effective treatment for gastrointestinal stromal tumors. In multiple myeloma, KIT expression has been detected by flow cytometry in about 33% of specimens, but no previous immunohistochemical assessment has yet been made of the expression pattern of KIT.Materials and methods We performed immunohistochemical analyses of 100 patients, including 72 with multiple myeloma (MM), 8 with lymphoplasmacytic lymphoma (LPL), 10 with monoclonal gammopathy of undetermined significance (MGUS) and 10 with reactive plasmocytosis. One KIT-positive MM was sequenced using polymerase chain reaction analysis.Results In MM, only 2 cases (2.8%) were KIT positive. The great majority of the cases (97, 2%) did not express the KIT receptor tyrosine kinase. No mutation of the c-kit gene was detected.Conclusions KIT expression is a rare event in MM and not detectable in MGUS and LPL. Therefore, treatment with imatinib is unlikely to be effective in these patients.     

Discordant morphologic characteristics of B-cell lymphomas in bone marrow and lymph node biopsies

Classification of non-Hodgkin's lymphomas in bone marrow biopsies may be hampered by several factors, including histotechnical artifacts. The authors compared the initial morphologic characteristics of B-cell lymphomas and leukemias in uniformly plastic-embedded biopsies of lymph nodes and bone marrow, using the Kiel classification and International Working Formulation (WF). Fifteen of 48 cases with multiple positive biopsies showed major differences, with 9 cases resulting in a discrepant malignancy grade. A discordant low grade was found in the bone marrow of seven cases. Two patients with rapidly progressive bone marrow insufficiency had a reversed pattern. One patient with a composite lymphoma also had disseminated high-grade lymphoma. In 4 of 22 patients with a follicle center cell lymphoma, bone marrow histologic characteristics suggested immunocytoma. The available data suggest that most differences between bone marrow and lymph node biopsies are caused by tumor heterogeneity. Morphologic evaluation of this heterogeneity may be of help in the treatment of individual patients.     

Use of an automated hematology analyzer and flow cytometry to assess bone marrow cellularity and differential cell count

The automated analysis of bone marrow aspirates was performed to estimate the bone marrow cellularity and differential cell count. Total nucleated cell count (TNC) was measured using an automated hematology analyzer. TNC of the bone marrow correlated well with the bone marrow cellularity estimated by microscopic examination (r = 0.590, p <0.001). The bone marrow cellularity was readily confirmed as <30%, from 30 to 70%, or >70% according to TNC. Differential count of bone marrow cells was done with the combination of CD45 monoclonal antibody and propidium iodide using flow cytometry. Excellent correlations were obtained for the population distributions of normoblasts, eosinophils, lymphocytes, and blasts between the results of flow cytometry and manual differential counts. The percentage of mature granulocytes in flow cytometry showed good correlation with the manual percentage of metamyelocytes + neutrophils. The percentage of immature granulocytes by flow cytometry showed good correlation with the manual percentage of blasts + promyelocytes + myelocytes. These results demonstrate that analysis of bone marrow aspirates using an automated hematology analyzer is valuable in the determination of bone marrow cellularity. Moreover, as flow cytometry provides objective findings for percentages of major cell populations, it can serve as a method to automate bone marrow differentials.     

Value and pitfalls of assessing bone marrow morphologic findings to predict response in patients with myelofibrosis who undergo hematopoietic stem cell transplantation

BackgroundAllogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative option for patients with myelofibrosis (MF). Bone marrow (BM) morphologic evaluation of myelofibrosis following allo-HSCT is known to be challenging in this context because resolution of morphologic changes is a gradual process.Patients and methodsWe compared BM samples of patients with myelofibrosis who underwent first allo-HSCT and achieved molecular remission by day 100 with BM samples of patients who continued to have persistent molecular evidence of disease following allo-HSCT.ResultsThe study group included 29 patients: 17 primary MF, 7 post-polycythemia vera (PV) MF, and 5 post-essential thrombocythemia (ET) MF. In this cohort there were 18 JAK2 p.V617F, 8 CALR; 1 MPL, and 2 patients had concurrent JAK2 p.V617F and MPL mutations. The control group included 5 patients with primary MF, one with post-PV MF, one with post-ET MF (5 JAK2 p.V617F; 2 CALR). Following allo-HSCT, both groups showed reduction in BM cellularity and number of megakaryocytes. The study cohort also less commonly had dense megakaryocyte clusters and endosteal located megakaryocytes and showed less fibrosis. There was no statistical difference in BM cellularity, presence of erythroid islands, degree of osteosclerosis, or megakaryocyte number, size, nuclear lobation, presence of clusters or intrasinusoidal location.ConclusionsFollowing allo-HSCT at 100 days, morphologic evaluation of BM in patients with MF cannot reliably predict persistence versus clearance of molecular evidence of MF. Disappearance of BM MF, dense megakaryocyte clusters, and endosteal localization of megakaryocytes are suggestive of disease response.     

An unusual and potentially misleading phenotypic change in a primary gastrointestinal stromal tumour (GIST) under imatinib mesylate therapy

We present a unique case of a 62-year-old female who was diagnosed with a huge gastric gastrointestinal stromal tumour (GIST). Core needle biopsy revealed a cellular spindle cell GIST with diffuse expression of CD117 and CD34. Four mitotic figures were counted in ten available HPFs, indicating a high-risk tumour. Computed tomography scan, performed after 8 months of neoadjuvant imatinib mesylate treatment (Glivec, 400 mg/day), revealed a partial response with reduction of tumour size from 20 × 15 × 15 cm to 13.3 × 8 × 7.6 cm. The patient underwent complete tumour resection. The tumour revealed extensive cystic changes and hyalinisation in 90% of the tumour mass. Multiple viable tumour clones, measuring up to 1 cm, showed highly anaplastic, large epithelioid cells with vesicular nuclei and prominent, centrally located nucleoli, strikingly mimicking the appearance of proximal-type epithelioid sarcoma, anaplastic carcinoma, melanoma or epithelioid angiosarcoma. These anaplastic tumour cells expressed pankeratin (KL-1) and vimentin, but they were completely negative for CD117, DOG-1, CD34, S100, desmin, α-smooth muscle actin, HMB45, CD30, CD45, CK7, CK20 and 34βE-12. Sufficient tissue for molecular analysis was available from the resected tumour. No mutations were detected in KIT exons 9, 11, 13, 17, PDGFRA exons 12, 14, 18, KRAS and BRAF. The patient was alive with no evidence of recurrence 28 months later. To our knowledge, this represents the first report on this unusual type of trans-differentiation in GIST under imatinib therapy. Awareness of this phenomenon would help to avoid diagnostic confusion when evaluating post-treatment resections from GISTs.     

Value of combined morphologic,cytochemical, and immunophenotypic features in predicting recurrent cytogenetic abnormalities in acute myeloid leukemia

To evaluate the reliability of previously described morphologic, cytochemical and immunophenotypic criteria for the identification of acute myeloid leukemias (AMLs) with t(8;21), inv(16)/t(16;16) and t(15;17), 300 cases were reviewed retrospectively. Eighteen AMLs with features of t(8;21), 31 with features of inv(16)/t(16;16), and 22 with features of t(15;17) were identified. Cytogenetic studies were available for 228 cases and identified 15 cases of t(8;21), 30 cases of inv(16)/t(16;16), 18 cases of t(15;17) and 11 cases 11q23 AML. The true positive rate for pre-cytogenetic evaluation was 95% for t(15;17), 88% for inv(16)/t(16;16) and 87.5% for t(8;21). No difference in 5-year survival was identified in the precytogenetic and corresponding cytogenetic disease groups. No specific features to predict 11q23 abnormalities were identified. This study confirms the reliability of a combined morphologic, cytochemical and immunophenotypic approach to the initial classification of AML. Cytogenetic studies are still needed on all cases to identify the small proportion of cases that will be missed by these methods and to identify other significant cytogenetic abnormalities in AML.     

In vitro bone marrow cell culture and cytogenetic analysis in a case of myelodysplasia.

A case of refractory anemia with sideroblastosis and a number of bone-marrow blasts slightly over the limit which separates the I/II and III FAB-subtypes of myelodysplastic syndromes is described. The leukemic-like type of in vitro growth and the multiple karyotypic changes observed in the bone-marrow cells at presentation were both indicators of the malignant nature of the disorder and underlined the importance of these studies in assessing diagnosis and prognosis in patients with preleukemic disorders. The role that the chromosome aberrations, del(11)(q14) and del(18)(q21), both found in 100% of the bone-marrow metaphases examined, may play in the pathogenesis of the disease is also discussed.