THE VALUE OF IMMUNOHISTOCHEMISTRY
IN THE ASSESSMENT OF BONE MARROW DISORDERS

Attilio Orazi, M.D., FRCPath. and Dennis P. O'Malley, M.D.

MYELODYSPLASTIC SYNDROMES AND MYELOPROLIFERATIVE DISORDERS The value of bone marrow biopsy in this group of disorders is generally well established. It is less known however, that immunohistochemistry can be used to increase the diagnostic accuracy of bone marrow biopsy. This is especially important when marrow fibrosis, by minimizing the quality & quantity of the aspirate sample, produces a serious underestimation of the blast count, which is an important independent determinant of survival and progression to acute leukemia in MDS. In these cases, flow cytometry and cytogenetics can also be unreliable. By allowing for an objective assessment of the frequency of blasts in the bone marrow, immunohistochemistry can be used to predict the clinical behavior in patients with MDS. Although various techniques to assess prognosis have been recently proposed, CD34 immunostaining of bone marrow biopsies remains the most widely used and practical approach. The technique can be used to quantitate the number of marrow blast cells in leukemic conditions characterized by a stem cell phenotype, (i.e. CD34 expression), such as MDS and CML . Myelodysplastic Syndromes Myelodysplastic syndromes are a clinically heterogeneous group of clonal hematopoietic disorders which are divided according to the FAB system into five major categories primarily based on the percentage of blast cells in the peripheral blood and bone marrow, the percent of ringed sideroblasts in the marrow, and the absolute count of monocytes in peripheral blood. These subgroups show different rates of progression to AML and overall survival. In particular, the two "low grade " types of refractory anemia (RA) and refractory anemia with ringed sideroblasts (RARS) are associated with much longer median survival and a lower incidence of progression to acute leukemia than the other three subtypes. However, the FAB classification has severe limitations. Several of the MDS groups are still prognostically heterogeneous and a more comprehensive approach that takes into consideration other parameters (e.g. cytogenetics) is at present being developed by the WHO classification committees. A preliminary WHO classification scheme was published in 1999 (Harris et al, J Clin Oncol, 1999). MDS is usually associated with topographic alterations in the bone marrow and bone marrow biopsy can be used to provide useful diagnostic and prognostic information in these disorders. A prognostically important morphologic finding in MDS is the presence of aggregates of immature myeloid cells in an abnormal central marrow cavity location. This abnormal localization of immature precursors (ALIP) is mainly present in the aggressive MDS subtypes and is associated with a poor prognosis and an increased incidence of progression to acute leukemia. Presence of ALIP, however, is not unique to MDS and has been reported in reactive hematologic conditions (e.g. status post bone marrow transplantation and post induction chemotherapy). In addition, the identification of the presence of ALIP may be compromised by using paraffin sections of excessive thickness or otherwise suboptimal morphology. CD34 can be used as a surrogate marker for the presence of ALIP. Both an increase in the percentage of CD34 positive cells and a tendency of positive cells to form aggregates have been shown to be reliable predictor of leukemic transformation and of survival in MDS cases, irrespective of their FAB subtype. The occurrence of large sheets of CD34 positive blasts can be proposed as a means of recognizing patients with MDS undergoing transition to AML, who are therefore candidates for early aggressive therapy. Immunostaining for CD34 is especially important in two subsets of patients with MDS: MDS with fibrosis (MDS-f) and MDS with hypocellular marrow (MDS-h). The presence of reticulin fibrosis or fatty changes in the bone marrow of these MDS patients, by causing hemodilution and poorly cellular smears, can make the FAB characterization very difficult or impossible. The often low cellular yield of the bone marrow aspirate may also be insufficient to obtain adequate cytogenetic analysis, an important diagnostic technique in the assessment of MDS patients. MDS-f In MDS-f the presence of increased CD34 expression in the marrow is often associated with an aggressive behavior. The use of antibodies reactive with megakaryocytes has shown that these patients have a higher number of these cells than either normal subjects or patients affected by MDS without fibrosis. Furthermore, primary and secondary MDS with fibrosis, although clinically and histopathologically similar, differ in terms of the number of megakaryoblasts which are significantly higher in the primary forms (Lambertenghi-Deliliers et al, 1991). For the differential diagnosis with acute myelofibrosis see previous section on acute leukemia. MDS-f needs also to be distinguished from chronic idiopathic myelofibrosis (dysplasia only in megakaryocytes, giant megakaryocytes, prominent splenomegaly, tear drops erythrocytes) and therapy-related MDS another aggressive MDS often characterized by variably cellular and fibrotic marrows. Therapy-related MDS P53 protein overexpression has been reported in aggressive MDS subtypes and, in particular, in therapy-related cases following alkylating agent chemotherapy. In these cases, p53 expression is associated with increased apoptosis and severe ineffective hematopoiesis. CD34 expression is almost always increased in this aggressive MDS subtype (Orazi et al. 1993, 1996). MDS-h CD34 in conjunction with PCNA (see also section on the use of proliferation associated markers in marrow biopsies) can be used to distinguish hypoplastic MDS from acquired aplastic anemia. The former disorder is characterized by high CD34 and PCNA expression as compared to aplastic anemia (Orazi et al, 1997). Chronic Myeloid Leukemia Chronic myeloid leukemia (CML) is a biphasic or triphasic disease, i.e. the stable phase (SP) either changes to an acute blastic phase (BP) or, more commonly, evolves into an accelerated phase (AP) that later progresses to the terminal blastic phase. The phases of CML are commonly separated according to the criteria established by the International Bone Marrow Transplant Registry, which defines accelerated phase and blastic phase as the presence of more than 10% and 30% blasts in the bone marrow aspirate (and/or peripheral blood) respectively. Cytogenetics often shows evidence of clonal evolution in these aggressive phases. The blastic phase is further subdivided in myeloblastic (60%), lymphoid (20-30%), megakaryoblastic (<10%), and rare subtypes. Applying CD34 to bone marrow biopsy, CML can be separated in its three phases in the majority of cases. The separation is based on the number of blasts as detected by CD34 staining. This approach works since CML is a disease characterized by the presence of a clonogenic population of early progenitors which express CD34. However, in 30% of the cases of acute myeloblastic transformation, the proliferating population is composed of fairly mature myeloblasts in which CD34 is not expressed and therefore CD34 staining is of little use. In addition, cases of megakaryoblastic-type blastic transformation are often negative by this technique. An adequate panel of markers which can be used to identify the type of CML blastic transformation using bone marrow biopsies should include myeloperoxidase, CD68 (PG-M1), TdT, CD79a, CD3, and vWF (or CD61). Chronic Idiopathic Myelofibrosis (CIMF; also termed agnogenic myeloid metaplasia) and Postpolycythemic Myeloid Metaplasia (PPMM) In the early phases of these diseases, the number of CD34 positive cells seen in the bone marrow is usually within normal limits. In some cases with advanced stage, CD34 may be increased, paralleling the increased number of myeloblasts. Between 10 and 20% of patients with CIMF transform to AML. In most of these cases, however, the findings on the marrow biopsy remain unchanged since the acute transformation is usually seen at least initially in extramedullary sites (e.g. spleen). 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