Evaluation of Immunostaining in Detecting Occult Lymphoma in Bone Marrow Biopsies

Abstract

Bone marrow biopsy (BMB) is a standard procedure in the evaluation of patients with NHL. One of the major advantages of BMB, compared to BM aspirates, is its distinct characteristic in identifying the histotopography or pattern of distribution of the lymphoid infiltrates. It is also of value in detection of small focal lesions accompanied by fibrosis that may not be readily detected in aspirate, thereby increasing the yield of possible diagnosis and becoming superior to aspirate study. Bone marrow biopsy, aided by immunohistochemistry, can also serve to establish or confirm a primary diagnosis as well as to determine the extent of disease dissemination for staging purposes. The availability of a broad panel of antibodies suitable for paraffin-embedded tissues, in conjunction with less damaging decalcification procedures, enables us to perform complete immunophenotyping on bone marrow trephines and allows for classification of lymphoma infiltrates. Immunohistochemistry is an indispensable procedure for establishing the diagnosis of lymphoma or recognizing a subtle, morphologically occult lesion, this is achieved by a well-fixed and processed BM material with optimal morphology combined with prudently applied and sensibly interpreted immunohistochemistry. It is also useful in distinguishing between reactive and neoplastic lymphoid infiltration. The aim of our study is to establish the role of IHC in detecting occult (histologically inapparent) lymphoma in routinely processed bone marrow trephine specimens, and to distinguish between reactive and neoplastic lymphoid infiltration. This study was carried on one hundred newly diagnosed and follow up adult cases having lymphoid neoplasm (group I= 80) or those suspected to have lymphoid neoplasm (group II= 20); 45 of them were males and 55 were females with a male to female ratio of 1:1.2. Their ages ranged from 20 to 81yrs. All patients were subjected to complete history taking, clinical examination for organomegaly and lymphadenopathy. Diagnostic workup for cases included: CBC, BM aspiration and flow cytometric IPT for infiltrated cases, BM trephine biopsy and immunohistochemistry using selected panel of monoclonal antibodies: CD20, CD79a, CD3 and Bcl-2. According to H&E results, patients were classified into: 49/100 cases with obvious lymphoid infiltration that were further subdivided by IHC into 30 cases with malignant lymphoid infiltration and 10 cases with reactive infiltration. Among 51/100 cases that showed no obvious infiltration by H&E: 49 cases showed concordant no infiltration by IHC while 5 cases with occult infiltration (3 malignant +2 reactive lymphoid infiltrations). IHC results were available in 91/100 cases, it showed involvement in 45 cases , of these 40 cases showed lymphomatous infiltration by both routine histology (H&E) and IHC . In the remaing 5 (5.5%) cases, IHC detected additional lymphomatous involvement compared with histology alone (occult). Particular interest was focused on the histotopography of lymphoid infiltration in combination with immunohistochemical reactions in providing practical guidelines for diagnosis, a uniform pattern with predominant B or T lymphocytes is consistent with malignant lymphoma. They were characterized by being multiple (≥2 patches) or with paratrabecular pattern. In contrast, benign lymphoid aggregates show mixture of B &T lymphocytes with normal ratio of T to B cells being up to 6:1, these cells are scattered randomly or arranged as small non trabecular aggregates in the bone marrow. Of all 45 infiltrated cases (33 malignant +12 reactive) by IHC, CD20 showed positivity in 37(82.2%) cases: 29(87.9%) of them were of malignant lymphoid infiltration and the remaining 8(66.7%) cases were of reactive lymphoid infiltration. CD3 showed positivity in 25(56.8%) cases: 12(100%) proved to be reactive lymphoid infiltration i.e. in all reactive cases and 13(40.6%) cases proved to be malignant lymphoid infiltration. The ratio between CD20 and CD3 was used to differentiate whether the lymphoid infiltration was reactive or malignant, together with morphological and topographic appearance. Normal ratio (1:1 up to 1:6) was found in 8(100%) cases i.e. all of reactive lymphoid cases. Decreased ratio i.e CD20<CD3 (<1:6) was found in only one (14.3%) case of T-NHL, while increased ratio i.e CD20>CD3 (>1:6) in 6 (85.7%) cases of B-NHL.