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Introduction

The role of the Bone Marrow Biopsy

Dr Fran�oise Delacr�taz, Privat-Docent, Institute of Pathology of the University Hospital (CHUV), CH-1011 Lausanne

Optimal Bone Marrow (BM) examination requires both Aspirate (BMA) and Core Biopsy (BMB).

BMA allows refined cytological examination (such as dysplastic changes, Auer rods, ring sideroblasts) as well as cytochemistry, flow cytometry, cytogenetics and BM cultures.

BMB definitively has some advantages over aspirate : examination of a greater volume of tissue with preserved architecture, assessment of cellularity and modifications of stroma and/or osseous trabeculae, detection of compact and/or fibrotic lesions, identification of granulomas and opportunistic micro-organisms and application of immunohistochemistry (IHC). BMB is mandatory when aspirate is not obtained ("dry aspirate" or "dry tap"). Molecular biology can be performed on both aspirate and biopsy.

The management of BMB requires refined processing so as to allow optimal preservation of morphological details. Plastic embedding - although expensive - is still advocated by some groups but most laboratories are now using paraffin-embedding. Both IHC and molecular biology can be performed on paraffin-embedded material. Gentle decalcification and thin sections (2-3 mu) are required.

The indications for the combined cyto-histological examination (BM aspirate and biopsy) can be summarized as follows :

Lymphomas
Multiple Myeloma (MM) and other gammopathies
Chronic Myeloproliferative Diseases (MPD)
Myelodysplastic Syndromes (MDS)
Acute leukaemia (AL)
Search for metastases
Search for infectious processes (granulomas)
Unexplained cytopenia

Guidelines for BMB examination are presented on Table 1.

BM examination is an important procedure in the diagnosis and management of patients with lymphoma . The role of BM biopsy in lymphomas can be viewed as follows : assessment of initial diagnosis, staging of an already identified tumour, evaluation of residual haemopoiesis, patient follow-up and detection of residual disease after treatment. Different patterns of BM infiltration are described in lymphoma: nodular paratrabecular, nodular random, interstitial, diffuse, and intrasinusoidal.

Nodular involvement is the most common pattern. Interstitial infiltration is usually associated with generalized BM involvement, even though normal haematopoietic tissue and fat are not greatly compromised. Intrasinusoidal infiltration (marginal zone lymphoma, gamma-delta T cell lymphoma) may be difficult to appreciate on routinely stained sections without the aid of immunohistochemical stains. Lymphoma infiltration may be fibrotic and compact, so that aspirate is often not representative. Bilateral biopsy is recommended by some authors.
A panel of antibodies (Ab) reactive on paraffin sections is available. The most commonly used Ab are presented on Table 2. Immunophenotyping by flow cytometry or Fluorescent Activated Cell Sorter (FACS) is an important advantage of BM aspirate over biopsy, if enough cells of interest are present in the BMA material. It is particularly useful for the characterization and classification of small B-cell lymphomas according to the REAL/WHO scheme (Table 3).

The usefulness of molecular biology for diagnosis and follow-up of lymphomas has been reported by several authors.

For proper management of the patient, an integrative approach of all available data is necessary - i.e. peripheral blood and BM morphology, immunohistochemistry (IHC), flow cytometry, molecular biology and clinical data.

The frequency of BM involvement in lymphoma depends on the histological types and immunophenotypes.

Among B-cell lymphomas, 3 groups can be distinguished.
1) B-cell lymphoma with constant BM involvement:
Chronic Lymphocyte Leukaemia (B-CLL), Prolymphocytic Leukaemia (B-PLL), Hairy Cell Disease (HCD).
2) B-cell lymphoma with a high frequency of BM involvement:
Immunocytoma (IC), Follicular (FL), Mantle Cell Lymphoma (MCL), lymphoblastic lymphoma
3) B-cell lymphoma with a low frequency of BM involvement:
Diffuse large B-cell lymphoma of the REAL/WHO classification (high-grade category in the Kiel classification) and Burkitt's lymphoma.

In category 3), disseminated disease with or without BM involvement represents a poor prognostic factor. The marrow is infiltrated in 15-30 % of cases in large B-cell lymphomas. In cases presenting with interstitial and/or minor marrow involvement, tumour cells may be overlooked if IHC is not applied.
T-cell and/or histiocyte-rich B-cell lymphomas are characterized by the presence of reactive cells, sometimes in considerable number, and may be confused with a reactive process, Hodgkin's disease or T-cell lymphoma, if IHC is not performed

Most patients with Peripheral T-cell lymphomas, unspecified and angioimmunoblastic T-cell lymphoma present with generalized disease. BM involvement represents a poor prognostic factor. The diagnosis of a T-cell neoplasm on BM biopsy may be a challenge for the haematopathologist. A biopsy from another site (lymph node) is often necessary to establish the diagnosis.

In contrast to B-cell lymphomas, non specific reactive changes are common features in T-cell lymphomas. They are probably induced by a variety of cytokines produced by neoplastic T-cells. These changes may be very prominent and obscure the neoplastic process. Tumour cells constitute only a minor part of an infiltrate including lymphocytes, plasma cells, histiocytes (occasionally granulomas), macrophages, neutrophils, eosinophils, increased vascularity, fibrosis, and necrosis.

According to our experience and results, TCR- g PCR represents an additional helpful tool for the detection of monoclonality and diagnosis of T-cell lymphomas in BM biopsies.

BM involvement has been reported in 7-30 % of cases Anaplastic Large Cell Lymphoma (ALCL). BM infiltration is indicative of worse prognosis. In some cases, the pattern of infiltration is interstitial with isolated lymphoma cells or very small clusters of tumour cells. The lymphoma infiltration may be overlooked on HE or Giemsa sections and the detection of tumour cells is significantly higher when IHC with CD30 Ab is employed.

In Hodgkin's disease , BMA is not representative and BMB is mandatory since the tumour infiltration is usually heterogeneous and classically associated with severe fibrosis and inflammation. Blocks should be cut at multiple levels.

The most frequent immunoproliferative diseases are Multiple Myeloma (MM), Monoclonal Gammopathy of Undetermined Significance (MGUS), lymphoplasmocytic lymphoma (Immunocytoma).
In Multiple Myeloma (MM) and other gammopathies, tumour load, pattern of infiltration are best appreciated on histological preparations. Immunophenotyping on sections is necessary in cases of non secreting MM (about 1 % of the cases) and in follow-up of MM patients (distinction between residual disease and reactive plasma cell population). According to Bain et al.(1996), non-diagnostic biopsies are obtained in 5-10 % of the cases (early disease, heterogeneous infiltration) so that aspirate and biopsy should be regarded as complementary.

The monoclonality of the plasma cell population can be evaluated according to the
" Light-chain Ratio " (LR) (Peterson et al. 1986).

N of plasma cells reacting for the predominant light-chain
LR = -----------------------------------------------------------------------------
N of plasma cells reacting for the minority light-chain

According to Peterson et al. (1986), a LR of 4 or more corresponds to monoclonality.
In our institution, BMB is also part of the work-up of patients with Monoclonal Gammopathy of Undetermined Significance (MGUS) at diagnosis and during follow-up (progression to MM). Search for amyloid deposits (Congo red staining) should be applied on every BMB performed for gammopathy.

BMB is of diagnostic and prognostic value in the assessment of Chronic Myeloproliferative Diseases (MPD) at presentation and during follow-up. MPD correspond to a group of clonal diseases including Chronic Myeloid Leukaemia (CML), Polycythaemia Vera, (PV) Myelofibrosis with myeloid metaplasia (MMM) (syn.: primary or idiopathic myelofibrosis, agnogenic myeloid metaplasia) and Essential Thombocythaemia (ET). CML is the only MPD characterized by a specific chromosomal abnormality (Philadelphia (Phi) Chromosome). Overlapping cases exist and MMM can supervene during the course of another MPD. BM hyperplasia and fibrosis are common features in MPD. Aspirate is frequently not obtained. "Dry tap" is typical for MMM. BM cellularity, distribution of haemopoietic cell lines, quantification of BM fibrosis and blasts are best appreciated on biopsy.

The diagnosis of MPD is based on a combination of data: clinical status, assessment of PB and BM smears and BM biopsy, cytogenetics (Phi chromosome in CML) and erythroid cultures (particularly important in PV).

The Myelodysplastic Syndromes (MDS) represent a group of clonal diseases characterized by ineffective haemopoiesis. MDS usually present with PB cytopenia of one or several cell lines with a hyper- or normocellular BM in most cases. The diagnosis of MDS relies on a combination of data including clinical status, morphologic evaluation of the peripheral blood (PB) smear, BM aspirate and biopsy as well as cytogenetic analysis. It is often a diagnosis of exclusion and BMB is necessary to exclude other conditions associated with cytopenia, such as Aplastic Anaemia (AA) or a metastatic process. About 12 % of MDS patients present with the hypocellular variant of MDS, thus raising a differential diagnosis with AA. The use of the FAB classification (1982) on sections has allowed a better correlation between cytology and biopsy. Over the last fifteen years, the introduction of BM biopsies in MDS has led to consideration of histological prognostic parameters such as cellularity, fibrosis, Abnormal Localization of Immature Precursors (ALIP) and CD34 positive blasts. The negative prognostic impact of histological parameters such as quantity of blasts, quantity of CD34+ immature cells, marrow fibrosis and ALIP has been demonstrated.
The differential diagnosis between MDS and MPD may be difficult and overlapping cases exist, such as Chronic Myelomonocytic Leukaemia (CMML).

In Acute Leukaemia (AL), BMB has to be performed in cases of unsatisfactory aspirates due to packed and/or fibrotic BM. One of the main roles of the biopsy is to rule out other malignant tumours involving the BM. In most cases a rough characterization of the blast cells can be established by IHC, but most often a precise application of the FAB classification is not possible. Flow cytometry is the most reliable technique to identify the phenotype of blast cells in AL but a panel of IHC markers can be usefully applied on BM sections, including TdT, MPO, CD34, CD68, CD79a, FVW.

Precursor lymphoid cells are positive for TdT (terminal deoxynucleotidyl transferase), whereas peripheral B- and T- cells are negative for that marker. The histological and cytological features and immunophenotypes are identical in lymphoblastic lymphomas and Acute Lymphoblastic Leukaemias (ALL). The distinction is usually based on the percentage of lymphoblasts in the marrow at time of diagnosis (25 % of BM blasts correspond to ALL).

Acute non lymphoid (myeloblastic or myelomonocytic) leukaemias (ANLL) are positive for myeloperoxydase (MPO) and/or for the myelomonocytic marker CD68, whereas lymphoblasts are regularly negative for MPO.

Aberrant expression of lymphoid markers may be observed in ANLL (e.g. aberrant positivity for B-cell markers). CD34 and CD45 can be expressed by both lymphoblasts and myeloblasts.

In daily practice, unexplained cytopenia represents one of the major indications for BM examination. Precise assessment of BM features such as hypocellularity or aplasia (e.g. aplastic anaemia), hyper-/or normocellularity (e.g. peripheral destruction, hypersplenism, inefficient haemopoiesis, HIV-positive patients, Myelodysplastic Syndromes), expanding process in BM (e.g. granulomas, tumour cells, MPD) has to be done on histological sections.

BMB is part of the routine clinical work-up of haematological and oncological patients. Optimal interpretation of BMB requires an integrative approach of all available data.


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