Cell proliferation in non-Hodgkin''s lymphomas. Digital image analysis of Ki-67 antibody staining.

Ki-67 is a monoclonal antibody to a nuclear antigen present in cycling human cells but not in resting cells. The authors have performed immunoperoxidase on non-Hodgkin's lymphomas using Ki-67 antibody in order to correlate proliferation rates with tumor grade and type, and compare Ki-67 staining with S-phase content as determined by flow cytometry. Ki-67 staining of 109 sections was quantitated using a digital image analysis system (CAS 100). There was a significant difference among mean overall Ki-67 staining values in Working Formulation low (13.7%), intermediate (42.6%), and high grade tumors (57.9%, P less than 0.00001). The level of significance improved when a revised grading system was formulated based on proliferative activity, with the inclusion of diffuse large cell lymphomas in the high grade category. Within nodular and a few diffuse lymphomas, there were well-defined proliferation centers in which Ki-67 staining showed no correlation with grade. Flow cytometric DNA determination was performed on 74 specimens, and there was a positive correlation between Ki-67 positivity and S phase content (r = 0.66). It is concluded that Ki-67 staining of tissue sections is an alternative to flow cytometric quantitation of cell cycle activity in lymphomas, and provides the advantage of revealing histologic patterns of proliferation. By including G1 phase cells, Ki-67 staining allows a more complete determination of total cell cycle activity in lymphomas.     

Immunophenotyping of acute lymphoblastic leukaemia in routinely processed bone marrow biopsy specimens

AIMS: To assess the value of immunophenotyping of acute lymphoblastic leukaemia (ALL) in routinely processed bone marrow trephine biopsy specimens and to establish a minimum panel of antibodies to assess lymphoid lineage and enable differentiation from acute myeloid leukaemia. METHODS: 45 routinely processed bone marrow biopsy specimens (formalin fixed, paraffin embedded and mildly decalcified in EDTA) reported to contain leukaemic infiltrates on the basis of cytomorphological and enzyme-cytochemical analysis of bone marrow smears (22 c-ALL, 11 T-ALL, 2 B-ALL, 10 u-ALL (unclassified)) were immunostained by the ABC method with a broad panel of 26 antibodies against various haemopoietic antigens. RESULTS: Staining with antibodies directed against myeloperoxidase and lysozyme showed that seven cases were either biphenotypic or mixed leukaemias (2), or of myelogenous origin (acute myeloid leukaemia (AML)-M1 (2); AML-M4 (2); AML-M5a (1)). Five of these seven cases had been diagnosed initially as u-ALL. Three further cases with no compact leukaemic infiltrates were excluded. ALL was confirmed in the remaining 35 cases. Because of revised diagnoses, the total numbers of ALL subtypes changed (23 c-ALL, 8 T-ALL, 2 B-ALL, 2 u-ALL). Immunostaining of more than 10% of blast cells in at least one case was found with 19 of the 26 antibodies. The most sensitive lineage specific antibodies for diagnosis were found to be anti-CD10 for c-ALL (22/23) and beta F1 for T-ALL (6/8). Expression of aberrant antigens was fairly common--for example, 7/23 cases of c-ALL stained with antibodies against T cell associated antigens. CONCLUSIONS: Immunohistochemical investigation of routinely processed bone marrow biopsy specimens enables reliable detection of ALL subtypes c-ALL and T-ALL. A minimum panel of antibodies, against TdT, CD34, myeloperoxidase, lysozyme, CD10, CD79a, and CD20, and the antibody beta F1, is proposed for the immunophenotyping of acute leukaemia.     

Bone marrow stromal cell changes in haematological malignancies.

Stromal cell numbers from subjects with no haematological disease and those with acute myeloid leukaemia (AML), chronic granulocytic leukaemia (CGL), acute lymphatic leukaemia (ALL) and non-Hodgkin's lymphoma (NHL) were compared to determine their role in malignancy. Frozen sections of trephine biopsy specimens from iliac crests were stained for endogenous alkaline phosphatase activity, endogenous acid phosphatase activity, and, using immunocytochemical methods, for endothelial cells (anti-factor-VIII related antigen) and macrophages and related cells (EBM/11). In granulocytic malignancies, whether acute or chronic, alkaline phosphatase positive reticulum cells (AL-RC) and vascular endothelial cells were generally increased. In lymphoid malignancies, the numbers of AL-RC were generally reduced. Numbers of vascular endothelial cells seemed to be normal in ALL but reduced in foci of NHL. Macrophages are numerous in normal marrow, and their numbers seemed to be normal in granulocytic lesions but were more variable and sometimes reduced in ALL and NHL. Lymphoid malignancies, therefore, have a destructive effect on some stromal elements; granulocytic malignancies are associated with normal or increased numbers of stromal cells. A possible consequence of depleted stromal cells might be slower reconstitution of normal haemopoiesis after treatment. The large numbers in granulocytic malignancies raises the possibility of synergistic stimulation between stromal and neoplastic cells.     

Assessment of cell proliferation in normal and pathological bone marrow biopsies: a study using double sequential immunophenotyping on paraffin sections

The proliferative activity of the haematopoietic and plasma cells in bone marrow was evaluated under normal and neoplastic conditions, by means of a sequential double immunostaining technique, using monoclonal antibody MIB-1 recognizing the cell proliferation-associated nuclear antigen Ki-67, and antibodies against glycophorin-C, myeloperoxidase, factor VIII-related antigen, and immunoglobulin light chains. Fifty-eight B5 fixed, paraffin-embedded bone marrow biopsies were analysed, including 11 normal controls, 10 cases of myelodysplasia, 14 cases of chronic myeloproliferative disorder, eight cases of acute non-lymphoid leukaemia, and 15 cases of myeloma. In normal marrows, the highest proliferative activity was noticed in the erythroid cells (75% to 95%; mean 90%), in comparison with myeloid precursors (15% to 80%; mean 38%), and megakaryocytes (10% to 20%; mean 14%); no Ki-67 positive plasma cells were found. In all investigated haematological disorders, the expression of MIB-1 by erythroid cells was similar to that observed in controls. Similarly, the percentage of MIB-1+ myeloid precursors in chronic myeloproliferative disorders and myelodysplasia largely overlapped the values observed in normals, and comparable values were also found in the blast cells from acute non-lymphoid leukaemia type M1 and M2. These findings suggest that the evaluation of either erythroid or myeloid proliferative activity is of little value in the differential diagnosis between these myeloproliferative disorders. By contrast, the obvious increase of Ki-67 expression of megakaryocytes in chronic myeloproliferative disorders, with labelling also of micro-megakaryocytes, might sustain the diagnosis in controversial cases. Since cases of mature myeloma showed less than 2% of Ki-67 positive cells, evaluation of proliferative activity is of no value in the differential diagnosis with reactive plasmacytosis. The sequential double immunophenotyping for Ki-67 antigen and for haematopoietic cell lineage-associated markers can be applied in a consistent manner to routine bone marrow biopsies to evaluate proliferating cells in normal and neoplastic conditions.     

Expression of Ki-67 nuclear antigen in B and T cell lymphoproliferative disorders.

AIMS: To determine whether the proliferation rates of tumour cells may relate to prognosis and reflect disease activity. METHODS: Blood mononuclear cells from 155 patients with B cell (n = 120) or T cell (n = 35) chronic lymphoproliferative disorders were tested with the monoclonal antibody Ki-67 by indirect immunoperoxidase or immunoalkaline phosphatase techniques. B cell diseases included chronic lymphocytic leukaemia (CLL), CLL in prolymphocytic transformation (CLL/PL), prolymphocytic leukaemia (B-PLL) and non-Hodgkin's lymphoma (B-NHL) in leukaemic phase. The T cell diseases comprised large granular lymphocyte (LGL) leukaemia, T-PLL, and T-NHL. RESULTS: These showed significantly higher proportions of Ki-67 positive cells in T cell (11.2%) than in B cell (2.9%) disorders (p < 0.001). The highest values were found in NHL of both B and T cell types, particularly when low grade disease transformed to high grade. The lowest percentages of Ki-67 positive cells were found in CLL (1.4%) and LGL leukaemia (1.7%); intermediate values were seen in B PLL (3.3%) and T PLL (5.8%). CONCLUSIONS: There is a positive correlation between prognosis and proliferation rates in chronic B and T cell lymphoproliferative disorders. Estimation of Ki-67 in circulating leukaemic cells could be used to determine prognosis in low grade malignancies.     

Angiogenesis in nodal B cell lymphomas: a high throughput study

AIM: To assess the biological significance of vascular endothelial growth factor (VEGF) A, VEGF receptor (Flk-1) and cyclooxygenase 2 (COX2) expression with respect to microvessel density (MVD), proliferative activity (Ki-67), expression of p53 and clinical presentation in a large cohort of nodal B cell lymphomas. METHODS: An immunohistochemical and morphometric study was performed on a validated tissue microarray containing 271 B cell lymphoma specimens, 197 of which included follow-up data. Statistical assessment was done by Pearson's chi(2) test, Spearman's rank correlation coefficient, analysis of variance and survival analysis. RESULTS: 266 (98%) cases were evaluable. Strong VEGF expression was observed in only 20 diffuse large B cell lymphomas (DLBCLs). Flk-1 and COX2 were expressed in 53 and 21 cases, respectively, mainly in DLBCLs, follicular lymphoma (FL) grade 3 and mantle cell lymphomas (MCLs), in a low proportion of cells. MVD decreased in the following order: DLBCLs, FLs, MCLs and small lymphocytic lymphomas/chronic lymphocytic leukaemia (SLL/CLLs). VEGF expression correlated with Ki-67, p53 and COX2 expression in the whole cohort and in DLBCLs. Flk-1 expression correlated with Ki-67 in the cohort and in SLL/CLL and FL grade 1 and 2. COX2 expression correlated with Ki-67 and p53. The analysed angiogenesis parameters did not correlate with clinical parameters or survival. CONCLUSIONS: Angiogenesis plays a differential role in various B cell lymphomas. Aggressive lymphomas express the potential molecular therapeutic targets VEGF and COX2, and have higher MVD. In a few low proliferation-fraction lymphomas, Flk-1 might have a role in proliferative advantage. Therapeutic strategies aimed at angiogenesis should take into account lymphoma heterogeneity.     

Relevance of Ki-67 expression in the classification of non-Hodgkin''s lymphomas: a morphometric and double-immunostaining study

The proliferation of reactive and neoplastic cells was retrospectively assessed in 92 cases of non-Hodgkin's lymphoma by morphometry using a double-immunoenzymatic technique including surface markers and the monoclonal antibody Ki-67. The findings were compared with the histological diagnosis. The overall Ki-67 positivity is not always a good measure of the corresponding corrected values and therefore we recommend that a correction should be made for the total number of complementary lymphocytes in the tumour. Taking the macrophages and the Ki-67 positivity of the reactive cells into account does not generally add any information. There was no difference in reactive cell content between follicular (counted within follicles) and diffuse lymphomas within the tumour areas. The value of the group mean for low-grade follicular (nodular) lymphomas was significantly higher than that of diffuse low-grade lymphomas, but not significantly different from that of intermediate-grade lymphomas. High-grade lymphomas exhibited significantly greater Ki-67 positivity than those of intermediate grade. In 76% of the cases there was significant agreement between malignancy grade (low/intermediate malignant versus high malignant) at 45% corrected Ki-67 counts.     

Expression of functional molecules in non-Hodgkin's lymphoma. Correlation with bone marrow involvement and serum LDH value.

It is well known that non-Hodgkin's lymphoma (NHL) cells express various antigens which are normally involved in a variety of functions. In addition, NHL is diverse in its proliferative capacity. To investigate the relation between these factors and the clinical picture, 45 cases of NHL were studied by immunohistochemistry using snap-frozen materials obtained before therapy. Reactivities with 27 monoclonal antibodies were examined and the results were correlated with clinical findings. The expression of surface mu and ICAM-1 in B-NHLs and CD25 in T-NHLs were significantly associated with bone marrow involvement. B-NHLs without expression of CD21(B2) and T-NHLs with CD25 were seen more frequently in cases with a LDH value of over 500 units/ml. The positivity rate of Ki-67 on B-NHLs was correlated with serum LDH value, NHL histologic classification, and overall survival. These data indicate that immunophenotyping and determination of the proliferative capacity of NHL are of value not only for confirmation of the histopathologic classification of the tumor but also for assessment of clinical behavior.     

Expression of Functional Molecules in Non-Hodgkin's Lymphoma

It is well known that non Hodgkin's lymphoma (NHL) cells express various antigens which are normally involved in a variety of functions. In addition, NHL is diverse in its proliferative capacity. To investigate the relation between these factors and the clinical picture, 45 cases of NHL were studied by immunohistochemistry using snap-frozen materials obtained before therapy. Reactivities with 27 monoclonal antibodies were examined and the results were correlated with clinical findings. The expression of surface μ and CAM-1 in B-NHLs and CD25 in T-NHLs were significantly associated with bone marrow involvement. B-NHLs without expression of CD21(B2) and T-NHLs with CD25 were seen more frequently in cases with a LDH value of over 500 units/ml. The positivity rate of Ki-67 on B-NHLs was correlated with serum LDH value, NHL histologic classification, and overall survival. These data indicate that immunophenotyping and determination of the proliferative capacity of NHL are of value not only for confirmation of the histopathologic classification of the tumor but also for assessment of clinical behavior.     

Erythropoietic activities in acute leukemia and in malignant lymphoma with or without bone marrow involvement

Erythropoietic activities and immature reticulocyte production were investigated in a total of 157 patients (81 men, 76 women, median age = 42 yr, range = 23 to 65 yr) with acute lymphoid leukemia (ALL, n = 31), acute myeloid leukemia (AML, n = 39), or non-Hodgkin's lymphoma (NHL, n = 87), based on assays of the hemogram, red cell indices, reticulocyte subpopulations, and intramedullary erythroid precursors. There were no significant differences in red blood cell (RBC) counts, blood hemoglobin levels, or erythroid precursors between ALL and AML patients. Reticulocytes in AML patients averaged 1.7 +/- 0.8%, which was higher than in patients with ALL (0.8 +/- 0.3%, p <0.01); the proportion of high-fluorescence reticulocytes (HFR) averaged 4-fold higher in AML versus ALL (p <0.01) and the reticulocyte maturity index (RMI) was higher in AML (20.8 +/- 8.3%) versus ALL (12.4 +/- 6.5%, p <0.01). The RMI was higher in NHL patients with bone marrow (BM) involvement (15.6 +/- 9.4%), compared to those without BM involvement (4.3 +/- 2.1%, p <0.01). The proportion of HFR averaged 11-fold higher in NHL with BM involvement versus NHL without BM involvement. In summary, erythropoietic activity is significantly more active in patients with AML compared to ALL and in patients with NHL with BM involvement, compared to NHL without BM involvement.     

Acute lymphoblastic leukaemia: correlation between morphological/immunohistochemical and molecular biological findings in bone marrow biopsy specimens.

BACKGROUND: Although numerous antibodies suitable for use on paraffin wax embedded sections are available for the subtyping of acute leukaemia (acute myelogenous leukaemia (AML) and acute lymphoblastic leukaemia (ALL)) in bone marrow biopsy sections, unequivocal identification of the cell line involved is sometimes impossible. METHODS: Forty eight formalin fixed, paraffin wax embedded bone marrow biopsy specimens that had been decalcified in EDTA were investigated, including 42 thought to exhibit ALL on the basis of bone marrow smears. Five specimens exhibited AML and one biphenotypic leukaemia, as diagnosed immunohistochemically in bone marrow biopsies. Immunostaining was performed with antibodies against relatively specific B and T cell antigens. The blasts were investigated for rearrangements of the immunoglobulin heavy chain (IgH) and the T cell antigen receptor (TCR) genes. RESULTS: Amplifiable DNA was obtained from all 48 specimens. An IgH gene rearrangement was detected in 20 of 23 c-ALL specimens. Four of seven T cell ALL (T-ALL) specimens had a TCR-gamma gene rearrangement, and the one B cell ALL (B-ALL) specimen exhibited a clonal IgH gene. Three of four cases of unclassifiable ALL could be assigned to the B cell lineage on the basis of gene rearrangement analysis. Seven cases originally diagnosed in smears as ALL were rediagnosed as AML (n = 5) or biphenotypic leukaemia (n = 2) because of immunohistochemical reactivity for myeloperoxidase or lysozyme. Two of these AML cases and two of three cases of biphenotypic leukaemia exhibited a monoclonal IgH gene rearrangement. CONCLUSIONS: Acute leukaemia can be subtyped in bone marrow sections with a limited panel of antibodies suitable for use on paraffin wax embedded sections (against CD3, CD10, CD20, CD79a, myeloperoxidase, and lysozyme). In patients with ALL and a diagnostically equivocal immunophenotype, gene rearrangement analysis might indicate whether the B or T cell lineage is involved.     

Bone marrow biopsy changes in acute myeloid leukaemia I: observations before chemotherapy

Pretreatment bone marrow trephine biopsy sections (BMB) from 34 patients with acute myeloid leukaemia (AML) were studied in parallel with bone marrow aspiration smears and peripheral blood films. In four cases marrow aspiration was inadequate and in five cases it was unsatisfactory. In two other cases hypoplastic AML was diagnosed, the aspirate in one suggested hypercellularity and in another it was unsatisfactory. Trephine biopsy was superior to aspiration for the evaluation of fat and marrow cellularity, pattern and extent of blast cell infiltration, homogeneity of the leukaemic infiltrate, frequency of mitoses, residual haemopoietic activity and presence of inflammatory cells. Of the various features studied in the sections, the presence of an increased number of plasma cells and considerable myelodysplasia (MD) appeared to be unfavourable prognostic features. We conclude that trephine biopsies are essential for the diagnosis of hypoplastic AML and are most useful when marrow aspiration is either inadequate or unsatisfactory. They also provide additional information about the bone marrow changes in AML and suggest that some histological features may also have prognostic significance.     

Value of bilateral bone marrow biopsy specimens in non-Hodgkin''s lymphoma.

A study of 260 patients with non-Hodgkin's lymphoma (NHL) who underwent bilateral bone marrow biopsy at initial diagnosis showed marrow disease in 99 (38%) cases. The highest incidence of disease (83%) was seen in small lymphocytic lymphoma (SLL) and the lowest (19%) in diffuse large cell lymphoma (DLCL). Among cases with positive marrows, disease was bilateral in all 15 cases of SLL but in only 10 of 20 (50%) of the DLCL cases. In 30 of 99 (30%) positive marrows disease was unilateral. Follicular lymphomas were strongly associated with a paratrabecular pattern, with 40 of 45 positive cases showing this. Discordant histology was seen in six of 20 positive cases of DLCL and two of 37 positive cases of follicular small cleaved cell lymphomas (FSCCL). A bone marrow aspirate was positive in only 56 of the 99 (57%) cases. Peripheral blood disease was present in 15% of the bone marrow positive cases and in 6% of the cases overall. The incidence of marrow disease varies with the histological subtype of lymphoma. The paratrabecular pattern is associated with follicular lymphoma, and bilateral biopsy specimens increase the positivity rate in most subtypes of NHL.     

Peripheral T-cell lymphoma: a clinicopathological study of 41 cases and evaluation of the prognostic significance of the updated Kiel classification

A total of 41 non-cutaneous peripheral T-cell lymphomas were classified following the updated Kiel classification. Of these, 20 cases belonged to the low-grade group (T-cell chronic lymphocytic leukaemia, 3; lymphoepithelioid, 5; angioimmunoblastic, 4; pleomorphic small cell, 8) and 21 to the high grade group (pleomorphic medium and large cell, 11; immunoblastic, 3; large-cell anaplastic Ki-1 positive, 7). Seventy per cent showed a CD4+/CD8-phenotype, 39% a defective phenotype and 88% an activation phenotype. Eighty per cent had B-symptoms, 63% hepatomegaly, 48% splenomegaly and 26% had involvement of more than three lymphoid areas. Bone marrow was infiltrated in 34% central nervous system in 4%, lung in 12% and skin in 14.6%. Seventeen per cent presented with extranodal disease and 82.8% had stage III/IV disease. Hypergammaglobulinaemia was found in 29%, hypercalcaemia in 7%, raised LDH serum levels in 58% and HTLV-I antibodies in only one case. Of the 37 treated patients 18 (48%) achieved a complete remission, but 33% relapsed. Mortality was 59% and actuarial overall survival at 38 months was 0.32. In the comparison of the clinical, analytical and immunophenotypic variables and outcome between low and high grade groups, only the average of bone marrow infiltration in the low grade and stage I–II, presence of defective phenotypes and higher Ki-67 positivity in the high grade group were significantly different. In the statistical studies, extranodal prentation and the failure to achieve a complete remission were the only variables that influenced mortality; there weere no significant differences in the general features of the low and high grade groups and only minor differences were found in the immunoblastic and angioimmunoblastic subgroups. There were no differences in the actuarial survival between the low and high grade groups, among the subgroups of the Kiel classification, among stages I to IV, between patients with or without B-symptoms, with or without defective phenotypes, Ki-67 positivity over or under 60%, or among different CD4/CD8 phenotypes. The updated Kiel classification did not separate groups with a prognostic significance.     

Determination proliferative activity of myeloma cells in histologic material

The assessment of proliferative activity of plasma cells in multiple myeloma has prognostic significance. The percentage of the proliferating plasma cells (plasma cell labelling index--PCLI) at the time of the diagnosis correlates with survival, and is increased during transition from the plateau phase to the relapse. Flow cytometry and immunofluorescence examination of bone marrow aspirates are used for the assessment of the PCLI. This study describes a method for evaluation of PCLI in formalin-fixed, paraffin embedded material. Bone marrow biopsies from 31 patients with relapsing (n = 10) and newly diagnosed (n = 21) multiple myeloma were examined by double immunohistochemical staining: anti-CD 138 (Syndecan 1) and anti--Ki-67. The percentage of plasma cells positive for Ki-67 were counted by an image analysis system. New cases of multiple myeloma showed 0.7 to 12.7% positivity of Ki-67 labelled plasma cells (median 4.75), the relapsing cases showed 4.4 to 22.4% positivity (median 7.75). Statistic analysis revealed prognostic significance (p = 0.046). This study presents a new method for assessment of proliferative activity of plasma cells, which can be used on archived formalin-fixed, paraffin embedded material.     

Proliferative rates of non-Hodgkin's lymphomas as assessed by Ki-67 antibody

The Ki-67 antibody, a monoclonal antibody that reacts with nuclei in actively proliferating cells, was used in an immunohistochemical study to assess the growth fractions of non-Hodgkin's lymphomas and related disorders. The lowest proliferative indices were found in small lymphocytic lymphoma/chronic lymphocytic leukemia and intermediate lymphocytic/mantle zone lymphoma. An intermediate proliferative index was seen in the follicular lymphomas and diffuse small cleaved cell and diffuse mixed cell lymphomas. A high index was seen in the diffuse large cell lymphoma and lymphoblastic lymphoma. The highest and most consistent proliferative index was seen in small noncleaved cell lymphoma. Cases of reactive follicular hyperplasia had a significantly higher proliferative index than those of follicular lymphoma. We conclude that the Ki-67 antibody has great utility in providing an estimate of the proliferative rate of non-Hodgkin's lymphomas. Prospective studies may show this information to have prognostic value independent of histologic classification.     

Expression of DNA-PKcs and Ku86, but not Ku70, differs between lymphoid malignancies

We have investigated the role of DNA-dependent protein kinase (DNA-PK) and related it to proliferation and maturation of different lymphoid malignancies. DNA-PK and Ki-67 protein content was investigated in tumour samples of lymphoid malignancies, obtained from patients with low- and high-grade lymphomas, acute lymphoblastic leukaemia and multiple myeloma. All patients were untreated before sampling. Normal bone marrow, reactive tonsillar tissue and ordinary lymph node tissue were used as controls. We show here that lymphoid malignancies display differences in DNA-PK protein expression. Low-grade lymphoma, appearing as chronic lymphocytic leukaemia (CLL) displayed a significantly lower frequency of cells staining positive for DNA-PKcs and Ku86, but surprisingly not for Ku70, compared with acute lymphoblastic leukaemia (ALL) cells. When material from individual CLL patients was investigated, cells from lymph nodes showed a higher frequency of positive cells with respect to all DNA-PK subunits, compared with CLL cells infiltrating the bone marrow. High-grade lymphoma lymph node samples showed an increased frequency of cells staining positive for DNA-PKcs, Ku86 and Ki-67 compared with lymph node samples from low-grade lymphoma patients. Again, no difference in the Ku70 levels between the two lymphoma entities was noted. In multiple myeloma, the frequency of cells with positive staining for DNA-PKcs was similar to that detected in ALL and high-grade lymphoma. We conclude that with the exception of multiple myeloma, expression of DNA-PK coincides with the degree of maturation of lymphoid malignancies. In low- and high-grade lymphoma, DNA-PK is associated with the proliferation rate.     

Focal lymphoid aggregates (nodules) in bone marrow biopsies: differentiation between benign hyperplasia and malignant lymphoma--a practical guideline.

AIMS: To provide practical guidelines for the differentiation between benign and malignant focal lymphoid aggregates (lymphoid nodules) in routinely referred bone marrow trephine biopsies, using a synoptic approach including clinical data and histological workup. METHODS: For easy identification of very small lymphoid infiltrates the chloroacetate esterase stain was applied as a screening procedure. This allowed the identification of 491 formalin fixed, paraffin wax embedded specimens with one or more lymphoid nodules. Examination of lymphoid infiltrates included such variables as histotopography, demarcation, cytology, reticulin fibres, and immunohistochemistry with a set of monoclonal antibodies (CD20, CD45R, CD45R0, CD3, CD43). Evaluation of clinical and morphological data was carried out independently. In case of malignant lymphomas, a correlation with corresponding lymph node findings was made. RESULTS: 352 patients had benign focal lymphoid aggregates usually associated with systemic autoimmune diseases, chronic myeloproliferative disorders, toxic myelopathy, and viral infections. Discrete nodular infiltrates of (small cell) malignant lymphomas (n = 93) simulating benign hyperplasia were found in chronic lymphocytic leukaemia, germinal centre cell lymphomas (CB-CC), and lymphoplasmacytic/cytoid lymphomas (LPI). In addition to immunoreactivity, certain histological variables proved distinctive. These were: (1) histotopography, that is, localisation of the lymphoid aggregates within the bone marrow space; (2) relation to the surrounding tissue: margination or interstitial spillage of lymphoid cells; and (3) increase in reticulin fibres. CONCLUSIONS: A combined diagnostic procedure identifying several distinctive features, in particular histotopography and immunohistochemistry, provides a most promising way of discriminating reactive from neoplastic lymphoid nodules in the bone marrow.     

Biological characteristics of multiple myeloma

On a series of thirty trephine bone marrow biopsies from patients with multiple myeloma, the authors evaluated expression of markers of cell proliferation or of its blockade (Ki-67, PCNA, topoisomerase IIa, cyclin D-1, AgNOR, and p27kip1) and markers indicating multidrug resistance (P-170 and Bcl-2). Expression of Ki-67 and of topoisomerase IIa was unfrequent. Marked positivity of PCNA was expressed in about one third of cases, negative staining was exceptional. No expression of cyclin D-1 was noted. Positivity of p27kip1 was frequent. P-170 was demonstrated in a small number of cases, Bcl-2 was strongly positive in most cases. The results characterise multiple myeloma as a tumour with low proliferation rate and, simultaneously, with high resistance to apoptosis.     

Reticulin fibre content of bone marrow infiltrates of malignant non-Hodgkin's lymphomas (B-cell type,low malignancy) — a morphometric evaluation before and after therapy

Summary A morphometric study was performed on bone marrow infiltrates of non-Hodgkin's lymphomas (B-cell type, low malignancy) to evaluate the content of argyrophilic (reticulin) fibres in the various subtypes before and after therapy. In congruence with the corresponding lymph node lesions, subtypes consisted of lymphocytic lymphoma — chronic lymphocytic leukaemia (CLL,n = 39), centroblastic-centrocytic lymphoma (CB-CC,n = 35), lymphoplasmacytoid immunocytoma (LPI,n = 22) and finally hairy cell leukaemia (HCL,n = 21). In comparison with control specimens, morphometric measurements on trephine biopsies (initial staging procedure) disclosed a borderline or minimal increase in reticulin in CLL and moderate fibrosis in CB-CC and LPI, whereas HCL had the greatest increase in fibres. The marrow surrounding focal or patchy lymphoma infiltrates of CLL and CB-CC displayed no relevant changes in fibre density with respect to the control samples. Following chemotherapy, repeated trephine biopsies (restaging procedure) were obtainable from 38 patients. There was no significant decrease in the fibre content of CLL, CB-CC and LPI infiltrates. In HCL an incomplete reduction was recorded after interferon treatment. So-called benign lymphoid lesions may be distinguished from focal-patchy infiltrates of CB-CC and LPI not only by showing a central localization, but also by the absence of significant amounts of reticulin. However, considering the density of the reticulin fibres, a clear-cut discrimination of these lymphoid aggregates from an early nodal-central growth pattern of CLL is not feasible in many cases.