Bryon E. Roberts

NON-HODGKIN'S LYMPHOMA: CASE CONTROL EPIDEMIOLOGICAL STUDY IN YORKSHIRE

This paper reports the results of a case control study of non-Hodgkins lymphoma in the Yorkshire Health Region. In all, 437 cases and 724 controls were interviewed. Risk factors associated with past skin conditions, family history of cancer and infectious mononucleosis, aspects of social life and contact with wood dust and epoxy glues all emerge. A comparison of high and low grade morphological forms of disease reveal contrasting risks and suggest separate aetiologies for these conditions.

Decreased T helper cells in the myelodysplastic syndromes

Summary. The myelodysplastic syndromes (MDS) are disorders in which the abnormalities are thought to be confined to cells of the myeloid series. However, examination of peripheral blood from 56 patients with MDS showed that the majority had low lymphocyte counts. In a detailed study of 25 patients, using OKT3, OKT4 and OKT8 monoclonal antisera together with SRBC rosettes, it was shown that there is a consistent and significant reduction in peripheral blood T lymphocytes. This decrease was primarily confined to the OKT4‐defined helper subpopulation. There is a consequent relative increase in the OKT8‐defined subset but in absolute numbers however, suppressor cells are reduced compared to normal particularly in those patients with lymphopenia. No correlations between T cell abnormalities and the different morphological groups of MDS were found. The possible implications of the reversed helper/suppressor ratio in the pathogenesis of MDS are discussed.

Aetiologic factors in lymphoid malignancies: A case-control epidemiological study

A prospective case-control study of the aetiologic factors involved in the production of lymphoid malignancies has been conducted within a defined geographical area covering six health districts in the Yorkshire Region. Among the aspects investigated were past medical events, occupations and certain social factors. A number of possible causal relationships have been identified including Jewish religion, past solvent exposure and ingestion of amphetamines, although the latter did not achieve statistical significance in this study. In addition several new associations have been identified, most notably with the occurrence of adult eczema/dermatitis and with treatment by radiation or steroids. The feasibility of conducting such a broadly based epidemiological investigation has been established.

Transient and persistent expansions of large granular lymphocytes (LGL) and NK‐associated (NKa) cells: the Yorkshire Leukaemia Group study

Summary. A survey of 870 different adult blood samples (primarily from patients with non‐haematological disorders) found that 269 (31%) had increased proportions (>25%) and/or absolute numbers (>1.0 × 109/l) of morphologically‐defined large granular lymphocytes (LGL), and/or pheno‐typically‐defined NK‐associated (NKa) cells. Of these, 112 were re‐analysed at least 6 months after initial presentation and were classified as‘persistent’(92/112) or ‘transient’(20/112) according to whether or not the original abnormality was still present. Lymphocyte counts in most patients with persistent abnormalities were within normal limits (18/92) or slightly increased (68/92), with only six having a lymphocytosis exceeding 10.0 × 109/l. With the exception of five persistent LGL expansions in which the granular lymphocytes did not express NKa determinants (designated LGL+NKa‐), the remaining 87 cases could be phenotypically grouped according to their primary abnormality as CD8+NKa+ (n = 33), CD4+ NKa+ (n=14), CD8dim+NKa+ (n=7) or CD8−NKa+ (n=33). TCR genotypic studies in 58 patients showed that the 16 patients with rearranged TCR components were restricted to the CD8+NKa+ group and that, in most of these, the CD8+ fraction showed abnormal relative CD16/CD56 expression. Persistent neutropenia (n=15) also appeared to be associated with primary abnormalities of CD8+NKa+ cells (12/15). with 10 of these additionally showing rearranged TCR genes. In contrast, persistently increased CD8dim+NKa+ and CD8–NKa+ components did not appear to phenotypically differ from their corresponding ‘counterparts’in normal bloods or in patients with transient LGL/NKa+ abnormalities. This survey has therefore established that persistent LGL/NKa+ abnormalities are considerably more common than suggested in published work, that a high proportion of patients with expanded CD8+NKa+ components, with quite diverse clinical histories, show evidence of clonal lymphoid populations, and that the clonal nature of such disorders appears to be associated with abnormal NKa phenotypic patterns.

Diagnostic and prognostic factors in acute monocytic leukaemia: an analysis of 51 cases

Summary. Diagnostic features (cytochemistry, immunophenotyping and serum biochemistry) were examined in 51 cases of acute monocytic leukaemia (AMoL). Peroxidase, Sudan black B and alpha naphthyl acetate esterase (ANAE) cytochemical reactions were unrelated to morphological (FAB groups M5a and M5b) or immunological subtype. ANAE cytochemistry, however, indicated that AMoL cases could be subdivided into those with typical (M‐type) reactions and those with insignificant staining or monocytic ANAE isoenzymes (defined by IEF). All cases were phenotypically CD13/CD33 positive and, with one exception, had > 30% HLA‐DR positive cells. Membrane CD14 expression was insignificant or variable in 33% of M5a cases in contrast to 23/24 M5b cases which showed high proportions of CD14‐staining cells with at least two monoclonal antibodies. Serum lysozyme, LDH and beta‐2 microglobulin (β2m) were increased in 88%, 68% and 81% of cases respectively but, with the exception of statistically higher lysozyme levels in CD14+ cases, were unrelated to the morphological, cytochemical or immunological diagnostic subgroups. Clinical and diagnostic features were also examined as possible prognostic indicators. The morphological, cytochemical and immunological subgroups of AMoL were not found to be of prognostic relevance but age (P=0.004), renal failure (P=0.005) and serum β2m levels (P=0.002) were related to patient survival. Moreover, renal failure and serum β2m remained significant (P=0.012 respectively) when age was taken into account and were shown to be independent prognostic variables.

Cytochemical and immunological characteristics of acute monocytic leukaemia

Summary. Immunological and cytochemical findings are presented from 12 cases of morphologically unequivocal acute monocytic leukaemia (AMoL). The results indicate considerable heterogeneity and three main non‐morphological subgroups were identified. The blast cells from half the patients were positive for the presence of both cytoplasmic alpha naphthyl acetate esterase (ANAE) and monocyte‐associated membrane determinants whereas the cells from three cases lacked detectable monocytic antigens despite the presence of strong cytochemical ANAE activity. A further three cases expressed monocytic antigens but were cytochemically unreac‐tive for ANAE. These cytochemical results, which were extended by electrophoretic studies of ANAE isoenzymes, suggest that the absence of significant cytoplasmic ANAE activity does not preclude the diagnosis of AMoL and that serum lysozyme estimations may be of value in the recognition of irnmunocytochemically‐atypical cases.

Yorkshire Case Control Study of Leukaemias and Lymphomas Parallel Multivariate Analyses of Seven Disease Categories

Between 1980 and 1986 a case control study of leukaemias aid lymphomas in Yorkshire conducted face to face interviews on 1362 cases and 2442 age and sex matched hospital controls. Case diagnoses were histopathologically confirmed and grouped into non-Hodgkins lymphomma (NHL), Hodgkins Disease (HD), malignant lymphocytic lymphoma (MLL.), chronic lymphocytic leukaemia (CLL), acute myeloid leukaemia (AML), chronic myeloid leukaemia (CML) and acute lymphoblastic leukaemia (ALL). Multivariate analyses were completed on each separate disease to evaluate risk factors relating to past medical history, occupation, environmental exposures and social contact variables. The results show a significant association (with OR adjusted for other risk factors) between a family history of leukaemia/lymphoma and HD (OR = 4.29), NHL (OR = 1.98) and AML (OR = 6.36). For HD other cancers in the family also conveyed a significant risk (OR = 1.61). Use of heart drugs l(and heart disease) was linked to the chronic leukaemias (CML, CLL). A previous cancer and NHL, CLL and AML were associated even after adjustment for radiotherapy. A complex set of risk factors including prior skin lesions and steroid use showed significant links with HD, NHL and CLL., Increasing risk of NHL was linked to small family size. A significant excess of NHL cases reported exposure to glues and similarly ALL cases with agricultural chemical exposure. There present data provide both confirmatory and novel results. Overall they concur with the hypothesis of a multifactoral aetiology encompassing both genetic and immunological components.

Flow cytometric analysis of membrane CD11b, CD11c and CD14 expression in acute myeloid leukaemia: relationships with monocytic subtypes and the concept of relative antigen expression

Abstract: Blast cells from 26 cases of acute myeloid leukaemia (AML) were examined, by single and “two‐colour” flow cytometry, for relationships between membrane CD11b (monoclonal antibody OKM1), CD11c (KB90) and CD14 (Leu‐M3). Increased expression of all three determinants was associated with myelomonocytic leukaemias, with their relative diagnostic value in discriminating monocytic (M4 and M5) from non‐monocytic (M1, M2 and M3) subtypes being CD14 > CD11c > CD11b. However, the results also indicated, because of the heterogenous expression of CD11c in particular, and to a lesser extent CD11b, that the patterns or histograms of fluorescent staining were potentially more informative than an empirical subdivision of blasts into positive and negative sub‐populations. In addition, analysis of phenotypic correlations by simultaneous two‐colour fluorescence showed that the expression of CD11b and CD11c determinants by leukaemic myeloid blasts was highly correlated, in contrast to the expression of CD14 and CD11c which were relatively independent. Consequently, CD11c + myeloid blasts almost always co‐expressed CD11b whereas CD14 + cases of AML often comprised CD14 + CD11c + and CD14 + CD11c‐ subpopulations. It is concluded from these observations that CD11c immunophenotyping is a useful supplementary investigation, particularly in CD14‐ cases of myelomonocytic leukaemia. However, it is also apparent that the presence of membrane CD11c per se is not lineage‐specific and that the level of expression is perhaps a more discriminatory factor.

Patterns of CD16 and CD56 expression in persistent expansions of CD3+NKa+ lymphocytes are predictive for clonal T-cell receptor gene rearrangements

Summary. Phenotypic characteristics, and correlations between the expression of membrane NK‐associated (NKa) determinants (CD11b, CD16, CD56 and CD57) and T cell receptor (TCR) genotypic patterns, were examined in 25 patients with persistent (>6 months) expansions of CD3+WT31+NKa+ (CD8+ and CD8dim+) lymphocytes. These studies showed that distinct NKa phenotypic profiles were restricted to cases with rearranged TCR configurations and that clonal CD3+NKa+ components could be predicted in most cases by assessing relationships between membrane CD16 and CD56 expression. For all normal NKa subpopulations, there was a high correlation (P<0·0001: n= 31) between the expression of these two membrane determinants. Markedly increased CD16 expression by CD3+NKa+ cells, in relation to CD56 (i.e. a high CD16:CD56 ratio), was found exclusively in cases with rearranged TCR (13/16 cases); 2/3 of the remaining cases showing significantly reduced CD16:CD56 ratios and high (>2·0) CD3+CD56+ absolute numbers. In contrast, 7/9 of the germline TCR cases had a normal CD16:CD56 ratio and 2/9 a decreased ratio with low (<1·0) CD3+CD56+ absolute numbers. A high ratio of CD16:CD56 expression by CD3+NKa+ lymphocytes was therefore informative for 82% of TCR rearrangements in this series: and analysis of CD16 and CD56 expression was predictive for germline and rearranged TCR configurations in 24/25 persistent CD3+NKa+ expansions.

Diagnostic differentiation of chronic B‐cell malignancies using monoclonal antibody L161 (CD1c)

The expression of membrane CD1c, as defined by monoclonal antibody L161, was examined on malignant lymphoid cells from 191 cases of chronic lymphoproliferative disease and on eight ‘normal’ enriched tonsil B‐cell extracts. Of 79 cases of chronic lymphocytic leukaemia (CLL) studied, 77 showed low (<20% positive cells) CD1c expression whereas 63/71 (89%) cases of B‐PLL, HCL and B‐NHL showed increased CD1c+ (but not CD1a or CD1b) components. In contrast, malignancies corresponding to terminal stages of B‐cell differentiation (immunocytoma and myeloma) generally showed low CD1c expression as did lymphoid cells from 10 cases of post‐thymic malignancy. Although there was some correlation between the expression of membrane CD1c and immunoglobulin (SIg) light chain densities (P<0.001), it is relevant in diagnostic terms that seven cases of B‐NHL, with low SIg staining intensities more typically associated with CLL were CD1c+. CD1c expression was not, however, correlated with the presence of CD23 or FMC7 determinants but did show a similar pattern of expression to that previously reported for beta‐2 microglobulin. Determination of cellular CD1c by APAAP immunocyto‐chemistry confirmed the presence of higher antigen densities in malignant B‐cells at intermediate/late stages of differentiation and this interpretation was further supported by the finding that the majority of phenotypically mature tonsil B‐cells were also CD1c+. The determination of CD1c expression by malignant B‐cells may therefore be of particular value in the diagnostic differentiation of chronic lymphoproliferative disorders.