Non-Hodgkin's Lymphomas

A current hypothesis related to non-Hodgkin's lymphoma states that the wide variety of cytologic types in this disorder reflects morphologic alterations during different stages (Gs, S, and G2) of the cell cycle involved in the blastogenic transformation of normal lymphocytes. In our investigations of biochemical and structural changes during lymphocyte transformation, we have used correlated stereologic morphometric analysis, assessment of chromatin organization, and autoradiography of human peripheral T-lymphocytes labeled with 3H-thymidine and stimulated with concanavalin A. These studies have confirmed that the characteristic

A current hypothesis related to non-Hodgkin's lymphoma states that the wide variety of cytologic types in this disorder reflects morphologic alterations during different stages (Gs, S, and G2) of the cell cycle involved in the blastogenic transformation of normal lymphocytes. In our investigations of biochemical and structural changes during lymphocyte transformation, we have used correlated stereologic morphometric analysis, assessment of chromatin organization, and autoradiography of human peripheral T-lymphocytes labeled with 3H-thymidine and stimulated with concanavalin A. These studies have confirmed that the characteristic DESPITE THE FACT that no one disputes the important role in surgical pathology that nuclear features play in categorizing non-Hodgkin's lymphomas, little attention has been focused on cellular events that influence or directly contribute to nuclear size and shape or the configuration and distribution of condensed chromatin masses within the nucleus in normal and neoplastic lymphocytes. Though it is acknowledged that the nucleus is the repository for genetic information controlling cellular differentiation, it is generally not appreciated that the nucleus is an organelle that itself undergoes modifications during differentiation. Quantitative assessments of nuclear organization are now being increasingly reported,1-9 some of which involve lymphocytes.1022 Such investigations may have relevance to the understanding of the morphologic heterogeneity of the lymphomas, particularly those studies dealing with large-scale alterations in chromatin organization during differentiation3'5 19,21,22 and the cell cycle. [7][8][9][23][24][25] Mechanisms involved in the differentiation of normal lymphocytes and the resulting morphologic ex-From the Canadian Tumour Reference Centre and the Department of Laboratory Medicine, Ottawa Civic Hospital, Ottawa, and the Departments of Biology of Carleton University and the University of Ottawa, Ottawa, Canada increase in nuclear size and disaggregation of condensed chromatin masses precedes and is independent of DNA synthesis. Since the full range of morphologic alterations observed in lymphocyte transformation can occur in the G, phase of this process, modifications to the above hypothesis are required. Assessment of the nuclear contour index following mitogen stimulation indicates that at least in this in vitro system, there is no cleaved or convoluted phase during the transformation of human peripheral T lymphocytes. (Am J Pathol 1981, 103: [10][11][12][13][14][15][16][17][18][19][20] pressions of this dynamic process in the cell and nucleus have important implications for the study of non-Hodgkin's lymphoma.
Current classifications of non-Hodgkin's lymphoma are based largely on postulated sequences of morphologic alterations occurring in mature lymphocytes following antigenic or mitogenic stimulation.26-30 To account for the plethora of cytologic types of non-Hodgkin's lymphoma, Taylor31-33 has proposed that each of the types of non-Hodgkin's lymphoma reflects a morphologic stage in the normal sequence of lymphocyte modification following activation. According to this theory, the accumulation of abnor-mal lymphocytes of particular morphologic type occurs as a result of a "block" or "arrest" at specific stages in the cell cycle of a transforming clone of neoplastic small lymphocytes. Such an event may occur prior to, during, or subsequent to DNA replication.
We have recently been investigating biologic mechanisms and morphologic nuclear changes involved in the activation of human peripheral T cell lymphocytes following concanavalin A (Con A) stimulation. The results of these studies indicate that Taylor's hypotheses31-33 may require modification, at least as they relate to the T cell lymphomas. Utilizing correlated stereologic morphometric analysis, assessment of chromatin organization, and autoradiography of 3H-thymidine-labeled Con A-stimulated lymphocytes, we have confirmed the findings of Milner24'25 that the resulting characteristic increase in nuclear size and disaggregation of condensed chromatin masses precedes and can be fully expressed prior to the onset of DNA synthesis. Additional studies by Setterfield et a18 confirm that these morphologic events are, in fact, not dependent on DNA synthesis. These observations show that in T lymphocytes undergoing blastogenesis, the full range of nuclear alterations can occur in the G1 phase of the cell cycle. As a result, abnormal expressions of cellular and nuclear events normally occurring during G1 would be sufficient to account for the cytologic varieties of neoplastic lymphocytes in non-Hodgkin's lymphoma.
Alterations in the nuclear shape of stimulated T cells have not previously been systematically documented. In this study, the results of assessment of the nuclear contour index indicate that, at least in this in vitro system, the nucleus of transforming T lymphocytes does not undergo a cleaved stage.

Cell Isolation and Culture
For the harvesting of human peripheral lymphocytes, venous blood was collected from healthy volunteers into heparinized syringes and mixed 3:2 with RPMI 1640 medium (Gibco). This was layered onto cold Ficoll-Hypaque (Pharmacia) in 50-ml centrifuge tubes and spun at 400g for 30 minutes in a Beckman J-6 centrifuge at 20 C. The lymphocytes at the serum/ Ficoll-Hypaque interface were removed, washed twice in RPMI 1640, and cultured at a concentration of 1.0-1.5 x 106 cells/ml at 37 C. Culture medium consisted of RPMI 1640 supplemented with 1007o fetal calf or autologous serum and penicillin-streptomycin (100 IU/ml-100 Mg/ml; Difco). Lymphocytes were stimulated with Con A at a final concentration of 25 Mg/ml and labeled with 3H-thymidine (Amersham; 5 . t Ci/ml; sp. act. 40 IA Ci/mM) for 1.5 hours prior to fixation.

Cell Processing and Microscopy
Lymphocytes were collected from the Ficoll-Hypaque gradient, washed and fixed sequentially in glutaraldehyde and osmium tetroxide, and embedded in Epon-Araldite as described previously.12 Sections 2 p thick, stained with 1 o methylene blue in 1 Wo borax, were used for light microscopic examination.
Random, non-overlapping areas of each lymphocyte sample were photographed (Kodak Panatomic X), and each frame was enlarged to an 8 x 10-inch format. Individual nuclei were classified as to chromatin morphotype based on the relative degree of chromatin aggregation, where Type 1 represents the large aggregated clumps of chromatin evident in the nucleus of the mature lymphocyte, Type 2 includes intermediate degrees of chromatin disaggregation, and Type 3, a marked degree of disaggregation of chromatin masses (Figures 1-6). This basic scheme of scoring nuclei as to morphotype has been used previously and has been found to be reproducible.7'8

Morphometry
Measurements of nuclear area and perimeter were carried out directly on photograph prints using a Zeiss MOP-3 Image Analysis System. Each nuclear profile in the various experimental samples was identified for correlation with its chromatin morphotype. Nuclear volumes were calculated using the value of the radius (in microns) derived from the area value obtained from the image analyzer tracings. Nuclei were assumed to be essentially spherical.

Contour Index
The contour index is a method of evaluating nuclear shape and the degree that it varies from a circle. The calculation of this parameter was based on the definition of Schrek,34 which consists of the ratio of the perimeter of the nucleus and the square root of the nuclear area. The latter values were obtained directly from image analyzer readings. The result for a circle is 3.56, and this value rises with the increasing eccentricity of the nuclear profile.
Lymphocytes were counted as having deeply indented nuclei if they had one or more deep cytoplasmic indentations greater than 1 M in length. Measurements were made directly on the enlarged micrographs and corrected for the enlargement to obtain lengths of the clefts in microns.

E-rosetting
Sheep red blood cells (SRBCs) were washed three times in PBS and resuspended in RPMI 1640 medium without fetal calf serum (FCS) at a final concentration of 0.5%. We prepared absorbed FCS by refrigerating (4 C) a suspension of equal parts of packed SRBCs and FCS for 12 hours, centrifuging the suspension, and collecting the supernatant.
To prepare rosetted cells, we added 2 ml of lymphocyte suspension (! 7 x 106/ml) to 2 ml of SRBC suspension (0.5%) and 0.4 ml of absorbed FCS. Following a 5-minute incubation at 37 C, the mixture was centrifuged at 150g. The resulting pellet was kept at 4 C for at least 4 hours, then fixed and embedded in plastic resin as previously described.' Autoradiography Sections 0.5 ,u thick were placed on gelatin-coated slides (0. I W/o gelatin), coated with Ilford L4 emulsion (diluted 1:1 with distilled water) and exposed for 1, 5, and 10 days. Slides were developed in Dektol and water (1:1) for 4 minutes and after fixation were stained with toluidine blue (pH 9.0).

Nuclear Volume
The progressive alterations in nuclear size and distribution of condensed chromatin masses following mitogenic stimulation are evident in Figures 1 to 6. A significant proportion of the mature lymphocytes with relatively small nuclei and prominent aggregates of condensed chromatin (Type 1) in the unstimulated sample ( Figure 1) and early phases of the poststimulation population show a gradual transition to intermediate stages with nuclei (Type 2) that are slightly to moderately increased in size and in which there is progressive separation and disaggregation of chromatin masses (Figures 2 and 3). At these early times after stimulation, some nuclei show irregularity of the nuclear contour, but only small numbers of profiles have a deep narrow indentation greater than 1 , in length ( Figure 1). Subsequent to 24 hours of incubation there are increasing numbers of large, round, smoothly contoured nuclei (Type 3) with prominent nucleoli and almost complete disaggregation of the large masses of condensed chromatin (Figures 4-6).
The distribution of nuclear volume in unstimulated (0 hour) and Con A-stimulated lymphocytes is shown in Table 1. Reference to the unstimulated population shows that the majority of mature lymphocyte nuclei (79%) occur in the small-volume class, only approximately one-fifth of this sample occurring in the intermediate-sized class. After the application of the mitogen, there is no shift in the population of lymphocyte nuclei in the intermediate and large-volume classes until 24 hours of incubation, when this combined group rises to 32.5%, the principal increase occurring in the nuclei of the large-volume class (Table  1). However, at this stage the mean nuclear volume of the 24-hour postmitogen sample is not significantly greater than control nuclei, and it is not until 36 hours that a major alteration in the percentage of intermediate and large-volume nuclei (59%; Table 1) occurs and the mean nuclear volume becomes significantly increased to 122 cu ,i. Subsequently, the mean nuclear volume rises to 140 cu , at 72 hours after Con A stimulation.

Assessment of Transformation and Relation to DNA Synthesis
The typical appearance of SRBC-rosetted lymphocytes (ie, T lymphocytes) 36 hours after Con A stimulation, in a plastic-embedded preparation (Figure 7), illustrates the disaggregated chromatin pattern and large nucleoli of the nucleus. In Figure 8, the silver grains over the nucleus, which was pulse-labeled with 3H-thymidine 1.5 hours prior to fixation, indicate that it is this class of cell that is involved in DNA synthesis. The percentage of 3H-thymidine-labeled cells in the unstimulated and Con A-stimulated samples of lymphocytes is tabulated in Table 2. It is apparent that significant DNA synthetic activity does not commence until 36 hours after mitogen stimulation and that the increase in DNA synthesis is rapid at some point between 24 and 36 hours. The number of stimulated T lymphocytes in S phase remains more or less constant from 48 to 72 hours. The unlabeled but structurally transformed lymphocyte nuclei in Figure  8 (arrow) confirm that the morphologic processes involved in this transformation are completed prior to the onset of DNA synthesis.
The nuclear volume distribution of lymphocytes (Table 1) shows shifts to nuclei of larger volume (many of which have dispersed chromatin) after 24 hours of mitogenic stimulation. However, this summary data does not allow detailed characterization of any alterations in distribution of condensed chromatin that may be occurring prior to or concomitant with these changes in nuclear volume. By classifying each nucleus into one of three types, based on gross organization of the condensed chromatin (as outlined in the methods) and relating this to classes of nuclear volume, we can make a more refined assessment of the metamorphosis of the nucleus of mitogenically stimulated lymphocytes (Table 3).
Essentially, the data in Table 3 indicate the progressive alteration in the condensed chromatin pattern of the nucleus of a certain proportion of human peripheral lymphocytes following Con A stimulation. As expected, these data show that the majority of unstimulated lymphocyte nuclei (O hour) in both the small and intermediate nuclear volume classes have the chromatin organization of mature lymphocytes (Type 1), with a small proportion of nuclei with an intermediate degree of chromatin disaggregation (Type 2). What was unexpected was the alteration in condensed chromatin pattern of lymphocyte nuclei 12 hours after mitogen stimulation. At this time, though the total percentage of nuclei in the small volume class remains unchanged from the unstimulated lymphocyte sample (Table 1), the percentage of Type 2 nuclei has risen from 10% in the unstimulated sample to 37% in the 12-hour sample ( Table 3). A similar type of shift is apparent in the intermediate nuclear volume class at 12 hours after mitogen stimulation, and in both the small and intermediate nuclear volume classes, the shift has resulted from modifications of the condensed chromatin masses of Type 1 nuclei. The data in Table 1 show that these alterations in nuclear morphotype are occurring prior to changes in lymphocyte nuclear volume. By 24 hours after mitogen stimulation, similar but larger shifts in the percentage of nuclei with Type 1 to Type 2 morphotype have occurred in the small and intermediate nuclear volume classes and in the large nuclear volume class, 8% of the lymphocytes now have nuclei of Type 3 morphotype. Reference to Table 2 shows that the process of metamorphosis of lymphocyte nuclei in terms of chromatin disaggregation is well established prior to the onset of DNA synthesis at about 36 hours following Con A stimulation, and as indicated above, also prior to large scale shifts to nuclei with larger volume.
Beyond 24 hours after mitogen stimulation, data in Table 3 indicate, there is a progression of small and intermediate-sized nuclei of Type 1 morphotype to increasing numbers of lymphocytes with intermediate and large-sized nuclei with Type 2 and Type 3 morphotypes. Eventually at 48 to 72 hours of incubation after Con A stimulation, approximately 35-55% of the lymphocytes have a Type 3 nuclear morphotype and are classifiable morphologically as lymphoblasts.

Contour Index
The values for the contour index were calculated from area and perimeter measurements of each nuclear profile in the unstimulated and post-mitogenstimulated lymphocyte populations used in the preceding sections. The results, distributed as to nuclear volume classes, are presented in Table 4. Comparison of the unstimulated sample (O hour) data with the post-mitogen-stimulated lymphocyte populations after from 12 to 72 hours of incubation shows no evidence of a population of lymphocytes with nuclear profiles of increasing eccentricity or with marked indentations, ie, with a contour index greater than about 4.5. This result is apparent for all three nuclear volume classes. In the unstimulated sample, summation of the lymphocyte nuclei in the small and intermediate nuclear volume classes with a contour index between 3.6 and 4.39 reveals that 847o of the nuclei occur in this range. Similar treatment of the 12-72-hour post-mitogen-stimulated samples shows that for all three nuclear volume classes in individual samples, the total percentage of nuclei with contour indexes in the range of 3.6-4.39 is 81, 83, 84, 90, and 84, respectively. Assessment of the number of lymphocyte nuclei having narrow, elongated indentations of the nuclear membrane greater than 1 * was made directly on the micrographs of unstimulated and Con A-stimulated lymphocyte populations (utilizing nuclear profiles in sections probably causes underestimates of the lymphocyte population with this feature). In the unstimulated population 14% of the cells had nuclei with an indentation greater than 1 p (Table 5), and this group of nuclei had a mean contour index of 4.72. The percentage of nuclei of this type in the 12-72-hour postmitogen samples shows no significant alteration ( Table 5). Assessment of the fraction of unstimulated B cell and T cell peripheral lymphocytes with deep nuclear clefts in SRBC-rosetted preparations indicates that a greater proportion of B cells than T cells have this nuclear feature. Thus it appears that lymphocytes with persisting nuclear clefts after Con A stimulation are primarily of B cell type. These results indicate that during mitogenic transformation induced in human peripheral lymphocytes by Con A, T cell nuclei do not undergo a "cleaved," or deeply indented, phase.   investigation quantitating and correlating cell and nuclear morphologic changes with the synthetic processes occurring during lymphocyte differentiation.

Discussion
The accumulation of such data is essential to understanding the morphologic complexities of the lymphomas of both B and T cell origin. Currently, emphasis is placed on the relationship of the various forms of non-Hodgkin's lymphoma to the lymphocyte cell cycle during blastogenic transformation.31-33 Thus, an important question to consider is the temporal relationship of lymphocyte transfor-mation to DNA synthesis. Data published by Mitrou et a139 can be interpreted as indicating a gradual shift from a diploid to a tetraploid complement of DNA as cultured germinal center cells progress from centrocytes to small, medium, and large centroblasts. However, the observations reported here and also by Milner, 24 Tokuyasu et Tables 3 and 4 show that in the 24-hour post-mitogen sample, 21% of the lymphocyte nuclei are of blastic type; yet only 0.5% of the total population of lymphocytes in this sample show evidence of DNA synthesis. Since the cytologic alterations involved in lymphocyte transformation can occur prior to and independent of DNA replication, it would not appear necessary for us to relate these changes to the various stages of the cell cycle, as proposed by Taylor.31-33 In fact, on the basis of current data, it would seem of greater relevance that we direct attention to cellular events occurring in the G, phase of blastogenic transformation in order to elucidate mechanisms that may be involved in the genesis of the diverse histologic types of non-Hodgkin's lymphoma. Of greater significance than DNA replication is the   temporal relationship between RNA and protein synthesis and T cell lymphocyte transformation. Addition of actinomycin-D8 41 or cycloheximide8 to the culture medium prior to 20 hours after stimulation prevents T lymphocyte transformation. The autoradiographic studies of Setterfield et al8 have shown that RNA transcription occurring in the 20-hour period after stimulation is followed by the appearance of new stable protein(s) in the nucleus. This latter event occurs more or less in concert with modifications of nuclear size and chromatin distribution. The dependence of mitogen-induced lymphocyte activation on de novo protein synthesis in the early post-stimulated period has also been reported by Varesio et al.42 Soren43 observed an increased nuclear mass due to protein accumulation occurring prior to the initiation to DNA synthesis in mitogen-stimulated human lymphocytes, and similar findings were obtained by Bolund et al44 in cell fusion experiments. These findings may have considerable relevance to the varied morphologic expressions present in the lymphomas. For example, a neoplastic clone of lymphocytes could have an abnormality of gene regulation or expression with a resultant excess production or absence of one or more nuclear proteins, which are normally involved in modifying the gross organization of chromatin within the nucleus. The nature, number, and/or concentration of these proteins would determine the eventual size and morphologic appearance of the nucleus. Since the abnormality might reflect a phase during the process of differentiation to the dormant mature lymphocyte or during the phases of antigen-induced transformation, a wide variety of lymphomas could result. Some such lymphomas could mimic normal cells at various stages of differentiation or activation, while in other cases derangements of normal processes involved in nuclear modification might result in clones of abnormal lymphocytes bearing no resemblance to any normal cellular stage, eg Sezary cells. Such a model would preclude the necessity of searching for populations of cells in each lymphoma representing phases in a maturation process to the point of a theorized "block."3"-33 The observations in this report relate primarily to T cell lymphomas with the use of T lymphocytes in this experimental system. However, the results are likely relevant to B cell transformation and B cell lymphomas, particularly since the various types of diffuse B cell neoplasms are also reported in T cell lymphomas.35 '45 An additional source of heterogeneity in non-Hodgkin's lymphoma might result from subpopulations of B and T cells, some of which have different or variable morphologic expressions, depending on the type of antigenic or mitogenic stimulus. Though it is evident from a number of studies46-48 that various phytomitogens have distinctly different morphologic expressions in transformed lymphocytes, in only one of these reports48 did mitogen stimulation result in a significant number of Sezary-type cells (with pokeweed mitogen, 6-8% and with phytohemagglutinin, 5-11 Wo). In the study by Douglas et al,4' who used cultured Con A-transformed lymphocytes, cells containing nuclei with convoluted or highly cleaved profiles were not reported. The nuclear features in this latter report are similar to those noted in the current study and the ultrastructural findings of Setterfield et al. 8 In fact, the lymphoblast type of T cell developed in the current in vitro system is indistinguishable from B cell lymphoblasts and B cell lymphoblastic neoplasms.49 These findings suggest that various antigens and mitogens may have highly selective effects on specific subpopulations of T lymphocytes and that one of these subpopulations could act as a precursor for neoplastic cells of the Sezary type.
There are considerable variations in the reported values for the contour index of normal lymphocytes, some of which may be accounted for by a wide range in the number of nuclei with indentations greater than 1 . in depth. This has varied from 13.27o to 40.5'Vo (mean = 30%) of the lymphocyte population in the report of Schrek,34 33.4% in the study of Woessner et al, 18 and 14% in the present study. The contour index of normal unstimulated human peripheral lymphocytes ranges from 4.1 to 4.5,34 3.5 to 4.5,14 and 3.6 to 4.4 in the present study. Based on these values, it is evident from the data in Table 5 that there is no change in the percentage of nuclei with deep indentations or marked irregularity of the nuclear outline in any of the samples assessed following mitogen stimulation. These results form the basis for the conclusion that there is no cleaved or convoluted phase during the Con A-induced transformation of human peripheral T lymphocytes. However, this conclusion may not encompass all T cell subpopulations, since T cell neoplasms, whether lympho-blastoid49 or Sezary cell syndrome,14 have highly characteristic structural nuclear features distinctly different from those of B cell lymphomas.
There is increasing support for the concepts expressed above, based on experimental evidence for a high degree of structural organization within the nucleus; this data was recently reviewed by Berezney.50 What has become apparent is that there is a largely protein framework in the nucleus, referred to as the nuclear matrix, within which and upon which the chromatin may be organized.5-53 The nuclear protein matrix is not simply supportive, however, but has dynamic properties, exhibiting in vitro synthesis and turnover, and, at present, a poorly understood role in DNA synthesis, RNA transcription, and intranuclear RNA transport.5" It is qualitative and quantitative alterations in nuclear matrix proteins which appear to be implicated in changes in nuclear volume and form during the cell cycle and differentiation.50 52 Since the process of neoplasia is often characterized by marked alterations in nuclear contour, size, and chromatin distribution, the recent observations of Berezney et al54 are relevant. This comparative analysis of matrix proteins from cultured hepatoma and normal liver cell nuclei revealed not only the presence of a unique protein but also quantitative differences in other proteins in the nuclear matrix derived from hepatoma cells. The relatively homogeneous cell populations in non-Hodgkin's lymphomas offer an additional system for further evaluation of the role that nuclear matrix proteins may have in establishing the varied nuclear configurations evident in this group of disorders.