Nuclear morphologic and morphometric analyses of nodular poorly differentiated lymphocytic lymphoma: Assessment of small cleaved nuclei

Original Contributions Nuclear Morphologic and Morphometric Analyses of Nodular Poorly Differentiated Lymphocytic Lymphoma: Assessment of Small Cleaved Nuclei IRVING DARDICK, MD, FRCP(C), DOUGLAS R. CALDWELL, BS, RT, DENIS B. BAILEY,ME), FRCP[C], ALEXANDER M. DARDICK, AND M. T. DIANE JEANS, ASRT C o m p a r a t i v e analytic m e a s u r e m e n t s of n u c l e a r p a r a m e t e r s in normal and neoplastic lymphocytes are limited. In the present morphometric study lymphocyte nuclear features in 21 cases of nodular poorly differentiated lymphocytic l y m p h o m a (NPDLL) were assessed with respect to the theoretical aspects of some nonHodgkin's l y m p h o m a (NtIL) classifications. T h e mean nuclear area of the lymphocytes in NPDLL is generally within the range of the areas of unstimulated (mature) lymphocytes of mantle and follicular regions of l y m p h nodes with reactive hyperplasia. O n this basis, the neoplastic lymphocytes in NPDLL do not reflect, at least cytologically, the antigen-activated, transforming lymphocytes of normal follicular centers. All measured nuclear parameters of small, unstimulated lymphocytes of neoplastic follicles suggest that major proportions of this component are also p a r t of t h e n e o p l a s t i c cohort. Sectional n u c l e a r p r o f i l e s in NPDLL are m u c h more irregular in shape a n d have a higher percentage of invaginations than normal lymphocytes. However, only 4 to 5 per cent of nuclear profiles in NPDLL are of sufficient depth to be termed clefted. Serial section reconstruction of both normal and neoplastic lymphocytes indicates the degree to which the numbers of invaginated or clefted nuclei are underestimated in the examination of histologic sections. For example, the 4 to 5 per cent of nuclear profiles with clefts in histologie sections of NPDLL actually represent about 25 to 30 per cent of the lymphocyte population. On the basis of computer modeling of stylized nuclei with simple invaglnatlons of varying depths and serial section reconstruction of normal a n d neoplastic nuclei, it is likely t h a t all l y m p h o c y t e n u c l e i h a v e some f o r m o f n u c l e a r m e m b r a n e invagination and that in poorly differentiated lymphomas these invaginations may be single and multiple discrete i n d e n t a t i o n s or linear, b r a n c h i n g grooves. Assessment of the ratio of nuclear invagination depth to nuclear diameter in normal a n d neoplastic lymphocytes indicates that transforming normal lymphocytes in follicular centers do not undergo a phase of increased nuclear cleftlng and that this ratio is somewhat greater in lymphocytes in NPDLL than in follicular center lymphocytes. However, the latter effect is not due to increased depth of nuclear invaginations in NPDLL, but rather results from the fact that mean nuclear diameter in this subtype of N H L is considerably smaller than that of normal lymphocytes. Morphometric analysis of nu-

clear parameters in eight cases of NPDLL characterized by prominent mantle zones reveals the marked similarity of nuclear features in lymphocytes f r o m the m a n t l e and follicular regions. From such results, it is a p p a r e n t that there are a n u m b e r of misconceptions about nuclear features in NHL. It should not be surprising or unexpected that lymphocytes in NHL might differ structurally from normal lymphocytes. Similarly, functional and immunologic characteristics of B-cell differentiation may not be uniformly matched with cytologic features in some subtypes of NItL. IIuM PATHOL 16:1187--1199, 1985.

Both of the classifications of non-Hodgkin's lymphoma (NHL) principally used in North America are based oil comparison of each neoprastic subtype with a specific phase of tile transformation and differentiation of normal B and T lymphocytes, t-5 However, such a premise remains largely hypothetical. Indeed, recent experimental and morphonletric evidence has shown that the postulated morphologic changes in lymphoc),te nuclei following antigenic or naitogenic stimulation are not a reality. 6,7 Such restths thtts suggest that the lymphocytes in certain N H L subtypes might not have normal lymphocyte counterparts. This would hardly be a surprising revelation since most other neoplastic processes have variants that cytologically and organizationally bear only minimal resemblance to their tissue of origin. Furthermore, current morphologic and morphometric assessment of large cell (histiocytic) N H L emphasizes the inadequacy of the foundation for lymphoma classification: not only do many large cell lymphonms have tumor cell nuclei that are smaller than those of the fully transfornted lymphocyte, but some o f these lymphomas have nuclei within the size range observed for the unstimulated (mature) lymphocyte. 8 Others have made a similar observation. 9 As is evident from the review by Crocker, l~ morphontetric image analysis is being applied increasingly to malignant lymphomas. Such studies are esReceived from the Canadian T u m o u r Reference Centre, Otsential if objective data concerning cellular and nutawa, and the Departments of Pathology, Toronto General tlosclear parameters in N H L are to be used eventually pital, Toronto, and the University of Ottawa, Ottawa, Ontario, to improve diagnosis and classification in this disease. Canada. Revision accepted for publication J u n e 6, 1985. Supported by a grant from the National Cancer Institute of To date, the morphometrically derived data for neoCanada. plastic lymphocytes in N H L have been presented in Address correspondence and reprint requests to Dr. 1. Dartwo formats. In the first, the mean nuclear paramedick: Canadian T u m o u r Reference Centre, National Cancer Inters tabulated are derived from mean values for each stitute of Canada, Clinical Studies Unit Building, 60 Ruskin Avcase, studied, 11-15 and although the mean nuclear enue, Ottawa, Ontario KIY 4M9, Canada. 1187

HUMAN PATHOLOGY

Volume '16,No. '12 (December '1985]

features analyzed may show statistically d i f f e r e n t values for each subtype of NHL, these values may be nfisleading. This becomes apparent from a review of data presented in the second format in which the mean values for each case analyzed morphometrically are presented. 8,16-2~ Only this latter presentation allows quantification o f the range of values for each morphologic feature of neoplastic lymphocytes in the various subtypes of N H L and assessment of the degree of homogeneity or overlap between these subtypes. It is this type of approach to morphometric analysis that may prove invahmble in establishing definitive histologic criteria for segregating varieties of N H L with differing biologic behavior. O n e major m o r p h o l o g i c feature, i.e., the "cleaved" nucleus, is an essential element in the classification of NHL. However, nuclei with single, deep infoldings or invaginations are not apparent in increasing numbers during normal lymt~hocyte transformations, 6,7 and such nuclear profiles are seen in only a very small proportion of the lymphocytes in germinal cente/'s of lymph nodes undergoing reactive hypert)lasia.21 However, some diffuse and nodular lymphomas are composed almost exclusively of neoplastic lymphocytes with so-called cleaved nuclei, and such lymphocytes are postulated to be the counterparts of the partially transformed lymphocytes of normal germinal centers. 3-5 Some previous morphometric studies included data for small cleaved cell lymphomas, 12,16,18,19 but neither the relation to germinal center cells nor the characteristics of the cleaved nuclei were examined in these studies. With this background, 21 cases of nodular poorly differentiated lymphocytic, or small cleaved cell, l y m p h o m a (NPDLL) were systematically analyzed by morphometric image analysis in an effort to determine the relation of the lymphocytes in such lymphomas to normal lymph node lymphocytes. In addition, serial section reconstruction was used to evaluate some aspects of nnclear shape and invaginations in neoplastic lympl~ocytes.

be classified as mixed, were excluded. Sections were examined independently by consuhant pathologists from the Lymphoreticular Panel of the Canadian T u m o u r R e f e r e n c e Centre, and only the cases in which there was a consensus diagnosis in the range of 75 to 100 per cent were used for morphometric studies. All 21 cases analyzed were n o d u l a r lymphomas; mantle regions in eight o f these cases were sufficiently prominent and distinct to allow separate morphometric studies of this region, in addition to the measurements of follicular areas performed in all cases. For comparative purposes, six lymph node biopsy specimens in which reactive hyperplasia had been diagnosed and in which wet tissue was available were also selected for morphometric analysis. The latter data were reported previously in detail. 21,23 Histiocyte nuclear p a r a m e t e r s were m e a s u r e d in prominent aggregates of this cell type in a lymph node invoh'ed with atypical lymphoid hyperplasia. In both the lymphomas and the lymph nodes with reactive hyperplasia, the majority of tissues had been fixed in ten per cent buffered tormalin, while tissues fi'om eight cases of NPDLL had been fixed initially with Karnovsky's solution. Wet tissue was processed for glycol nlethacrylate resin embedding (Sorvall e m b e d d i n g medium, Dupont Instruments, Wilmington, Delaware), and 1 I-tin sections were cut on a Sorwdl JB4 microtome with glass knives and stained with Gill's h e m a t ~ (Fisher Scientific, Pittsburgh, Pennsylvania) and eosin. Tissues fixed in Karnovsky's solution has been previously embedded in Epon-Araldite resin; with an LKB IV ultramicrotome, 1 ttm sections were cut and stained with toluidine blue. Morphometry

For morphometric analysis, random but representative regions of the tumors and representative areas of mantle zone and germinal centers from reactive lymph nodes were photographed in nonoverlapping frames on 35-ram film with an oil iinmersioi~ objective (100 x ). Each frame of the negative file was projected onto a digitizer pad (Summagraphics CorMATERIALS AND METHODS poration, Fairfield, Connecticut) at a final magnifiTissue Samples cation of 2 , 5 0 0 x . Calibration of the magnification was performed by photographing a scale of 10-1.tm Material for this study was selected from cases units with the oil immersion lens, projecting this negthat had been accessioned at the Canadian T u m o u r ative onto the digitizer, and adjusting enlargement Reference Centre, Ottawa, and the Department of on the digitizer pad and the scale factor in the comPathology, T o r o n t o General Hospital, T o r o n t o , puter program until the distance between any two Canada, with a diagnosis of NPDLL; wet tissue or lines of individual units measured on the digitizer plastic r e s i n - e m b e d d e d tissue was available in all registered approximately 10 lamaon the computer discases. By criteria established in the literature, 22 neoplay screen. plasms that were composed prinmrily of small lymIn each frame all well-outlined lymphocyte nuphoreticular cells and had the characteristics and cyclear profiles, except obvious glancing sections, were tologic details of small cleaved cell lymphorna were traced; the data w e r e collected and analyzed by an segregated for morphonaetric analysis. In routinely MSA II program (Atlantis Scientific Systems Group, prepared histologic sections, these tumors were basOttawa, Canada) and an Apple II Plus microcomically composed of neoplastic lymphocytes with irregputer (Apple Computer, Cupertino, California). The ularly contoured nuclei, inconspicuous nuclenli, and digitized information was used to calculate the area, muhiple, m o d e r a t e l f large clumps o f c o n d e n s e d perimeter, volume and contour index of each nuclear chromatin. Lymphomas tlmt had any significant comprofile. Profiles that had well-defined angulated or ponent of large noncleaved cells, and that might thus 1188

NODULAR POORLYDIFFERENllATEDLYMPHOCYTICLYMPHOMA[Dardick et al.)

FIGURES t (left) and 2 (fight).

Glycol methacrylate sections from two cases of nodular poorly differentiated lymphocytic lymphoma [NPDLL) (case 1, fig. 1; case 2, fig. 2} showing the distribution of nuclear phenotypes in follicular and mantle [m) regions, with examples of the three nuclear morpho~ypes designated in case 2. Nuclear invaginations (arrows) of varying forms and depths are evident in case 2. (Gill's hematoxylin stain. Fig. 1, x 550; fig. 2_ x 1,200.] To illustrate similarities and differences between lymphocyte populations in the two cases of NPDLL the mean [• for the nuclear contour index (NCl) and the depth of nuclear invaginations (NiNV) are detailed below [Student's t-test at 95 per cent confidence levels].

Type 1 Nuclei NCl

Case 1 N = 1,000 P Case 2 N = 1,011

Type 2 Nuclei

NINV (.am)

4.35 • 0.-t3

0.81 • 0.31 (5.7%)

NS 4.50 • 0.61

NS 0.92 • 0.43 (6.0%)

NCI

NINV (.am)

4.51 • 0.41 0.87 • 0.37 (44.7%) <0.001 <0.001 4.72 • 0.62 1.14 • 0.53 (53.1%)

n a r r o w i n d e n t a t i o n s were identified, a n d the d e p t h o f this i n d e n t a t i o n was m e a s u r e d f r o m the m a r g i n s o f the n u c l e a r m e m b r a n e on e i t h e r side o f the invagi n a t i o n to its a p e x . S e p a r a t e c r i t e r i a w e r e u s e d to identify two types o f indentations: 1) nuclear invaginations (INV) w e r e distinct notch-like o r n a r r o w indentations with an a n g u l a t i o n o f 45 ~ o r less a n d a m i n i m u m d e p t h o f 0.4 g m (the m o d e o f collection o f these data and their analysis were detailed previously21; 2) nuclear clefts (CL) were single linear indentations that m e t the criteria for I N V but were at least o n e t h i r d o f t h e n u c l e a r d i a m e t e r in depth.24, 25 T h e s e data were o b t a i n e d f r o m a histog r a m plot o f the distributi6n o f the d e p t h o f nuclear indentations f o r a particular s a m p l e a n d the m e a n nuclear d i a m e t e r for the s a m e sample, as outlined

Type 3 Nuclei NCl

NINV (.am)

4.26 • 0.52 0.88 • 0.32 (.t9.6%) <0.001 <0.00 ! 4.63 • 0.53 1.08 • 0.51 (40.9%)

previously. 21 T h e c o n t o u r index is a s i z e - i n d e p e n d e n t m e a s u r e m e n t o f the s h a p e o f a profile in which increasing irregularity o r eccentricity o f a profile results in h i g h e r values (a circle has a c o n t o u r index o f 3.54). Chromatin organization of normal and neoplastic nuclei was assessed by descriptive criteria rep o r t e d previously. 6,7,~1,23 Essentially the l y m p h o c y t e nuclei are divided into t h r e e types (for e x a m p l e s , see figs. 1 a n d 2): m o r p h o t y p e 1 nuclei contain the p r o m inent a n d c o m p a c t masses o f c o n d e n s e d c h r o m a t i n seen in u n s t i m u l a t e d small lymphocytes; m o r p h o t y p e 3 nuclei h a v e the m a r k e d l y d i s a g g r e g a t e d c o n d e n s e d c h r o m a t i n a n d p r o m i n e n t nucleoli characteristic o f the large, fully t r a n s f o r m e d lympocyte; and m o r p h o type 2 nuclei, with n u m e r o u s , small to m o d e r a t e l y sized c l u m p s o f c o n d e n s e d c h r o m a t i n , show the in1189

HUMAN PATHOLOGY

Volume 16, No. 12 (December 1985)

diameter in depth; 4) cleaved (CL)--single or multiple linear indentations, with the depth o f ' o n e or more of these invaginations equal to or greater than one third of the nuclear diameter; 5) convoluted ( C O N V ) - - t w o or more deep linear or complexly organized indentations. For the assignment of each, three-dimensional lymphocyte nucleus to a particular case, the complete sequence of sections was assessed. Thus, if a particular nucleus generally had a smooth three-dimensional contour but one profile had a significant indentation, the nucleus was placed in the invaginated or cleaved category. FIGURE 3. Stylizedthree-dimensional nucleus used to determine the percentage of sectional profiles containing invaginations.The sphere has a conical indentation with angle a and depth b and radius ~ Invagination depths used In the model were proportional to bl2c, where the nuclear diameter (2c] has a value of 1.

termediate degree of condensed chromatin disaggregation a p p a r e n t in partially transformed lyinl)hocytes. N u c l e a r Serial Sections

The following tissues were used to assess threedimensional lymphocyte nuclear form: 1) a paratracheal lyinph node biopsy specimen with primary follicles, p r o m i n e n t paracortical regions, and no evidence of neoplasia; 2) a spleen resected from a child with congenital spherocytosis in which tissue sections revealed n u m e r o u s reactive germinal centers bordered by prominent mantle zones; and 3) a cervical lymph node biopsy specimen involved with malignant lymphoma, diffilse poorly differentiated lymphocytic type (DPDLL). Serial sections (1 p.m thick) obtained from each of the lymphoid tissue samples were similarly oriented, and the same regions in each serial section were photographed with an oil immersion (100• objective. Individual frames were printed at a final magnification of 2,400 • in some cases and 3,500 • in others. Complete lymphocyte nuclei, spanning some portion of the serial sections, were numbered, and consecutive nuclear profiles, were identified for each nucleus. The outline of each such profile was traced from the print, cut out, and arranged in a linear sequence in approximately tile same orientation as in the original sections. This procedure produced a perspective of the three-dintensional form of each lymphocyte nucleus (see figures in the Results section). To categorize three-dimensional nuclear form, the following classes were a d a p t e d and modified from similar definitions used to characterize two-dimensional nuclear profiles24: 1) smooth ( S M ) - round, oval, or elliptical with minor irregularities; 2) irregular (IRR)--wavy, irregular, or angulated profiles or profiles with lnuhiple shallow indentations; 3) indented (IND)--single or multiple linear or angulated indentations less than one third of the lmclear

Computer Modeling

A simple model o f a spherical nucleus with a conical indentation (fig. 3) was used to obtain some information about tile following questions: 1) From what sort of three-dimensional indentations do the cross-sectional nuclear invaginations arise? 2) What percentage of the nuclei in a cell population contain such indentations? Computer-generated information was obtained from 30 random slices taken perpendicular to the axis of the invagination aligned with the center of the spherical nucleus (fig. 3). The invagination axis was then rotated 3 ~ about the center of the sphere, and 30 additional slices were taken' perpendicular to the original inwlgination axis. This slicing process was repeated at 3 ~ intervals until the invagination axis was at 90 ~ to its original position. To simulate experiinental conditions, in which only nuclear profile indentations equal to or greater than 0.4 p.m were measured, tile computer was programmed to exclude invaginations with a depth less than 10 per cent of the nuclear diameter and nuclear sections less than 10 per cent of the area o f the largest cross section of the nucleus. Nuclear sections containing portions of invaginations that were completely enclosed were treated as unindented, since such profiles would not be recognized as indented in routine histologic preparations. Using a series o f conical model invaginations with an angle of 30 ~ and depths representing increasing percentages of the nuclear diameter (i.e., increasing ratio of invagination depth to diameter), the mean invagination d e p t h that would be obtained from the above cross sections and the percentage of profiles with invaginations (Pi) can be computed in this model system with the assumption that all nuclei have such invaginations (table 1). Using normalized model invaginations (Im) in the range of 0.1 to 0.5 (i.e., 10 to 50 per cent of the nuclear diameter), the c o m p u t e r - c a l c u l a t e d cross-sectional invagination depths (Ic) p r o d u c e d a linear slope when hn was plotted against Ic (regression analysis). Thus, for a minimum cross-sectional invagination depth of 10 per cent of the nuclear diameter, the equation of this line was given by:

1190

Ic = (0.'t8 Ira) + 0.05.

(1)

NODULARPOORLYDIFFERENTIATEDLYMPHOCYTICLYMPHOMA[Dardick et al.)

From this information Pi can be calculated by using the slope of the line obtained when Pi is derived from the above computer-generated model with various invagination depths. Thus, Pi = [(0.67 Im) - 0.07] x 100.

TABLE 1. Percentage of Nuclear Profiles Containing Invaginations with a Minimum Normalized Depth of 0.10"

Normalized Invagination Depth (Im)

(2)

O.lO 0.15 0.20 0.25 0.30 0.35 0.'t0 0.45 0.50

Since the value for Pi was calculated with the assumption that all nuclei in the population were invaginated, the fraction (F) of any lymphoid sample in which the percentage o f profiles with indentations (Pf) have actnally been analyzed is given by: F = Pf/Pi.

Mean Cross-sectional lnwlgi,mtion Depth (Ic)

(3)

0.55

0.60 RESULTS

Figures i and 2 illustrate typical follicular regions in two of the cases of NPDLL as seen in glycol methacrylate secti,ons. These cases indicate the range o f nuclear shape and i n d e n t a t i o n s in this type o f NHL, with the nuclear shape less irregular and the inw~ginations more shallow in case 1 (fig. 1), titan in case 2 (fig. 2). Figure 2 illustrates the patterns o f chromatin distribution used to segregate neoplastic nuclei into the three nuclear morphotypes comparable to those of normal lymphocytes.7,23 In NPDLL, it is the morphotype 2 and 3 nuclei that are'nsually considered to be the neoplastic population, while morphotype 1 nuclei are believed to represent a nonneoplastic population.

O.lO 0.12 0.15 0.17 0.10 0.22 0.24 0.26 0.28 0.31 0.34

Per Cent Profiles (Pi) 0.2 3.9 6.5 10.1 14.4 17.9 17.9 21.2 94.5 29.9 33.2

* I)erived from a spherical model in which all nuclei have a conical indentation with an angle of 30~. in the six specimens measured ranged from 3.70 to 4.03 (a circle has a CI o f 3.54). In contrast, in the majority of cases of NPDLL the nuclear profiles were considerably more irregular, with mean CI usually greater than 4.20. However, in a few cases of NPDLL populations of lymphocytes were observed in which the nuclei were rounder and more regular resuhing in a mean CI of approximately 4.00. All mean CI values for morphotype 1 nuclei ih NPDLL, rather than being within the ranges found for morphotype 1 nuclei in mantle and germinal centers, were elevated and, notably, within the ranges for both morphotype 2 and 3 neoplastic nuclei (fig. 5).

Nuclear Area

A scattergram distribution o f mean nuclear areas for the morphotype I nuclei of mantle lymphocytes, the three nuclear morphotypes of germinal center lymphocytes from normal lymph nodes, and the comparable morphotypes o f neoplastic lymphocytes in NPDLL is presented in fig. 4. There were three principal findings: I) the range of mean areas for morphotype 2 and 3 nuclei in NPDLI. (PD2 and PD3 in fig. 4) was fairly narrow (11.9 to 24.2 lain2); 2) mean nuclear areas in morphotype 2 and 3 lymphocytes in NPDLL were distinctly smaller than those of comparable nuclei in normal germinal centers (GC2 and GC3 in fig. 4), with mean nuclear area in some cases within the range o f that for normal unstimulated lymphocytes (MI and GCI in fig. 4), while the nuclei in other cases were distinctly smaller than normal morphotype 1 nuclei; and 3) morphotype 1 mean nuclear areas in NPDLL (PDI in fig. 4) were also tightly grouped and distinctly smaller than the type 1 mean nuclear areas of both mantle and germinal center lymphocytes (fig. 4). Contour Index

As shown in figure 5, lymphocyte nuclei in mantle and follicular center regions of normal lymph nodes had only minor deg~'ees of irregularity and eccentricity, since the mean contour index (CI) values

Nuclear Invagination s

A s m i g h t be expected from the CI results, all classes of nuclei in NPDLL lind greater percentages of nuclear profiles with one or more invaginations equal to or greater than 0.4 lun, compared with tile 6 to 20 per cent of the profiles in normal lymphocytes with similar indentations (fig. 6). This feature was appreciated more readily at the magnification used for morphometric tracings (2,500 • ) than at the usual magnifications used in routine microscopy (figs. 2 and 3). Again, the ranges for the percentage of nuclei with invaginations were sitnilar in all three morphotypes in NPDLL (fig. 6). Nuclear Invagination Depth

M e a s u r e m e n t s of the mean d e p t h of nuclear membrane invaginations (~0.4 la.m) in normal lymph node lymphocytes showed a progressive increment from morphotype 1 to morphotype 3 profiles (fig. 7). Such increasing mean depth of invaginations was not evident in the three morphotypes of neoplastic lymphocytes in NPDLL. In fact, compared with that of normal lymphocytes, nuclear invagination depth was distinctly smaller in all classes of neoplastic lyml.)hocytes, with the range of mean depth of invaginatmns in morphotype I nuclei paralleling that of the other two inorphotyi)es (tig. 7). Tile nlean percentages of

1191

HUMAN PATHOLOGY

Volume 16, No. '12 (December 4985] 5.0 4.8

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PD1 GC2 PD2 GC3 NUCLEAR MORPHOTYPE

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FIGURE4 (top left). Mean nuclear area. Each point in figures 4 to 7 represents the mean value of morphometric measurements of lymphocyte nuclei in selected regions of reactive 10 lymph nodes (solid symbols] and in nodular poorly differentiated lymphocytic lymphoma [NPDLL][open symbols]. Abbre9 I W 1 I I I I 0 viations: mantle zone, morphotype 1 lymphocyte nuclei, M1; M1 GC1 PDI GC2 PD2 GC3 PD3 H germinal center, morphotype 1, 2, and 3 lymphocyte nuclei, NUCLEAR MORPHOTYPE GCl, GC2, and GC3, respectively;, poorly differentiated lymphocytic lymphomas, morphotype 1, 2, and 3 nuclei, PDI, PD2, and PD3, respectively. Both PD2 and PD3 lymphocyte nuclei have smaller mean nuclear areas than either partially (GC2] or fully [GC3] transformed follicular center cells. Open circles Indicate lymphomas fixed in formalin: open diamonds, lymphomas fixed in Karnovsky's solution. FIGURE 5 [top right]. Mean nuclear contour Index [NCl], Normal lymphocytes of all morphotypes have nuclei that show minor degrees of deviation from the shape of a circle [NCl of circle, 3.54] and profile irregularities, resulting in a mean NCl In the range of 3.7 to 4.0. Mean NCl for the majority of cases of NPDLL is considerably greater than the normal values, and the ranges of mean NCl in NPDLLare similar for all three morphotypes. FIGURE 6 (bottom left). Nuclear profile indentations. Each point represents the percentage of individual nuclear profiles with indentations of 0.4 H.m or more in depth. The relatively low level of such nuclear membrane Indentations in all lymphocyte morphotypes in reactive lymph nodes contrasts with elevated levels in the majority of the 21 cases of NPDLL The range of values for "normal, residual" [PD4] lympocytes is similar to that of the neoplastic lymphocytes with morphotype 2 and 3 nuclei. FIGURE 7 (bottom right]. Mean nuclear invagination depth. The depths of the nuclear membrane invoginations in the three lymphocyte morphotypes in NPDLL span a similar range, and such indentations are more shallow than those in their morphologic counterpart in follicular centers of reactive lymph nodes. 2O

i.i

.,.

nuclear profiles with invaginations, derived by pooling and analyzing the data for each of tile six specimens of normal lymph node and the data for each of the 21 cases of NPDLL are presented in table 2. A similar analysis for the mean percentage of nuclear profiles with indentations sufficiently deep to be categorized as clefted is also presented in table 2. This analysis reveals tile relatively low proportion of nuclear profiles with significant indentations and the negligible fraction of profiles that can be categorized

as clefted in normal lymph node germinal centers (table 2). In poorly differentiated lymphocytic lymphoma, despite the considerably higher percentage of nuclear profiles with indentations (fig. 6 and table 2), the mean percentage of nuclear profiles qualifying as clefted ranged from only 4.2 to 5.4 (table 2). Cleaved Nuclei

One of the factors instrumental in producing the impression of increased indentation in nuclear pro1192

NODULARPOORLYDIFFERENTIATEDLYMPHOCYTICLYMPHOMA(Dardick et al.) TABLE 2.

Nuclear Invaginations and Clefts in Reactive Hyperplasia and Malignant Lymphoma, Poorly Differentiated Lymphocytic Type* Nuclear Morphotype: Malignant Lymphoma, Nodtdar Poorly Differentiated Lymphocytic (Neoplastic Follicles)

Nuclear Morphotype: Reactive Hyperplasia (Germinal Centers)

N % Nuclear profiles with invaginations (~SI)) % Nuclear profiles with clefts (-'-St)) Mean inwlgination depth (gm -- SD) Mean nuclear diameter (Wn • SD) Mean invagination depth/ mean nuclear diameter (•

1

'2.

3

I

2

3

979

2,998

639

7,967

20,979

16,356

12.9 • 3.4

17.8 -4- 3.02

14.6 -4- 3.5

60.6 -4- 10.2

66.9 • 13.4

63.3 • 14.7

0.5 • 0.,t

1.2 -- 0.6

0.7 -- 0.6

5.3 - 2.9

5.4 -- 2.9

4.2 • 2.5

0.85 • 0.41

1.07 -'- 0.56

1.41 -'- 1.19

0.86 • 0.07

0.97 • 0.10

0.98" • 0.09

5.06 --- 1.52

6.48 • 2.53

8.27 • 3.03

3.87 • 0.26

4.94 - 0.33

5.13 -'- 0.38

0.17 --- 0.08

0.16 • 0.08

0.17 • 0.14

0.22 • 0.02

0.20 • 0.02

0.19 • 0.02

Statistical analysis (Student's t-test) shows that all differences between the same morphotype in reactive hyperplasia and malignant lynlphoma, nodular poorly differentiated lymphocytic, are significant (P < 0.05), except for morphotype 1 values for mean invagination depth. * Data is pooled for lymphocyte nuclear measurenlents o f 6 l)'mph node biopsies with reactive hyperplasia and 21 lymph node biopsies with lymphoma:

files was the increased ratio of mean nuclear invagination depth to mean nuclear diameter in NPDLL (0.19 to 0.22) compared with gernfinal center lymphocytes (0.16 to 0.17) (table 2). However, this effect was p r o d u c e d not by a greater mean invagination depth in NPDLL but rather by significantly smaller mean nuclear diameters in all three morphotypes coinpared with those of germinal center lymphocytes (table 2). From tile computer-generated (see Materials and Methods section) percentages of nuclear profiles with invaginations in spherical models (table 1), it was possible to obtain information concerning the appearance of indentations in intact lympocyte nuclei in germinal centers and NPDLL. The ratio of mean invagination depth to nuclear diameter in germinal center lymphocytes was 0.16 to 0.17 (table 2). In a model system in which all nuclei have indentations equal to or greater than 10 per cent of the nuclear diameter, such a ratio would mean that about 10 per cent of the resulting cross-sectional profiles would show indentations (table 1). The actual percentages of invaginated nuclear profiles in germinal center lymphocytes which were found to range from 12.9 to 17.8 (table 2), would suggest that all normal lymphocytes in tiffs location had some degree of nuclear profile indentation. In NPDLL, the ratio of mean invagination depth to mean nuclear diameter ranged from 0.19 to 0.22 (table 2), values that would result in 14 to 18 per cent of nuclear profiles with indentations in the model system in which all nuclei had a single conical invagination (table 1). However, the percentages of nuclear profiles with significant indentations in the NPDLL ranged from 60.6 to 66.9, values three to four times greater than expected (table 2). Such a result would suggest that 0aany of the nuclei in this subtype of malignant lymphoma had more than one indentation, linear or branclfing grooves, or a corn-

bination of these two features. In a similar fashion, it is possible to estimate the percentage of nuclei (threedimensional) that are cleaved in germinal centers and the follicular areas of NPDLL from the two-dimensional data derived from nuclear prdfiles in histologic sections. T h e data in tables 1 and 2 suggest that the range of such nuclei would be approximately 3 to 7 per cent (0.5/18 to 1.2/18) for germinal center lymphocytes, and 23 to 30 per cent (4.2/18 to 5.4/18) for NPDLL lymphocytes. Nuclear Reconstruction

T h e tissue samples (paracortex from normal lymph nodes, mantle and follicular center areas from normal spleen and DPDLL) used for assessment of tile three-dimensional form of lymphocyte nuclei were also analyzed morphometrically in histologic sections. T a b l e 3 shows that mean invagination depths and tile percentage of indented and clefted nuclear profiles measured in histologic sections from these samples were similar to tile values for these parameters f o u n d in normal germinal centers and NPDLL (table 2). Serial section reconstruction o f a total o f 333 lymphocyte nuclei was used to assess both the general topography of normal and neoplastic nuclei and the configuration of nuclear m e m b r a n e invaginations (figs. 8 to 11). Using such an indirect method, and obviously only a partial technique for visualizing nuclear membrane indentations in normal tissues, the serial section reconstruction revealed the presence of considerably larger numbers of nuclei with invaginations (31.4 to 62.0 per cent; table 4) than can be inferred from the examination of histologic sections (7.5 to 22.4 per cent; table 3). In malignant lymphoma, estimation of nuclear form from sections is also inadequate. In tile case of DPDLL analyzed, in1193

HUMAN PATHOLOGY SM

SM

IRR

IRR

IND

IND

CL

Volume 16, No. 12 [December 1985] SM

CL

IRR

9

IRR

IND

IND

IND

CL

9

C~

SM

IRR

IRR

IND

IND

CL

CL

IRR

IRR

IND

IND

IND

CL

CL

CL

G# q5)

@

(5 FIGURE 8. (top left). Representative reconstructed lymphocyte nuclei from paracorfex of normal human paretracheal lymph node. Nuclei shown in figures 8 to 11 were categorized by three-dimensional surface form as SM, smooth; IR~ irregular;, IND, indented; and CL, clefled. Some IND end CL nuclei hove more than one invagination, some [nvaginafions extend over o considerable portion of the surface end the grooves v a t / i n depth. FIGURE 9. (fop right]. Representative reconstructed lymphocyte nuclei from mantle zone of a reactive germinal center in the spleen of a child with congenital spherocytosis. The majority of nuclei in this situation had minimal surface irregularities, and few had deep indentations. [table 4]. FIGURE t0. [bottom left}. Representative reconstructed nuclei from a germinal center in the case illustrated in figure 9. The nuclel assessed were primarily those of partially and fully transformed lymphocytes, and, as illustrated, major portions of the surface of many nuclei were devoid of irregularities even in some IND forms. FIGURE 11, (bottom right). Representative reconstructed nuclei from a diffuse poorly differentiated lymphocytic lymphoma. The greater degrees of nuclear irregularity and angularity in this type of NHL are immediately apparent when compared with the normal lymphocyte nuclei illustrated In figures 8 to 10. Some of the neoplastic nuclei show Increased degree, complexity, and multiplicity of nuclear membrane invaginations.

3

vaginated profiles were observed in 33.8 per cent of the nuclei in histologic sections (table 3), whereas this figure rose to 81.3 per cent when three-dimensional nuclei were evaluated (the standard errors of the differences between the percentages in table 3 and 4 are all highly signiticant). It is possible that some of the nuclei categorized as irregular (IRR) represent the shallow indentations.of the nuclear membrane seen adjacent to centrioles and the Golgi apparatus in electron micrographs o f normal lymphocytes. '-'6,27 Fig-

ures 8 to I I show the wide variety of nuclear form found in both normal and neoplastic lymphocytes; few nuclei were truly sphcrical, but many were spheroidal, conical, or irregular. It is evident from figure 1 1 and table 4 that the tliree-dimensional lymphocyte nuclei in DPDLL were considerably more variable and irregular in shape than tile nuclei of normal lymphocytes (figs. 8 to I0). As suggested by the data derived from two-dimensional nuclear profiles (table 3) and the results of 1194

NODULAR POORLYDIFFERENTIATEDLYMPHOCYTICLYMPHOMA[Dardick et al.]

TABLE 3. Morphometric Image Analysis Data Derived from Sectional [Two-dimensional] Nuclear Profiles in Lymphoid Tissues Per Cent Indented Nuclear Profiles

Per Cent Clef ted Nuclear Profiles

Per Cent Invaginated (Indented + Clefted) Profiles

Tissue

N

Mean Invagination Depth • SEM (p.m)

Lylnph node, paracortex Spleen, mantle Spleen, follicle Lymphoma, diffilse poorly differentiated lymphocytic

517 855 1061

0.86 • 0.02 0.83 • 0.01 1.13 _ 0.02

18.5 6.1 15.8

3.9 1.4 1.1

22.4 7.5 16.9

5.23

1.00 _ 0.03

29.0

4.8

33.8

computer modeling of clefted nuclei shown in table 1, nuclear invaginations in tissue iymphocytes might be expected to vary in form and to be more complex than a simple cone of varying angulation. Indeed, some o f the nuclei in both normal and neoplastic tissue were indented and clefted with single or multiple, finger-like indentations o f different depths (best seen in fig. I 1), while others had one or more shallow to deep linear gooves traversing either a portion or nearly the entire length of the nucleus (figs. 8 to 1 I). Comparison of nuclear form in DPDLL (fig. 11) with the lymphocyte nuclei from the three regions of normal lymphoid tissues (figs. 8 to 10) revealed the scant resemblance of the neoplastic lymphocyte nuclei to those in normal tissues, including lymphocyte nuclei in follicular centers, at least in these particular samples. Mantle Versus Follicular Parameters

Among the 21 cases of NPDLL, the mantle regions in eight were sufficiently well developed and discrete to allow separate morphometric analysis and comparison with nuclear parameters derived from the neoplastic follicles. From the mantle regions in these eight lymphomas, a total of 3,958 nuclei were measured (mean _+ SD, 495 - 165; range, 277 to 731), while a total of 20,655 nuclei were measured within follicles in these same lymphomas (mean -4SD 2,582 • 441; range, 2062 to 3598). "Fable 5 shows the mean values for the distribution of lymphocytes for both mantle zones and follicular centers by nuclear features (morphotype), nuclear area, nuclear contour index, percentage of nuclear profiles with invaginations, and depth of the invaginations. Other titan differences in the distribution of lymphocytes by nuclear morphotype, the

m0rphometrically derived data for the nuclear parameters revealed no statistical differences (paired Student's t-test) between these two populations (table 5). Ahhough the mean nuclear areas in neoplastic mantle zones and follicular regions were found to be similar, the possibility remained tlmt both of these lymphocytic regions contained a certain proportion of normal lymphocytes. T o determine the possible extent of the latter population, histogram distributions o f nuclear area from c o m b i n e d samples o f mantle zone unstimulated (morphotype 1) lymplmcytes from five lymph node biopsy specimens with reactive hyperplasia (N = 2,525) and from eight lymph nodes with NPDLL (N = 1,,t64) were superimposed (fig. 12). T h e mean nucgear area of neoplastic lymphocytes (11.9 • 3.9 Ixm2) was significantly different (P < 0.0001; Student's t-test) from that of normal lymphocytes (18.9 • 3.7 ltm'-'). To obtain an upper limit for the size of neoplastic lymphocytes in NPDLL, the mean nuclear area of neoplastic lymphocytes plus one standard deviation and the mean nuclear area of normal lymphocytes minus one standard deviation, a value of approximately 16.0 p.m 2, was chosen. By measuring the area under the lfistogram curve from 16.0 to 22.5 p.m 2 for mantle zone lymphocytes of NPDI,L, it was calculated that 22 per cent of the neoplastic population lind a nuclear size similar to those of normal mature mantle zone lymphocytes (fig. 12). DISCUSSION

Two major premises pervade the N H L classification systems used in North America. The first relates the lymphocytes in lymphoma subtypes m o r -

TABLE 4. Percentage of Nuclear Morphologic Types in Lymphoid Tissues Based on Assessment of Serial Section Three-dimensional Reconstruction Nuclear

"type (%)

Tissue

N

sM

IRR

IND

CL

Lymph node, paracortex Spleen, mantle Spleen, follicle l.ymphoma, diffuse poorly diffet'entiated lymphocytic

100 54 67 112

I.t.0 48.1 16.4 2.7

23.0 20.4 29.9 13..1

44.0 27.7 35.8 53.6

18.0

1.0

3.7 1.t.9 27.7

0.0 3.0 2.7

ABBREVIATIONS: SM, smooth; IRR, irregular; IND, indented; CL, cleaved; CONV, convoluted.

1195

CONV

hwaginated (IND + CL) Nuclei (%) 62.0 31.4 50.7 81.3

HUMAN PATHOLOGY

Volume 16, No. 12 (December 1985)

TABLE 5. Comparison of Lymphocyte Mean (_SD] Nuclear Parameters in Mantle and Follicular Regions of Nodular Poorly Differentiated Lymphocytic Lymphomas (N = 8) Parameter

Type 1

Type 2

M o r p h o t y p e (%) M F

35.5 • 14.2 19.0 • 8.1

42.0 • 9.4 47.2 • 4.4

22.4 • 6.8 33.8 • 8.4

A r e a (Ixm2) M F

11.9 • 3.9 11.7 • 4.2

17.4 • 4.8 18.9 • 6.7

19.3 • 8.4 21.2 • 10.6

Contour index M F

4.40 • 0.55 4 . 4 0 • 0.52

4.35 • 0.55 4.35 • 0.55 4.41 • 0.55 4.35 •

I n v a g i n a t e d profiles (%) M 63.3 • F 61.7 •

Type 3

17.2 11.9

65.3 • 12.8 60.1 • 12.8 66.3 • 14.3 60.8 • 16.6

I n v a g i n a t i o n d e p t h (rtm) M 0.85 +- 0.39 F 0.86 • 0.35

0.97 • 0.49 0.97 • 0.45 0.96 • 0.45 0.98 • 0.46

ABBREVIA'I:'IO,\S: M, m a n t l e zone; F, follicular region.

phologically to specific counterparts evident in germinal centers of lymphoid tissues. The second goes beyond this concept and couples structural and functional phenotypic aspects of lymphocytes, suggesting that if immunologic characteristics in NHL tend to parallel those expressed during lymphocyte transformation and differentiation, then this same principle must apply directly to cytologic details in NHL. This presumably definitive correlation between morphology and immunology has become the main basis in the Lukes-Collins 3-5 and Lennert 28 classifications for segregating NHL into B- and T-cell types. It is this reasoning that has also been instrumental in suggesting that nodular B-cell lymphomas must be of follicular center cell origin. The current study was designed to test just how closely the neoplastic lymphocytes in NPDLL (follicular, small cleaved cell lymphoma) reflect their supposedly normal counterpart in germinal centers, the partially transformed lymphocyte. 3-5 T h e neoplastic lymphocytes with irregularly shaped nuclei in nodular lymphomas of the poorly differentiated lymphocytic type are said to be of the size o f normal lymphocytes or slightly larger. 29,29 Using the nucleus o f unstimulated lymphocytes in mantle zone and germinal centers as a standard, we showed by morphometric analysis that this is not entirely correct. Although in some NPDLLs neoplastic lymphocytes had mean nuclear areas in the lower range of the normal values for unstimulated lymphocytes, in many lymphomas mean nuclear area were smaller than those of unstimulated lymphocytes (fig. 2). Furthermore, in all NPDLLs measured, mean nuclear areas were far smaller than those of the partially t r a n s f o r m e d lymphocyte, their proposed n o r m a l counterpart.3-5, 28, 30~3] We would suggest that neoplastic lymphocytes in NPDLL appear to be larger than normal lymphocytes because of the abnormally

small size of the so-called "residual" lymphocytes, i.e., tile lymphocyte population in neoplastic follicles with nuclei morphologically resembling tile unstimulated lymphocyte in normal germinal centers and mantle regions. Undoubtedly, normal B and T lymphocytes are scattered among the neoplastic population, 32-36 but it would appear that a major proportion of the residual lymphocytes in NHL are part of the tumor cell cohort. T h e parallel range of mean values for nuclear contour index, depth of invaginations, and percentage o f nuclear profiles with indentations of the nuclear membrane in morphotype 1, 2, and 3 nuclei in NPDLL further supports this suggestion. It can only be concluded that any correlation between immunologic expression in NPDLL and the physiologic surface and cytoplasmic markers of some component of normal follicular center cells~7,38 isnot necessarily coupled with structural organization of the lymphocyte, particularly of the nucleus. In addition, some neoplastic lymphocyte markers and structural features are not always as expected. Investigations have revealed both molecular rearrangements of immunoglobulin genes, indicating B-cell differentiation, but without surface or cytoplasmic immuno-. globulin expression, 39-41 and T-cell markers synthesized in B-cell lymphomas. 42 Similarly, abnormally convoluted and T-cell-like nuclei have occurred in a B-cell lymphoma. 43

50-

40-

lZ 30tu tr tu n

~

1

20-

0

I 10

20 AREA (Fm 2)

I 30

! 40

FIGURE 12. Distribution of nuclear profile areas of mantle zone unstimulated lymphocytes (morphotype I) in reactive hyperplasia (solid symbol) and nodular poorly differentiated lymphocytic lymphoma (NPDLL) (open symbol). The mean nuclear area values are indicated by solid and open arrows, respectively. The hatched area, 22 per cent of the NPDLL nuclear area distribution, represents the lymphocyte nuclei in the mantle regions of NPDLLwith an area greater than the mean nuclear area +1 SD of lymphocytes in mantle zones of NPDLLand the mean nuclear area - 1 SD of mantle lymphocytes in reactive hyperplasia (i.e~ approximately 16.0 p.m2J.

1196

NODULAR POORLY DIFFERENTIATED LYMPHOCYTIC LYMPHOMA (Dardick et al.)

In the Lukes-Collins classification of NHL, is the designation small cleaved cell lymphonla appropriate? Based on the original proposition that transtorming lymphocytes undergo a specific phase of increased nuclear clefting, this label does not appear to be correct. 7 Furthermore, the data in the present report relating the mean depth of nuclear invaginations to mean nuclear diameter in normal and neoplastic lymphocytes (tahle 2) reveal that the premise for increased clefting in NPDLL is incorrect. It is not that nuclear invaginations are deeper in lymphocytes in N P D L L ~ t h e y are, in fact, significantly more shallow than those in normal lymphocytes (fig. 7 and table 2 ) - - b u t rather that the nuclei o f neoplastic lymt)hocytes are considerably smaller than those of normal lymphocytes, and the depth of the grooves thus occupies a slightly greater proportion of the nuclear diameter. However, it is necessary to take other factors into account prior to dismissing the terminology of cleaved cells entirely. Information can be obtained from the theoretical model of a population of cells, each bearing a conical indentation of specific depth. This model and the n u m b e r o f invaginated and cleaved nuclear profiles in normal and neoplastic lymphocytes can be used to gain some idea of the percentage of the subpopulation with indented nuclei and the number and form of these indentations of the nuclear memhrane. Such calculations suggest that all normal lymphocytes have some form of nuclear indentation. This also holds true fox"NPDLL. However, with a rate of indentation in three-dimensional lymphocyte nuclei in NPDLL about 300 per cent greater than expected from the model system, it might be anticipated that, in addition to some degree of nuclear memb r a n e indentation in all neoplastic nuclei, many would have m o r e than one indentation, linear grooves of varying depths, or a combination of these two features. Three-dimensional reconstruction of nuclei in a case of DPDLL reveals this supposition to be true (fig. 11). Enumeration of such profiles in histologic sections also underestimates the population of lymphocytes with three-dimensional nuclei defined as cleaved, i.e., those with indentations with a depth greater than or equal to one third of the nuclear diameter. The resuhs of the present study suggest that this underestimation is approximately fivefold to sixfold for lymphocytes in both germinal centers and follicular regions of NPDLL. Thus, in NPDLL about 25 to 30 per cent of the neoplastic lymphocytes have a nuclear form that can be labelled cleaved. The data in tables 3 and 4 support such assumptions. On this basis, the designation of small cleaved cell lymphoma is not inappropriate, ahhough it does not reflect the mechanism originally proposed. 3-s Given the considerable underestimation of the true proportion of intact nuclei with nuclear indentations in sectional profiles, the frequent failure to recognize this feature in routine histologic sections is understandable. It must be realized that these results do not mean that the current segregation of a class of N H L designated as poorly differentiated lymphocytic is not

valid. Indeed, tile naorphometric data reveal tile homogeneity of the NPDLL subgroup and indicate that it is probably a distinct entity light microscopically. This situation contrasts with morphometric data for the mean nuclear area in a group of 20 cases of diffuse histiocytic (large noncleaved) lymphonaas. 8 In these lymphomas a wider range of nuclear size is encountered with some values actually within the size range for unstinmlated lymphocytes. 8 Lack of clearcut segregation of follicular lymphomas on the basis of nuclear size was also reported by Donhuijsen et al. 44 I n d e e d , the mean nuclear areas in a small number of cases of diffuse histiocytic (diffuse, large noncleaved) lymphoma 8 fall within the size range found for lymphocyte nuclei in NPDLL in the current study. It is possible that nuclear size is not a central criterion for the segregation o f histiocytic lymphomas into a class with relatively uniform biologic features, clinical presentation and prognosis. The latter aspects are somehow coupled to the factors that control nuclear condensed chromatin organization, resulting in the reasonably uniform appearance of the nucleus in large cell lymphomas. With this understanding o f both poorly differentiated lymphocytic and histiocytic lymphomas, the advantage of or, in fact, the necessity for relating morphologic aspects of N H L to specific phases of lymphocyte differentiation and transformation must be questioned. It is more i m p o r t a n t to appreciate that N H L s involve complex alterations in morphology that must be fully delineated if pathologists are to improve their skills in diagnosing these tumors. In view of some reports of the monoclonal nature of tile process of transformation of NPDLL to a high-grade, large cell variety of NHL, "t5-47 it is of interest to specnlate as to the presence and role of the lymphocyte population with transformed (morp h o t y p e 3) nuclei in neoplastic follicles in NPDLL. 22,'-0,4s In the 21 cases of NPDLL examined morphometrically, the percentages o f lymph0cytes with morphotype 3 nuclei in neoplastic follicles varied from 11.2 to 59.8 (unpublished observations). Based on the improved resolution of glycol methacrylate and E p o n - A r a l d i t e plastic sections, m o r p h o t y p e 3 nuclei are more abundant than can be readily appreciated in routine histologic sections from typical cases of NPDLL. T h e ranges Of mean nuclear parameters for morphotype 2 and 3 nuclei in NPDLL are similar (figs. 4 to 7), suggesting that the latter component is part of the tumorous cohort. Despite lack of knowledge of the true relationship between lymt)hocytes with morphotype 2 and 3 nuclei in NPDLL, it is possible that the shift to a biologically more aggressive form of N H L is somehow coupled to production of increased numbers of a morphologic subset present in the original NPDLL, a possibility evidenced by the retention of the clonality of the original lesion when some NPDLLs terminate as histiocytic lymphonm: 15-'t7 T h e relation of mantle zones to normal or neoplastic follicles, which need not be the same, x'enmins an enigma. Evidence from immunologic ~2,33'4~176and morI)hometric studies 23 suggests that normal nmntle 1197

HUMAN PATHOLOGY

Volume 16. No. t2 (December 1985)

zone lymphocytes may be a celltdar population diff e r e n t f r o m c o m p a r a b l e l y m p h o c y t e s in g e r m i n a l centers. Functional studies of nodtdar lymphomas h a v e s u g g e s t e d t h a t in t h e m a j o r i t y o f t u m o r s with d i s t i n c t m a n t l e z o n e s , t h e l y m p h o c y t e s in this r e g i o n a r e n o n n e o p l a s t i c . 33 H o w e v e r , this r e s u l t d o e s n o t p r e c l u d e at l e a s t s o m e p r o p o r t i o n o f m a n t l e z o n e l y m p h o c y t e s b e i n g n e o p l a s t i c , 5~ a n d t w o l y m p h o m a s in t h e s t u d y o f H a r r i s a n d D a t a 33 a p p e a r e d to b e c o m p o s e d p r i n c i p a l l y o f t u m o r cells i m m u n o l o g i c a l l y i d e n t i c a l to t h e f o l l i c u l a r c o m p o n e n t . T h e c u r r e n t morphologic and image analytic resuhs indicate that a m a j o r p r o p o r t i o n o f t n a t l t l e z o n e s in at least s o m e N P D L L s a r e c o m p o s e d o f l y m p h o c y t e s with n u c l e a r p a r a m e t e r s i d e n t i c a l to t h o s e o f t h e l y m p h o c y t e s in t h e f o l l i c u l a r c o m p o n e n c T h e size r a n g e o f l y m p h o c y t e n u c l e i in m a n t l e r e g i o n s o f N P D L L l y m p h o m a s , however, shows that a component of normal lymphocytes is p r o b a b l y a d m i x e d with t h e n e o p l a s t i c l y m phocytes. Interestingly, the ranges of the percentages o f T cells i n ' n o r m a l m a n t l e z o n e s (14 to 21) a n d g e r m i n a l c e n t e r s (16 to 28), a n d in follicles o f f o l l i c u l a r l y n a p h o m a s (14 to 29) r e p o r t e d b y D v o r e t s k y et al. ~4 a r e v e r y s i m i l a r to t h e c a l c u l a t e d p e r c e n t a g e (22) o f l y m p h o c y t e s w i t h n u c l e i o f n o r m a l sizes in t h e m a n t l e z o n e s o f f o l l i c u l a r l y m p h o t n a s in t h e p r e s e n t r e p o r c

10. I1. 12. 13.

14.

15. 16. 17. 18. 19. 20.

Acknowledgments. T h e authors thank the following pathologists who reviewed the nmtcrial for inclusion in this stud}': Drs. M. AI-Jabi, Ottawa, Ontario; G . O . Bain, Edm o n t o n , Alberta; B. Burns, Ottawa, Ontario; l a n Carr, Winnipeg, Manitoba; W. Corbett, Kingston, Ontario; G. Hogg, Winnipeg, Manitoba; W. T. E. McCaughey, Ottawa, Ontario; J. Martin, Calgary, Alberta; D. Pantalony, T o ronto, O n t a r i o ; and A. H. Pontifex, New Westminster, British Columbia.

21. 22.

23.

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NODULAR POORLYDIFFERENTIATEDLYMPHOCYTICLYMPHOMA(Dardick et al.)

34. 35. 36. 37.

38.

39. ,t0. 41.

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