Pathology International 1994; 44: 779-784

Original Article Clinicopathological and immunophenotypic studies on 12 cases with B cell chronic lymphocytic leukemia

Naoya Nakamura,l Sayuri Suzuki,l Hideo Segami,' Yoshihiro Nozawa,' Kunihiko Tominaga,' Eiko Wachi,' Hiroshi Hojo,' Masafumi Abe,' Hideo Sakuma2 and Haruki Wakasa' Department of Pathology, Fukushima Medical College, Fukushima and 'Pathology section, Ohta Nishinouchi

General Hospital, Koriyama, Japan

To clarify the histogenesis of B cell chronic lymphocytic leukemia (BCLL), clinicopathological and immunophenotypic studies were performed using a large panel of monoclonal antibodies on 12 cases with BCLL including three caes with prolymphocytic/chronic lymphocytic leukemia (CLL/PL). lmmunophenotypically, CD19 and CD20 were positive for all cases of this series and CD5, CD21, CD22, CD23, CD25, CD38, Leu-8, KB-61, and bcl-2 protein were expressed in variable proportion from case to case. CD10, however, did not react. No alkaline phosphatase (ALP) positive cases were found. The phenotype of BCLL was similar to that of B cells of the mantle zone (MZ) of secondary follicle in the lymph node. It is therefore postulated that the neoplastic cells of BCLL in these cases might be derived from B cells of the MZ. Moreover, the cells possibly originated from the lymphocytes located in the inner layer of the MZ, since ALP+ B cells are usually observed in the outer layer of the MZ. The pseudofollicular (PF) pattern was observed in four biopsied lymph nodes among five cases tested, but no such a pattern in an aspiration clot of bone marrow. These four cases consisted of three cases with CLL and a case with CLL/PL. The immunohistochemical study showed that there were many proliferating cells showing Ki-67' in the PF area of the lymph nodes. In these cases, leukemic cells might have developed from the PF area of the lymph node.

Key words: alkaline phosphatase, B cell, chronic lymphocytic leukemia, immunophenotype, pseudofollicule

B cell chronic proliferative disorders (BCPD) consist of many disease entities: B cell chronic lymphocytic leukemia (BCLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HCL),

Correspondence: Naoya Nakamura, MD, Department of Pathology, Fukushima Medical College, 1 -Hikarigaoka, Fukushima 960-12, Japan.

Received 2 May 1994. Accepted for publication 6 July 1994.

Waldenstrom macroglobulinemia, multiple myeloma, plasma- cytic leukemia, and leukemic manifestations of lymphoma including intermediate lymphocytic lymphoma and follicular lyrnph~ma.'-~ All these disorders except BCLL have their own characteristic clinical course, morphology and immuno- phenotype, but BCLL is not a homogeneous Orfao et at. demonstrated that each of five morphologically sub- classified groups reveals different clinicopathological char- acteristics; particularly, the patients of the prolymphocytic/ chronic lymphocytic leukemia (CLUPL) group (1 1-55% of prolymphocytes) have a poor prognosis.6 The working group of the National Cancer Institute (NCI) does not subclassify CLL, but they suggested that further research will be needed for the CLUPL group, because the patients with CLUPL had a different prognosis in comparison with those with other CLL.4 According to FAB classification, CLL was subclassified CLL and CLL of mixed cell type in which CLUPL is in~ luded.~ A large number of immunophenotypic studies have revealed that BCLL cells generally express CD5 and weak surface immunoglobulin, although the immunophenotype of neo- plastic cells is heterogene~us.'~-~ There are two hypotheses concerning the histogenesis of CD5' BCLL. The first one is that CD5' BCLL is consistent with the cells in the immature B cell stage from pre-B cells in bone marrow and mature-B cells in peripheral lymphoid The other one is that CD5' B cells belong to a separate B cell lineage.9,'o However, there is no definite evidence for either. BCLL is a rare disease in Japan'' in contrast to Western countries in which the disease manifests a relatively high proportion of case^.^^'* The reason is controversial, although genetic factors in the ethnic and racial variations might be in~olved.~ In order to clarify the histogenesis of BCLL cells in Japan, 12 cases of BCLL, including five biopsied lymph nodes, were studied using a large panel of monoclonal antibodies.

780 N. Nakamuraetal.

MATERIALS AND METHODS

Peripheral blood andlor bone marrow for morphologic and immunophenotypic studies were obtained from all of the 12 cases. Biopsied lymph nodes of five cases (cases 1,2,3,10 and 12), aspiration clot materials from bone marrow (cases 2, 3 and 12) and autopsy materials (case 11) were also available for the study. Tonsils were used to detect the localization of alkaline phosphatase (ALP) + B cells.

Morphology

Peripheral blood and bone marrow aspirate smears were evaluated after May-Grunwald-Giemsastaining . Morphologic diagnosis and classification (CLL and CLUPL) were accord- ing to the guidelines of the NCI-sponsored working group.4 An estimation of biopsied lymph nodes was done according to the Working Formulation.' Hematoxylin-eosin stained slides from formalin-fixed tissues of the lymph nodes, aspiration clot materials and autopsy were examined by routine light microscopy.

lmmunophenotype

Monoclonal antibodies used in this study are listed in Table 1. Mononuclear cell fragment was obtained from peripheral blood by standard Ficoll-Hypaque density gradient cen- trifugation.'3 Surface markers were analyzed by indirect immunofluorescein technique. Fluorescein-isothionate (FITC)- conjugated F(ab')2 goat anti-mouse IgG and anti-mouse IgM (Tago, Burlingame, CA, USA) were used as the secondary antibodies. Cells were examined by fluorescence microscopy (Axiophota Zeiss, Oberkochen, Germany) and/or on a flow cytometry by FACScan (Becton-Dickinson, San Jose, CA, USA), and positive cells were determined by epi-illumination with Axiophota. For immunohistochemistry, cytocentrifuged mononuclear cells from peripheral blood and frozen sections of biopsied lymph nodes were placed on albumin-coated slides and immunostained by streptavidin-biotin complex methods.14 The reactivity of alkaline phosphatase (ALP) was histochemically detected as described before. i5

RESULTS

Clinical course

The main clinical and laboratory findings are summarized in Table 2. The median age of the patients was 63 years, with a range from 48 to 72 years. Nine were male and three were female. Lymphocyte counts of peripheral blood in all

Table 1 Panel of monoclonal antibodies (mAb) used

mAb name ~~

CD number Source Specificity ~

anti-lgM anti-lgD anti-lgG anti-lgA anti - kappa anti-lambda anti-HLA-DR anti434 anti-B1 anti-B2 anti-B3 anti-H107 anti-IL9R anti-Leu-1 anti-CALLA anti-Leu8 anti-Leu-MS

anti-MY4 anti-Leu-4 anti-DRC-1 anti-Ki-67 anti-PCNA Bcl-2 proteinT6 KB61 l7

19 20 21 22 23 25

5 10

I l c

14 3

BD BD BD BD BD BD BD CI CI CI CI NI BD BD BD BD BD

CI

DA DA DA * *

mu heavy chain delta heavy chain gamma heavy chain alpha heavy chain kappa chain lambda chain MHC class II antigen Pan B cell Pan B cell C3d receptor B cell subset Fc E receptor lnterleukin 2 receptor T cell and B cell subset Common ALL antigen T cell and B cell subset Monocytes, granulocytes, NK cells, hairy cells Monocytes, macrophages Pan T cell Follicular dendritic cells Proliferating cells Proliferating cells Bcl-2 protein Mantle zone B cell

BD, Becton Dickinson, USA; CI, Coulter Immunology, Sweden; NI.

* Bcl-2 protein and KB-61 were kindly provided by Professor DY Mason. Nichirei, Japan; DA, Dako, Denmark.

cases except case 5 were above 12000/cmm and below 100000/cmm. Lymphadenopathy was observed in eight cases and hepatosplenomegaly in three cases at the time of initial onset of the disease. Three cases (3, 11 and 12) had both lymphadenopathy and hepatosplenomegaly. In case 8, splenomegaly was not seen at initial onset, but it progressed during the course of the disease. Three cases were accom- panied with autoimmune diseases, all of which were diag- nosed before initiation of CLL. All patients except case 2, 11 and 12 are alive as long as 13 years after onset. Three patients died of infection, but an autopsy was only performed in case 11.

Morphology

These 12 cases with BCLL were morphologically subclassi- fied into nine cases of CLL and three cases of CLUPL. No case showed a transformation to another leukemia group or another type of lymphoma during their clinical course. The number of prolymphocytes in cases with CLUPL were 11,17 and 25%, respectively. All biopsied lymph nodes of five cases showed non-Hodgkin's lymphoma, small lymphocytic type.

B cell chronic lymphocytic leukemia 781

Table 2 Clinical course of 12 cases with chronic lymphocytic leukemia

Case Age Sex Lymphocytes/ Lymph node Hepatomegaly Splenomegaly AID Prognosis Subclass of no. (years) cmm swelling sites lymphocytes

- - - 1 59 M 12 000 Systemic Alive, 6y 10m CLL 2 69 M 15 000 Cervical + Dead, 5y CLL 3 47 M 76 000 Cervical + Alive, 3m CLL 4 65 M 22 900 Axillar + Alive, 9y 4m CLL 5 70 F 170 000 Cervical, axillar Alive, 12y 1 l m CLL 6 73 M 48 800 Alive, 3y 2m CLL 7 71 F 23 200 Alive, 6y 8m CLL 8 66 M 30 900 -++ Alive, 6y 8m CLL 9 83 M 15 000 Alive, 2y 8m CLL

10 56 M 48 900 + Alive, 9y 3m CLUPL

12 71 F 16 700 Systemic + 3+ - Dead, 4y CLL/PL

- -

- -

- -

- - -

- - - -

- - ~ -

- - -

- - - -

- ~ -

11 55 M 41 000 Systemic + + + Dead, 4y 2m CLUPL

AID, Autoimmune disease (case 2 with systemic lupus erythematosus, case 10 with rheumatoid arthritis, and case 11 with cryoglobulinemia). Case 8: triple cancer of CLL, uterine cancer and colon cancer. Morphologic classification is according to guidelines of NCI-sponsored working group.

Histologically, the normal architecture of the lymph node was replaced by a diffuse proliferation of small lymphocytes containing nodular areas, called a pseudofollicule (PF) (Fig. 1) in four cases (1, 2, 3 and 12). The sinus structures were completely obliterated in all four cases. Pseudofollicular areas consisted of blastoid cells with oval nuclei containing one eosinophilic nucleolus, small lymphocytes, and intermediate cells between blastoid cells and small lymphocytes. A few mitotic cells were also seen. In an imprintfrom the cut surface of the lymph node, these blastoid cells (prolymphocyte-like cells, PL-like cells) had distinct features resembling pro- lymphocytes in the peripheral blood as fo l l~ws: ’~ large oval nuclei with fine chromatin, an inconspicuous nucleolus and abundant cytoplasm (Fig. 2). In case 11, the lymph node showed a diffuse proliferation of small lymphocytes without PF pattern. Pseudofollicular-like cells and mitotic cells were scattered throughout the lymph node. The sinus structures were not identified. Aspiration clot sections of bone marrow (cases 2, 3 and 12) showed a diffuse infiltration or a few nodular areas of small lymphocytes without PF. The autopsy (case 11) revealed a diffuse infiltration of small lymphocytes in the liver, spleen, generalized lymph nodes and bone marrow. Many PL-like cells were scattered in areas of lym- phocyte infiltration, but PF pattern was not observed.

lmmunophenotype

Alkaline phosphatase reactivity was detected in lymphoid cells of the mantle zone (MZ) of the secondary follicle of the tonsil, especially prominent in the outer layer and scattered

for CD19 and CD20. Surface immunoglobulins were detected (May-Grunward-Giemsa stain).

782 N. Nakamura eta/.

Figure 3 lrnrnunoperoxidase staining of biopsied lymph node (case 2). Ki-67 reacts with nuclei of some prolymphocyte-like cells and large cells. Ki-67 positive cells were found in the pseudofollicular area, but rare in surrounding area.

in seven cases (IgM 4, IgM+D 2, IgG 1). A variable number of positive reactions was present for CD5 (8/12), CD21 (8/12), CD22 (3/6), CD23 (9/11), CD25 (2/12), Leu-8 (Wl), CD38 (2/11), C D l l c ( O h ) , and CD14 (0/4). No cases ex- pressing CD10 or CD3 were found. Alkaline phosphatase reaction was not detected (0/11). B ~ l - 2 ’ ~ and KB61 l 7 protein reacted with the three cases (1, 2, 11) examined. The per- centage of Ki-67’ cells in mononuclear cell fragments from peripheral blood was below 10% except in case 11. In the biopsied lymph nodes, Ki-67” reacted with the nuclei of a small proportion of the neoplastic cells. Clusters of Ki-67’ cells were found in four cases (1, 2, 3 and 12), and corre- sponded to PF area (Fig. 3). In each case, a proportion of Ki-67’ cells in the PF areas was higher than that of the surrounding area. DRC-1 failed to stain the PF areas. These four cases consisted of three cases with CD5’ CD23’ and a case with CD5- CD23-. In case 11 (CD5’ CD23+), Ki-67’ cells were scattered. No different irnmunophenotype between lymphocytes and PL-like cells in five lymph nodes was observed.

DISCUSSION

Clinicopathological and immunophenotypic studies were performed on 12 cases with BCLL including three cases of CLL/PL. lmmunophenotypically, various proportions of sur- face antigens CD5, CD21, CD22, CD23, CD25, CD38 and Leu-8 were expressed in BCLL cases in this series. The high incidence of CD5, CD21, CD23, and surface immunoglobulin (Slg) M and/or D indicated little difference in immunopheno- type between the present cases and those of Western coun- tries.’ However, there was no CD11 c+ case examined and

a a -I

A

B cell chronic lymphocytic leukemia 783

Table 4 Proliferation rates and pattern of BCLL

Case no. Peripheral blood Biopsied lymph node Aspiration of bone marrow Distribution of Ki-67’ cells (“h) Histologic pattern Distribution of Histologic pattern

proliferating cells proliferating cells

1 2 3 4 7 8

11 12

NT 9.6 0.5 1.6 0.2 1.4

15.2 NT

Many in PF Many in PF Many in PF

NT NT NT

Scattered Many in PF

NT Diffuse Diffuse

NT NT NT

Diffuse Diffuse

NT Rare

Scattered NT NT NT NT

Scattered

Proliferating cells were detected by immunohistochemical studies using Ki-67 on cytocentrifuged mononuclear cells from peripheral blood and

PF, pseudofollicular pattern; diffuse, diffuse proliferation of cells without pseudofollicular pattern; NT, not tested. frozen sections of biopsied lymph nodes (LN), and PCNA on paraffin sections of LN and bone marrow.

it has been reported that CDI 1 c is frequently expressed in BCLL in Western countries.zo,2’ CD5+ B cells have been considered as important counterpart cells of BCLL because of a high prevalence of CD5 in the disease.’-5 At the fifth international CLL workshop, it was suggested that only CD5’ BCLL cases are considered true CLL until more information about CD5- BCLL is known.” So CD5’ cases were mainly addressed; however, CD5- cases were not excluded in the present study since CD5- cases, like case 12, revealed similar biological behavior to CD5’ cases. CD5 is expressed on a small population of B cells in normal peripheral lymphoid tissues and many cases with low grade nodal non-Hodgkin’s l y r n p h ~ m a . ~ ~ - ~ ~ In contrast to that CD5+ low grade lympho- mas are rare in extranodal lymphoid tissue.23 The present authors previously reported that CD5, SlgM and/or D, CD25, and Leu-8 were immunohistologically expressed in B cells of the primary follicle and of the MZ of the secondary folli- cle.’5,26~27 CD23 is expressed on activated normal B cells with SlgM and DZ8 and is immunohistochemically detected on B cells in the MZ and follicular dendritic cells in the germinal center. This result indicates that the immunophenotype of BCLL is similar to these B cells, so it can be proposed that many cases of BCLL might be derived from B cells of the MZ in normal B cell differentiation. It is recognized that B cells of the MZ are the normal counterpart cells of diffuse small cleaved cell lymphoma (DSCCL), intermediate lymphocytic lymphoma (ILL), and mantle zone lymphoma (MZL) because of their morphologic, immunophenotypic and molecular ~ h a r a ~ t e r i ~ t i ~ ~ . ’ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ Banks et a/. proposed the term of ‘mantle cell lymphoma’ (MCL) for these entities. Both BCLL and MCL reveal a similar phenotypic profile except for surface immunoglobulin expression, which on MCL is stronger than on BCLL.30 The present authors reported that ALP is one of the B cell markers in the MZ and expressed

on some cases with MCL.26,27 In the present report, it was demonstrated that ALP+ B cells were mainly present in the outer layer (= marginal zone3’) and rare in the inner layer of the MZi3 and there were no ALP+ cases with BCLL. It is speculated, therefore, that the difference in ALP activity is an additional clue to distinguish CLL from MCL, and the ALP- B cell population in the MZ is considered to be important when the normal counterpart cells of BCLL is concerned. The interpretation of these results is quite close to the hypothesis of Gobbi eta/.32 that the normal equivalent cells of BCLL are present at the edge of the germinal center.

Lymph nodes (LN), aspiration clot materials of bone mar- row (BM) and an autopsy were also studied morphologically and immunohistochemically. Pseudofollicular (PF) pattern, which is regarded as one of the features of CLL,33 was observed in four LN among five cases examined. The PF area contained many Ki-67+ cells,’8 which were mostly iden- tical to large cells and blastic cells, but such a pattern was not seen in BM examined. Swollen lymph nodes are seem- ingly considered to be the source of leukemic cells in those cases. Moreover, it is interesting that PF pattern was seen in cases with CLL (313) and CLL/PL (1/2). These data indicated that there might be similar biological behavior between CLL and a part of CLUPL. A few reports have been published on the relationships between the subtype of BCLL and morphol- ogy of lymph nodes. For example, Ben-Ezra et a/. observed a 40% PF pattern in 268 cases with small lymphocytic lymphoma including many cases with CLL; however, they did not mention the proportion of CLUPL.34

In conclusion, the neoplastic cells of BCLL may be derived from alkaline phosphatase-negative cells in the MZ of the secondary follicle, which are mainly present in the inner layer of the MZ, and the lymph node must be a source of the leukemic cells in some cases with BCLL.

784 N. Nakamuraetal.

ACKNOWLEDGMENTS

This work was supported in part by Grant-in-Aid for Scientific Research (02404029) from the Japanese Ministry of Educa- tion. We are grateful to Drs Shin Matsuda, Tetsugoro Tanaka, Hiroyuki Kambayashi (Ohta Nishinouchi Hospital), Rokuo Abe, Kazuo Hichishima, Takashi Shiga (Fukushima Medical College), Toshio Sai (Iwaki Kyoritsu Hospital), Masayuki Mita (Hoshi Hospital), Hideo Kimura (Hobara Chuo Hospital) and Yoshiko Saito (Tohoku University) for providing materials and clinical information. We are also grateful to Professor D. Y. Mason for providing monoclonal antibodies.

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