Multiparameter Analysis for Discreet Differential Diagnosis of by ert634

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									in vivo 18: 437-442 (2004)




                Multiparameter Analysis for Discreet Differential
                Diagnosis of Mucosa-associated Lymphoid Tissue
                           Lymphoma in the Intestine
   EIKI NOMURA1, SHO TAKAGI1, RYO ICHINOHASAMA2, TATSUYA KIKUCHI1, MANABU SHIRAKI1,
       SHINYA OOMORI1, HIROSHI YOKOYAMA1, KYOKO UTSUNOMIYA1, KENICHI NEGORO1,
   HIROYUKI AIHARA1, SEIICHI TAKAHASHI1, YOSHITAKA KINOUCHI1 and TOORU SHIMOSEGAWA1

                 1Division   of Gastroenterology, Tohoku University Graduate School of Medicine, Sendai;
               2Division   of Oral Pathology, Tohoku University Graduate School of Dentistry, Sendai, Japan



Abstract. Background: The diagnosis of mucosa-associated            (1, 2). The intestinal lesions are reported to have rare typical
lymphoid tissue (MALT) lymphoma in the intestine is                 LELs (3). Macroscopically, MALT lymphoma in the intestine
occasionally difficult from histological examination on small       usually presents as a solitary tumor, but it occasionally shows
biopsy specimens obtained by endoscopy. This study focused          as a morphological feature of multiple lymphomatous
on unusual cases of reactive lymphoproliferative disorders in       polyposis or diffuse erosions (4, 5). In addition, it is sometimes
the intestine in order to make a differential diagnosis of MALT     difficult to make a differential diagnosis from other
lymphoma. Materials and Methods: Five patients were                 lymphoproliferative disorders such as reactive lymphoid
examined with regards to clinical symptoms, endoscopic              hyperplasia or benign lymphoid polyp. The histological
findings and multiparameter analysis (the morphological             findings of MALT lymphoma are often indistinguishable from
examination using routine hematoxylin and eosin staining by         them, for the reason mentioned above, especially in
light microscopy, immunophenotyping by flow cytometry               endoscopically obtained small biopsy specimens. There is a
(FCM), immunohistochemistry and genotyping of extracted             case report where the lesion was initially suspected as MALT
DNA). Results: All cases showed an aggregation of                   lymphoma of the large intestine based on the examination of
lymphocytes and one case showed similar features to                 endoscopic biopsied specimens, but was finally diagnosed as
lymphoepithelial lesions. Analyses of FCM and genetic               lymphoid hyperplasia from gene rearrangement analysis on
rearrangements denied the monoclonality in all cases.               postoperative specimens (6). The distinct diagnosis between
Consequently, we considered that all cases should be                them is one of the most controversial topics in histopathology.
diagnosed as reactive lymphoid hyperplasia and inflammatory            This study focused on a group of lymphoproliferative
change. Conclusion: Multiparameter analysis is useful in            disorders of the intestine, including MALT lymphoma, and
making an exact diagnosis of MALT lymphoma and therefore            stressed the importance of discreet differential diagnosis
contributes to prevent unnecessary overtreatment.                   between them by means of multiparameter analysis.

Mucosa-associated lymphoid tissue (MALT) lymphoma in the            Materials and Methods
intestine is a rare subset of low-grade malignant neoplasms. It
                                                                    Subjects. Five patients were enrolled in this study. All of them were
belongs to extranodal marginal zone lymphoma, characterized
                                                                    referred to our hospital because of diagnosis or suspicion of
histopathologically by centrocyte like cells (CCLs),                intestinal MALT lymphoma from physical, endoscopic and
lymphoepithelial lesion (LEL) and follicular colonization etc.      histopathological findings from forceps biopsy specimens. All
                                                                    patients underwent reexamination of the clinical symptoms,
                                                                    laboratory data and endoscopic findings and then multiparameter
                                                                    analysis was performed. The details of the multiparameter analysis
Correspondence to: Eiki Nomura, MD, Division of Gastroenterology,   were described elsewhere (7, 8).
Tohoku University Graduate School of Medicine, 1-1 Seiryo, Aoba,
Sendai 980-8574, Japan. Tel: +81-22-717-7171, Fax: +81-22-717-      Micromorphological examination. One-third of the respective
7177, e-mail: enomura@int3.med.tohoku.ac.jp                         specimens obtained from multiple biopsies or EMR of the lesions
                                                                    was fixed in 20% non-buffered formalin. Paraffin-embedded
Key Words: Intestinal mucosa-associated lymphoid tissue (MALT)      specimens were stained with routine hematoxylin-eosin for
lymphoma, flow cytometry (FCM), monoclonal gene rearrangement.      histological diagnosis.



0258-851X/2004 $2.00+.40                                                                                                             437
                                                           in vivo 18: 437-442 (2004)

Table I. Clinical and endoscopic features of 5 patients.

                                                                   Laboratory data

Case Age
no. (yrs) Gender         Clinical symptoms                   CRP       CEA       sIL-2R                 Endoscopic findings

1     15      f      Right lower abdominal pain               -*         -           -           Multiple protrusions and erosions in
                                                                                                 the terminal ileum and total colon
2     68      m      Upper abdominal pain, diarrhea        1.0 mg/dl     -     1279 U/ml         Multiple protrusions in the ileum
3     29      f      Anal bleeding                              -        -         -             Multiple protrusions and erosions in the rectum
4     52      f      Lower abdominal pain, diarrhea            -         -         -             Erosions and redness in the rectum
5     31      m      Diarrhea                                   -        -         -             Solitary polypoid lesion in the rectum

f: female; m: male; CRP: C reactive protein; CEA: carcinoembryonic antigens; sIL-2R: soluble interleukin-2 receptor
*-: within normal limit




Figures 1, 2. Endoscopic views after dye (0.2% indigocarmine) spraying showing multiple protrusions in the terminal ileum (1) and multiple erosions in
the ascending colon (2).




Two-color flow cytometry. The monoclonal antibodies (CD5, CD10,              HindIII endonucleases. Gene rearrangements were examined using
CD19, CD20, CD21, CD23, CD35, immunoglobulins etc.) were                     32P-probes for J gene of the immunoglobulin heavy chain (IgH),
                                                                                             H
used in two-color flow cytometry (2-FCM). The cell suspension                human T cell receptor ‚, Á and ‰ chain genes and BCL-2
obtained from the unfixed material was stained with a set of two             (Biomedical Laboratories, Kawagoe, Japan).
kinds of various monoclonal antibodies listed above. The
immunostained cells were analyzed by a FACScan (Becton-                      Results
Dickinson Immunocytometry Systems, Mountain View, CA, USA).

Immunohistochemistry. Small pieces of the specimens were fixed in            Table π shows the clinical and endoscopic features of the 5
PBS with 1% paraformaldehyde and preserved in PBS with 20%                   patients. All the patients had some symptoms. They had no
sucrose. Then the material was mounted in the OCT compound,                  ulcer, cancer, malignant lymphoma or H.pylori infection in the
quickly frozen and was sectioned by a cryostat. Frozen and paraffin
                                                                             stomach. A high level of soluble interleukin-2 receptor (sIL-
sections were stained with the unlabeled monoclonal antibodies,
followed by avidin-biotin peroxidase method.
                                                                             2R) was observed in case 2. Concerning the intestinal
                                                                             endoscopic findings, three cases had multiple protrusions and
Genotyping. DNA samples were extracted from the material and                 erosions in the large intestine or terminal ileum. In the other
were digested with the restriction enzymes BamHI, EcoRI and                  two cases, one had a solitary lesion of polypoid appearance in


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                                       Nomura et al: Diagnosis of the Intestinal MALT Lymphoma


Table II. Result of multiparameter analysis and diagnosis.

       Micromorphological                   Immunophenotyping                      Genotyping
          examination

Case                                                                              Monoclonal
 no.     CCLs        LEL           2-FCM and/or immunohistochemistry            gene rearrangement          Final diagnosis

1         ±*          ±*           Mixed pattern of B and T lymphocytes                   -                 Reactive lymphoid hyperplasia
2          -           -           Mixed pattern of B and T lymphocytes                   -                 Reactive lymphoid hyperplasia
3          -           -           Mixed pattern of B and T lymphocytes                   -                 Reactive lymphoid hyperplasia
4          -           -           Mixed pattern of B and T lymphocytes                   -                 Dense infiltration of inflammatory cells
5          -           -           Mixed pattern of B and T lymphocytes                   -                 Reactive lymphoid hyperplasia

CCLs:Centrocyte like cells; LEL:Lympho-epithelial lesion
*Hypertrophic lymphoid follicles with well-formed germinal centers were observed. Occasionally, crypts were infiltrated by lymphocytes, similar
to the LEL in MALT lymphoma.




Figures 3, 4. (3) Low-power view showing diffuse proliferation of lymphoid follicles and wide marginal zone in the mucosal and submucosal layer (HE).
(4) High-power view showing the lympho-epithelial lesion (LEL)-like lesion (HE).




the rectum, while the other had diffuse erosions and redness                IgH, BCL-2 or human T cell receptor ‚, Á and ‰ chain genes
in the rectum. The endoscopic finding of case 1 is shown in                 rearrangements showed negative results for any monoclonality
Figures 1 and 2. The results of the multiparameter analysis are             in all cases (Figure 6). Finally, four cases were diagnosed as
summarized in Table II. In multiple biopsied or EMR                         reactive lymphoid hyperplasia and one as inflammation
specimens of the intestine, all cases showed an aggregation of              change. The symptoms of all cases subsided spontaneously
lymphocytes in the mucosal and submucosal layer and only                    and they have been followed-up periodically. The intestinal
case 1 showed wide marginal zone and LEL like lesion                        lesions disappeared within six months in three patients, but
(Figures 3, 4). The distinct CCLs and LEL were not observed                 they remained without apparent changes in two patients.
in the other four cases. As a result of immunophenotyping by
FCM, a mixed pattern of B and T lymphocytes, that is, a                     Discussion
reactive pattern was observed in all cases. Figure 5 shows the
FCM of case 1. Immunohistochemical examination showed                       Lymphoproliferative disorders in the intestine are
that many of the lymphocytes were positive for CD20 and                     relatively rarely observed. Intestinal disorders involving
negative for both CD5 and cyclinD1 in case 1. Analysis of                   lymphocytes are divided into two major groups, that is,


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                                                         in vivo 18: 437-442 (2004)




Figure 5. Examples of the flow cytometry (FCM) showing mixed pattern of
B and T lymphocytes.




malignant lymphoma including MALT lymphoma, which
represents monoclonal neoplastic proliferation of
lymphocytes and reactive lymphoid hyperplasia, which
shows hyperplastic and polyclonal aggregation of
lymphocytes. Although MALT lymphoma and lymphoid
hyperplasia are distinct clinicopathological entities, they
can frequently cause serious problems in routine
                                                                          Figure 6. IgH gene rearrangement in fresh materials of the protrusions and
histological differential diagnosis.
                                                                          erosions showing negative for rearrangement. 1: BamHI. 2: EcoRI. 3:
   The cases we described in the current report closely                   HindIII (restriction nuclease).
resembled MALT lymphoma when examined on the
endoscopic findings and histological features. Specifically,
the infiltrating cells of the lymphoid hyperplasia of one
case were similar in size and morphology to CCLs of                       which are considered to be reactive changes. Genotyping to
MALT lymphoma. The distribution of proliferating                          detect clonal immunoglobulin, T cell receptors and BCL-2
lymphocytes may also make the diagnosis difficult. There                  revealed no monoclonal proliferation. Therefore, we
was focal infiltration of crypt epithelium like LEL, which                concluded that all current cases should be diagnosed as
was difficult to distinguish from MALT lymphoma.                          reactive and inflammatory lymphoid changes rather than
   The most important procedures to distinguish between them              MALT lymphoma. The symptoms of all cases have subsided
are     combined      multiparameter       analysis including             spontaneously and none of them showed an aggravation. In
immunophenotyping using FCM and/or immunohistochemistry                   addition, the level of sIL2-R in case 2 decreased gradually.
and monoclonal gene rearrangement (7, 8). The diagnostic                  Three patients presented disappearance of the lesions
criteria of MALT lymphoma has been proposed to be the                     endoscopically. These findings also support that the disorders
characteristic of tumor cells, positive for CD19, CD20, CD21              can be considered as reactive changes.
and CD35 and negative for CD5, CD10, CD23 and cyclinD1                       The etiology of lymphoid hyperplasia and inflammation
(2, 9). In addition, it has been recently reported that                   changes are still not clear in detail. Immunological factors
monoclonal gene rearrangement, including IgH and                          such as hypogammaglobulinemia or an isolated IgA
t(11;18)(q21;q21) chromosomal translocation, are frequently               deficiency, which are frequently observed in patients with
observed in MALT lymphoma (6, 10). All current cases clearly              diffuse lymphoid hyperplasia, may play some role in the
exhibited the mixed pattern of B and T lymphocytes in FCM                 etiology (11-13). Intestinal parasitosis or viral infection


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                                    Nomura et al: Diagnosis of the Intestinal MALT Lymphoma


may also be implicated as a possible cause of lymphoid               10 Ott G, Katzenberger T, Greiner A et al: The t(11;18)(q21; q21)
hyperplasia (14-17). However, stool culture tests of the                chromosome translocation is a frequent and specific aberration
current cases were negative for harmful bacteria.                       in low-grade but not high-grade malignant non-Hodgkin’s
                                                                        lymphomas of mucosa-associated lymphoid tissue (MALT-)
   There are several reports of "intestinal MALT
                                                                        type. Cancer Res 57: 3944-3948, 1997.
lymphoma" that underwent operation and/or chemotherapy               11 Hermans PE, Huizenga KA, Hoffman HN et al:
without multiparameter analysis (6, 18, 19). Here we                    Dysgammaglobulinemia associated with nodular lymphoid
described 5 cases of reactive lymphoid hyperplasia and                  hyperplasia of the small intestine. Am J Med 40: 78-89, 1966.
inflammatory changes in the intestine which should be                12 Ajdukiewicz AB, Youngs GR and Bouchier IA: Nodular
distinguished from MALT lymphoma. Multiparameter                        lymphoid hyperplasia with hypogammaglobulinaemia. Gut 13:
analysis including FCM and gene rearrangement was useful                589-595, 1972.
                                                                     13 Gryboski JD, Self TW, Clemett A et al: Selective
in making discreet differential diagnosis between these
                                                                        immunoglobulin A deficiency and intestinal nodular lymphoid
benign lesions and MALT lymphoma, suggesting that it                    hyperplasia: correction of diarrhea with antibiotics and plasma.
might contribute in preventing overtreatments for those                 Pediatrics 42: 833-837, 1968.
lymphoproliferative disorders.                                       14 Ranchod M, Lewin KJ and Dorfman RF: Lymphoid hyperplasia
                                                                        of the gastrointestinal tract. A study of 26 cases and review of
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