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Supplemental Digital Content
Practical approach to the histologic diagnosis of gastrointestinal lymphomas through the first-line marker battery of CD20, CD3, CD30, and Epstein-Barr virus-encoded RNAs
Eun Kyung Kim1,2, Woo Ick Yang1, Hyang Joo Ryu1, JI Hee Lee1, Sun Och Yoon1
1Department of Pathology, Yonsei University College of Medicine, Severance Hospital, Seoul 03722, South Korea
2Department of Pathology, National Health Insurance Service Ilsan Hospital, Goyang, 10444, South Korea
Contents
Appendix 1. Approaches to T and NK cell lesions: intraepithelial lymphocytosis by T cells Appendix 2. Approaches to low grade B cell non-Hodgkin lymphomas in the GI tract Appendix 3. Approaches to MALT lymphoma in the GI tract: lymphoepithelial lesions by marginal zone B cells
Appendix 4. Detailed legends to Figures of Fig.1 to 7: including brief case summaries Supplementary Tables: Table A.1 to A.2
Supplementary Figures: Fig. A.1 to A.12
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Appendix 1. Approaches to T and NK cell lesions: intraepithelial lymphocytosis by T cells In the GI tract, from the esophagus and stomach to the small and large intestines, T cells are usually infiltrated within the epithelial cells of mucosal glands, which is described as intraepithelial lymphocytosis (IEL). IEL is noted in a variety of clinical circumstances, details are summarized in Supplementary Appendix, and fewer than 20 T lymphocytes per 100 enterocytes in the duodenum is thought to be within the normal physiologic condition. When increased IEL is observed in the GI tract mucosa, primary and secondary IEL, such as lymphocytic colitis, food protein intolerance, autoimmune disease, graft-versus-host disease, Crohn disease, ulcerative colitis, viral enteritis, syphilis, Helicobacter pylori infection, medication, and celiac sprue are considered, depending on a variety of clinical indications.1
With regard to lymphoma, IEL should be considered as a clue for a subtype of the T/NK cell lymphomas, especially when the following features are noted: primarily small bowel lesions;
macroscopically mass-forming lesions, ulcers, or perforations; microscopically monotonous proliferation of small to medium-sized lymphocytes with a non-inflammatory background (monomorphic epitheliotropic intestinal T cell lymphoma; MEITL); or medium to large-sized lymphocytes admixed with polymorphic inflammatory cells (enteropathy-associated T cell lymphoma; EATL) (Supplementary Fig.A.6 and Table 3). In the stepwise approaches after the initial application of CD20, CD3, CD30, and EBER for the various subtypes of T/NK NHLs and benign to indolent T/NK cell LPDs that show IEL patterns (Table 3), considering the immunophenotype of the IELs can be helpful. Physiologically, most (85–90%) intraepithelial T cells have the CD3+, CD4-, CD8+, and CD103+ cytotoxic T cell phenotype with an αβ T cell receptor (TCR), and a minority population of intraepithelial T cells have the CD4- and CD8- T cell phenotype with a γδ TCR. A CD56+ IEL is also known, but it is virtually undetectable in
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normal mucosa. IELs increase in number and show aberrant phenotype (CD8- and αβ TCR) according to progression of refractory celiac disease and EATL.On the other hand, IELs show CD8+, CD56+, and γδ TCR in MEITL.2-4
IELs are quite characteristic of intestinal T-cell lymphoma, especially classic EATL (type 1 EATL in the 2008 WHO classification) and MEITL, which was changed in the revised 4th WHO classification from type 2 EATL.2 Usually, MEITL is thought be more prevalent among Asian people and unassociated with celiac disease, whereas classic EATL (type 1 EATL) is known to be more prevalent among people of European descent and associated with celiac disease. In those two entities, IELs in adjacent normal-looking mucosa distant from the main masses are characteristic features. Because initial diagnosis is usually made using endoscopic biopsy specimens, the microscopic features of IELs in grossly normal-looking mucosa can be an important clue to suspect MEITL or EATL when a mass-forming lesion is not identified, especially in the early phase of the disease (Fig.6) or before undergoing systemic work-up studies.
Although IEL is not as characteristic of other subtypes of T/NK cell lymphomas involving the GI tract as it is to MEITL and EATL, other relatively prevalent subtypes of GI T/NK cell lymphomas, such as ENKTL and indolent T/NK cell LPDs, can be also considered in the context of differential diagnosis (Table 3 and main article sections).
Appendix 2. Approaches to low grade B cell non-Hodgkin lymphomas in the GI tract When lymphoma, such as MALT lymphoma, is histologically or clinically indicated despite presenting as small polyps, the next stepwise immunohistochemistry testing would use Bcl2, Bcl6, CD10, and Ki-67. In particular, the expansion or nodular/follicular localization pattern of Bcl2+
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B cells in CD20+ B cell proliferating areas could help detect low grade B cell lymphoma because most low grade B cell lymphomas are Bcl2-positive (Fig.2, Supplementary Figs. A.2 and A.8).
Mantle cell lymphoma (MCL) occasionally involves long segments of the small and large intestines and presents as lymphomatous polyposis. This characteristic presentation occurs when either primary GI tract MCL develops or nodal MCL involves the GI tract. In addition, MCL can present as a subtle lesion in the GI tract in the form of small lymphoid aggregates. For nodal MCL that secondarily involves the GI tract, subtle lymphoid infiltrates in the initial or follow-up endoscopic biopsy are not uncommon, more often in a gastroduodenal biopsy than a lower GI tract biopsy. As explained above, however, benign lymphoid aggregates or benign reactive immune cell reactions are generally nonspecific in GI tract biopsy samples, so small lymphoid aggregates might not attract pathologists’ interest in daily practice. In addition, endoscopic findings for such subtle infiltrates are usually nonspecific. Therefore, the suspicion of pathologists might only be aroused when clinically indicated. Applying cyclinD1 (and SOX11) seems to be the simplest and most helpful step when encountering suspicious cases, although histologic findings have been described for subtle infiltrates of nodal MCL: small lymphocytes with irregular nuclear contours and penetration of lymphoid cells among glandular structures without invading them (Supplementary Fig.A.3).5
Follicular lymphoma can present as polyps or polyposis in asymptomatic patients, limited to the second portion of duodenum; duodenal type follicular lymphoma can thus be considered when the lesion is localized to the duodenum and confirmed as Ann-Arbor stage IE or IIE disease under the criteria of the revised 2017 WHO classification (Supplementary Fig.A.4).2, 6
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Appendix 3. Approaches to MALT lymphoma in the GI tract: lymphoepithelial lesions by marginal zone B cells
A lymphoepithelial lesion (LEL) can have diagnostic implications for MALT lymphoma. A LEL is defined as the infiltration of epithelial structures by three or more neoplastic B cells. LELs also include features of eosinophilic degeneration of the glandular epithelium and the destruction or distortion of glandular architectures. LELs are considered to be specific to the stomach, but they can be nonspecific to other sites (such as the ileum or rectum) that physiologically contain MALT, such as a Peyer patch or tonsil tissues. Therefore, LEL-like features in the presence of physiologic MALT or a Peyer patch should not be considered true LELs. In a true LEL of MALT lymphoma, the tumor cells expand into the lamina propria and infiltrate the glandular epithelium away from lymphoid follicles.2, 7-11
Tumor cells of MALT lymphoma, called centrocyte-like cells (CCL) or marginal zone cells (MZC), show the cytologic features of small irregular nuclei with a distinct rim of clear cytoplasm.
Overt gastric MALT lymphoma necessarily shows diffuse infiltrates of tumor cells, as well as prominent true LELs. The formation of reactive lymphoid follicles or follicular colonization could be admixed with MALT lymphoma. Plasma cell infiltration/plasmacytoid differentiation is occasionally admixed with tumor B cells with CCL or MZC features, which could generate a lymphoplasmacytic appearance in MALT lymphoma. This plasma cell infiltration/plasmacytoid differentiation occasionally contains Dutcher bodies in the form of intranuclear eosinophilic inclusions, which is considered to show the monoclonal trait of plasma cells. Scattered large cells, centroblasts or immunoblasts, could be accepted when they compose less than 10% of the tumor cells and don’t form sheets or large clusters. If the large cells go beyond that proportion, the
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transformation to diffuse large B cell lymphoma from MALT lymphoma should be considered (Supplementary Fig.A8, A.9 and A.10).2, 7-11
Most gastric MALT lymphoma develops from Helicobacter pylori infection, so the main benign mimicker is Helicobacter gastritis. Helicobacter gastritis also presents with florid lymphoid follicles, focal LELs, and plasma cells, which mimic gastric MALT lymphoma, but gastritis lesions do not generally include an expansile, destructive mass. Considering the diverse histologic spectrum, from Helicobacter gastritis, chronic active gastritis, and follicular gastritis to overt MALT lymphoma, a histologic scoring system could be used for differential diagnosis. The Wotherspoon scoring system is the most widely accepted system of histologic scoring for gastric MALT lymphoma (Supplementary Table 1),12 although it does not consider plasma cell infiltration/plasmacytoid differentiation. Although histopathology is required to confirm the diagnosis, a clonality assay of immunoglobulin gene rearrangement via PCR and a kappa/lambda light chain restriction pattern analysis could be helpful. Kappa and lambda light chain restriction pattern analyses are frequently difficult in small mucosal biopsies because the monoclonal plasma cell aggregates are frequently mixed with polyclonal, reactive plasma cells. In the smaller endoscopic biopsy samples that contain even fewer plasma cells, determining the monoclonal restriction versus the polyclonal pattern of plasma cells is even more difficult. Recently, immunoglobulin gene rearrangement PCR assays using Biomed-2 IGH and IGK multiplex primers have shown high sensitivity in the detection of monoclonality in MALT lymphoma.12 Abnormal CD43 expression on B cells can be detected by immunohistochemistry in up to 50% of MALT lymphoma cases; therefore, CD43, an indication of neoplastic B cells, also would be helpful in the diagnosis of MALT lymphoma.13 After therapy, the response rate is determined by lymphoid infiltration of B cells and plasma cells, LELs, and stromal changes. Posttherapy grading systems
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have also been introduced, such as the GELA (Groupe d’Etude des Lymphomes de l’Adult) system (Supplementary Table 2 and Supplementary Fig.A.11).12
Intestinal MALT lymphoma is rarer than gastric MALT lymphoma. In the intestine, LELs are not diagnostic, making an expansile, destructive mass formed by the solid proliferation of tumor cells with CCL or MZC features and a compatible immunophenotype more important in diagnosis (Supplementary Fig.A8 and A.12). As the benign or indolent counterpart of overt lymphoma, the atypical marginal zone hyperplasia found in native MALT of appendix tonsils or Peyer patches in pediatric patients or in immunoproliferative small intestinal disease in the small intestines of young adults might need to be considered in the clinical context when encountering non-destructive but proliferative B cells and plasma cells in the MALT zone of intestines.2, 7-11
Appendix 4. Detailed legends to Figures: including brief case summaries
Fig.1. Examples of various macroscopic/gross/endoscopic features of gastrointestinal lymphomas:
large mass lesions of ulcerofungating, ulceroinfiltrative, or annular constrictive gross types (A, diffuse large B cell lymphoma; B, anaplastic large cell lymphoma; C, mantle cell lymphoma; D, monomorphic epitheliotropic intestinal T cell lymphoma; E, classic Hodgkin lymphoma), near- normal looking features (F, monomorphic epitheliotropic intestinal T cell lymphoma), small polyp-like features (G, lymphoid polyp in colon; H, extranodal marginal zone B cell lymphoma of MALT type in colon), multiple small polyps limited to short segments of the intestines (I and J, duodenal type follicular lymphoma), multiple variable-sized polyps in long segments from the lower to upper gastrointestinal tract (K, mantle cell lymphoma), multiple erosions with a cobblestone-like appearance (L, involvement of nodal mantle cell lymphoma in stomach), atrophy
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and erosions (M and N, lymphomatoid gastropathy), large necrotic ulcer and perforation (O, extranodal NK/T cell lymphoma of nasal type), and small abrupt perforation (P, extranodal NK/T cell lymphoma of nasal type).
Fig.2. The pictures in the first row are of a 2.5cm rectal polyp. Secondary follicles are prominently developing, and the germinal centers show well-organized polarity of light and dark zones and a well-demarcated mantle layer. CD20 expression is localized to the follicles. Neither thick expansion of Bcl2+ B cells in the mantle/marginal zone nor follicular localization of Bcl2+ B cells was noted. This case was diagnosed as a rectal tonsil. Due to prominent germinal center development, CD20 expression seems to be solid or diffuse; however, thin layers of Bcl2 expression in the mantle zone, as well as CD10 and Ki-67 expression patterns in the germinal centers, could help to define the benign property of this lesion.
The pictures in the second row are of a 6mm single polyp arising in the rectum. In this case, CD20 expression looks nodular and is localized to the follicles. Neither thick expansion of Bcl2+ B cells in the mantle/marginal zone nor follicular localization of Bcl2+ B cells was noted. This case was diagnosed as a lymphoid polyp.
The pictures in the third row are of a 6mm single polyp arising in the descending colon. In this third case, expansile proliferation of monotonous lymphocytes with clear cytoplasm, so called centrocyte-like or marginal zone cells, is noted. In the immunohistochemistry, diffuse, solid, dense expression of CD20, as well as thick expansion of Bcl2+ marginal zone B cells were observed.
CD3 and CD10 (and Bcl6) could additionally help to discriminate the Bcl2+ CD20+ marginal zone B cell expansion with follicular colonization by tumor cells. The Ki-67 index is low in the expanded marginal B cell zone, suggesting the low grade B cell trait (supplementary Figure 3).
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This case was diagnosed as extranodal marginal zone B cell lymphoma of the MALT type after additional immunoglobulin gene rearrangement tests.
Fig.3. A case of a 62 year old female patient complaining of abdominal pain for one month (A–F).
A large ulcerofungating mass was noted in the stomach (A). The biopsied samples revealed monomorphic infiltration of medium-sized lymphocytes (B and C). The tumor cells were CD3+
(D), EBER+ (E), and TIA1+ (F). Following radiologic tests revealed multiple mesenteric lymphadenopathy and splenomegaly, but nasopharyngeal/nasal/sinonasal lesions were not noted.
This case was diagnosed as extranasal extranodal NK/T cell lymphoma of the nasal type.
A case of a 53 year old female referred from an outside hospital under the initial diagnosis of T cell lymphoma (G–L). In the gastric endoscopy, small, superficial, erosive, atrophic, and nodular lesions were multiply noted from the antrum to the fundus (G and H). Microscopically, multifocal aggregates of lymphocytes showed enlarged, irregularly angulated nuclei and eosinophilic cytoplasm. The atypical lymphocytes diffusely infiltrated into the lamina propria (I and J). The infiltrated lymphocytes were CD3+ (K), CD56+ (L), and EBER- and determined to be of an NK cell lineage. T cell receptor gamma, beta, and delta chain gene rearrangement tests were negative.
The gastric lesions were diagnosed as lymphomatoid gastropathy (NK cell gastropathy). After three years of follow-up observation without any further treatment, the lesion disappeared.
Fig.4. When encountering a proliferation of large, pleomorphic cells with diffuse, solid, sheet-like growth patterns in the gastrointestinal tract, undifferentiated or poorly differentiated carcinoma might be the first suspect. After excluding carcinoma (cytokeratin, CK), hematologic malignancies (CD45), melanoma or similar clear cell/perivascular epithelioid cell lineages (HMB45), or other round cells such as a desmoplastic small round cell tumor (desmin) would be other possibilities.
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Although CD43 is not expressed in normal B cells, CD45 and CD43 could be expressed and would help detect most hematologic malignancies of various cell lineages. This primary process using CK, CD45 (and CD43), HMB45, and desmin could be necessary (or not) depending on the histopathological and clinical contexts. If hematologic malignancies are suspected, the first line marker battery of CD20, CD3, CD30, and EBER could help narrow the possibilities among lymphomas that arise in the gastrointestinal tract. Certain subtypes of lymphoma, such as anaplastic large cell lymphoma, plasmablastic lymphoma, Hodgkin lymphoma, or highly undifferentiated lymphoma, might lose CD45; however, CD30, CD138, MUM1, and other markers of hematopoietic cell lineages such as CD68 and myeloperoxidase could help identify cell lineages for further differential diagnosis. When diagnosing such rare, unusual subtypes, it would be better to apply multiple markers to cross-check the accuracy of the diagnosis. Abbreviations:
DLBCL, diffuse large B cell lymphoma; BL, Burkitt lymphoma; HGBCL, high grade B cell lymphoma; NHL, non-Hodgkin lymphoma; ALCL, anaplastic large cell lymphoma; MPO, myeloperoxidase.
Fig.5. As shown in the pictures in the first row, when large, pleomorphic cells with a diffuse, solid, sheet-like growth pattern show diffuse and dense expression of CD20, the next step is differentiating among aggressive B cell lymphomas: diffuse large B cell lymphoma, Burkitt lymphoma, and high grade B cell lymphoma (DLBCL/BL/HGBCL). In B cell lymphoma, reactive T cells might be variably infiltrated in the tumor background in scattered, diffuse, or patch patterns.
Although T cell lymphoma might be combined with B cell lymphoma, this circumstance is very rare, and the present review focuses on the subtypes prevalent in the GI tract. The Ki-67 proliferation index might not be needed in cases such as this one, which shows a diffuse proliferation of large tumor cells; however, in cases with medium to large cells, especially focally
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sampled from an endoscopic biopsy, Ki-67 could help to distinguish aggressive B cell lymphoma from indolent/low grade B cell lymphoma.
In T/NK cell lymphomas, as shown in the pictures of the second row, CD20-positive B cell areas take the forms of peripheral displacement, residual germinal centers, or individual scattering, whereas most tumor T cells reveal the diffuse expression of CD3. A highly increased Ki-67 proliferation index might be helpful, but interpretation could be difficult due to the background of activated immune cell infiltration or residual germinal centers.
When tumor cells are negative or only focally positive for CD20 and CD3 with a high Ki-67 proliferation index, as shown in the pictures in the third row, CD30 or EBER could help detect lymphomas such as anaplastic large cell lymphoma or extranodal NK/T cell lymphoma because those types occasionally show sparse expression of CD3 (Table 2). In addition, CD20-negative DLBCL/BL/HGBCL might also be indicated when tumor cells are negative or only focally positive for CD20 or CD30. Other B cell markers, CD79a, PAX5, BOB.1, and OCT2, might help define B cell lineages. For tumors of plasma cells or plasmablastic differentiation, such as plasma cell myeloma involvement or HHV8-associated plasmablastic lymphoma, CD138 and MUM1 can be helpful (pictures in the fourth and fifth rows), as well as EBER.
Fig.6. A case of MEITL in a 60 year old Asian female complaining of watery diarrhea for three months but showing normal looking endoscopic features in the small and large intestines (A). The duodenal mucosa (B–E) revealed increased IELs with CD3+ (D), CD8+ (E), CD56-, and EBER- phenotypes, and the colon mucosa also revealed increased CD3+ IELs (F and G). The tumor cells are small to medium-sized. Other mucosal changes such as villous atrophy, chronic or mixed inflammation, and plasma cell infiltration were not prominent (B, C, F). Clinically, the patient had
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no evidence of celiac sprue, such as a history of gluten-sensitivity. T cell receptor gamma gene rearrangement was determined to be monoclonal. Patient underwent chemotherapy under the diagnosis of MEITL. However, mucosal IELs of the duodenum (H) and colon didn’t change six months after chemotherapy, and the symptom of diarrhea persisted. Seven months later, an emergency jejunum segmentectomy was performed due to bowel perforation (I). Macroscopically, multiple ulceroinfiltrative masses with multifocal perforations were noted (I). Microscopically, the mass lesion revealed diffuse proliferation of medium to large lymphocytes (J and K), and the adjacent mucosa revealed IELs (L). The tumor cells were CD3+ and CD8+ (M and N).
Fig.7. A case of EBV-associated diffuse large B cell lymphoma. On H&E staining, poorly formed granulomas and polymorphic, heterogeneous immune-inflammatory cells are predominantly observed, whereas large tumor cells are individually scattered or loosely aggregated with lower cellularity than that of the background immune-inflammatory cells. If the tumor cells are masked by the background cells, this can be misdiagnosed as granulomatous inflammation, such as tuberculosis. The large tumor cells are strongly positive for CD20 and EBER, partially express CD30, and are surrounded by background CD3+T cells.
Fig.8. An 8cm necrotic ulcer was noted in the endoscopy, which was clinically suspected to be advanced gastric cancer. The initial diagnosis from the gastric biopsy did not identify lymphoma (A and B). One month later, erythema developed on the back skin, which was not identified as lymphoma until then (C and D). Two months later, an emergency operation was performed due to gastric wall perforation. Grossly, a large, necrotic ulcer with perforation was noted without a definite mass formation (E). On microscopic examination, coagulative, transmural necrosis was noted in the gastric wall (F and G). A multifocal aggregation of large pleomorphic tumor cells within the necrotic gastric wall (H) and angiocentricity (I) was observed. Tumor cells were CD3+,
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EBER+, and CD56+ (J–L); therefore, this case was diagnosed as extranasal extranodal NK/T cell lymphoma of nasal type.
A small perforation was observed in the jejunum, which was emergently resected due to the unexplained perforation (M). Although the perforation site was small and covered with mixed inflammatory exudates (N), EBER-positive lymphoid cells were frequently observed in the perforated site (O). Four months later, extranodal NK/T cell lymphoma occurred in the nasal cavity (P). This case might show an example of subtle GI tract involvement in ENKTL that initially presented as bowel perforation before the overt growth of a nasal tumor, or it might show the disease spectrum of chronic active EBV infection.
14 Supplementary Tables
Supplementary Table A.1. Gastric marginal zone (MALT) lymphomas: Histologic scoring by the Wotherspoon scoring system12
Grade Description Histologic Features
0 Normal Plasma cells in LP No lymphoid follicles
1 Chronic active gastritis Lymphocyte clusters in LP No follicles, LELs
2
Chronic active gastritis with florid lymphoid follicle formation
Prominent follicles with surrounding mantle zone and plasma cells No LELs
3
Suspicious lymphoid infiltrate in LP, probably reactive
Follicles surrounded by lymphocytes that infiltrate diffusely in LP and +/- epithelium
4
Suspicious lymphoid infiltrate in LP, probably lymphoma
Follicles surrounded by CCL cells (MZC) that infiltrate diffusely in LP and epithelium
5 Marginal zone (MALT) lymphoma
Dense diffuse infiltrate of CCL cells in LP with prominent LELs
Abbreviations: CCL, centrocyte-like; MZC, marginal zone cell; LEL, lymphoepithelial lesion; LP, lamina propria
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Supplementary Table A.2. GELA grading system for the posttreatment evaluation of gastric MALT lymphoma12
Score Lymphoid Infiltrate LELs Stromal Changes Significance Complete histologic
remission (CR)
Absent or scattered plasma cells and small lymphoid cells in the LP
− Normal or empty LP and/or fibrosis
CR
Probable minimal residual disease (pMRD)
Aggregates of lymphoid cells or lymphoid nodules in the LP/muscularis mucosa and/or submucosa
− Empty LP and/or
fibrosis
CR
Responding residual disease (rRD)
Dense, diffuse, or nodular extending around glands in the LP
+/− Focal empty LP and/or fibrosis
PR
No change (NC) Dense, diffuse, or nodular +/− No changes SD or PD Abbreviation: GELA, Groupe d’Etude des Lymphomes de l’Adult; LEL, lymphoepithelial lesion;
LP, lamina propria
16 Supplementary Fig.A.1.
Supplementary Fig.A.1. A case of diffuse large B cell lymphoma arising in the ileocecal area, clinically presented as intussusception in a 33 year old female. This is one common presentation of gastrointestinal lymphoma. Grossly, a 4cm mass is noted in the endoscopy (A) and surgical resection specimen (B). Microscopically diffuse infiltration into the bowel wall by large, pleomorphic cells is noted (C and D). The lineage of the tumor cells was determined by CD20 immunohistochemistry to be B cells (E). Ki-67 revealed a high proliferation index (F). The CD10 (G), Bcl6 (H), and MUM1 (I) expression patterns suggest germinal center B cell-like type lymphoma.
17 Supplementary Fig.A.2.
Supplementary Fig.A.2. Pictures of a 2.5cm rectal polyp. Secondary follicles are prominently developing, and the germinal centers show well-organized polarity of light and dark zones and a well-demarcated mantle layer. CD20 expression is localized to the follicles. Neither thick expansion of Bcl2+ B cells in the mantle/marginal zone nor follicular localization of Bcl2+ B cells was noted. This case was diagnosed as a rectal tonsil. Due to the prominent germinal center
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development, CD20 expression seems to be solid or diffuse; however, thin layers of Bcl2 expression in the mantle zone, as well as the CD10 and Ki-67 expression patterns in the germinal centers, could help to define the benignity of this lesion.
19 Supplementary Fig.A.3.
Supplementary Fig.A.3. A case of mantle cell lymphoma that initially presented as lymphomatous polyposis. Numerous, variable-sized polyps are noted in a long segment of the gastrointestinal tract from the stomach to the distal colon (A–D). In addition to individual polyps, a multinodular mass is also forming in the ileocecal area (C). The polyp obtained from the colon showed nodular lymphocytic aggregates composed of monomorphic, small, cleaved CD20+ B cells (E–H), and the tumor B cells expressed cyclinD1 and CD5 (I and J). Although this case showed strong expression of CD5, many other cases of mantle cell lymphoma show dim CD5 expression, weaker than that of normal T cells. In another case of nodal mantle cell lymphoma that subtly involved the stomach (K and L), a patient with generalized lymphadenopathy complained of mild abdominal discomfort. The endoscopic findings looked nearly normal, so a blind biopsy was done. After confirming nodal mantle cell lymphoma, the retrospective application of cyclinD1
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immunohistochemistry to the gastric biopsy specimen revealed the subtle involvement of mantle cell lymphoma (L and M).
21 Supplementary Fig.A.4.
Supplementary Fig.A.4. A case of duodenal type follicular lymphoma in a 45 year old female.
During a heath checkup, multiple small polyps were noted in the 2nd portion of the duodenum. On microscopic examination, each polyp had one to two lymphocytic nodules composed of monotonous, small, cleaved CD20+ B cells. Similar to nodal follicular lymphoma, CD20 and Bcl2 expression was noted within both the follicle and the perifollicular area. Co-expression of Bcl2 and CD10 (and Bcl6) could confirm the usual immunophenotype of follicular lymphoma. The Ki- 67 index is low in the germinal center, suggesting the neoplastic property of this germinal center as well as the low grade lymphoma trait. After systemic work up, the lesion was defined as duodenal type follicular lymphoma.
22 Supplementary Fig.A.5.
Supplementary Fig.A.5. Under endoscopy, multiple large, ulcerofungating masses are noted (A), which are clinically suspected to be advanced gastric carcinoma. Microscopically, large, pleomorphic tumor cells are infiltrating between the mucosal glands (B). The nuclei of the tumor cells are kidney-shaped, Hodgkin Reed-Sternberg cell-like, namely hallmark cells (C). The tumor cells are negative for CD20, partially positive for CD3 (D), diffusely and strongly positive for CD30 (E), and also positive for CD4 (F). Therefore, the masses in the stomach are diagnosed as anaplastic large cell lymphoma, AKL-negative type.
23 Supplementary Fig.A.6.
Supplementary Fig.A.6. A case of celiac sprue in a 27 year old Caucasian male (A–E). The 2nd portion of the duodenal mucosa revealed villous atrophy (A–B) and increased plasma cell infiltration in the lamina propria (B and C: CD138). Within the glandular epithelium, increased infiltration of CD3+ and CD8+ T cells (D and E), so called intraepithelial lymphocytosis, is noted.
A case of MEITL (F–O) in a 66 year old Asian male complaining of dyspepsia, diarrhea, and weight loss for three months. Multiple ulcers were noted in the descending colon (F). The colon mucosa revealed highly increased IELs within the glandular epithelium. Tumor cells were small to medium-sized with dark angulated nuclei and clear cytoplasm. The tumor cells also infiltrated the lamia propria (G and H). The tumor cells were CD3+, CD8+, CD4-, CD56-, and EBER- (J–M) and diagnosed as MEITL. A 2nd biopsy revealed more prominent lymphoid proliferation, with IELs of CD3+ T cells (N and O).
24 Supplementary Fig.A.7.
Supplementary Fig.A.7. A case of classic Hodgkin lymphoma. On H&E staining, lymphoid nodules and perinodular fibrosis are noted within the colon wall. Large, pleomorphic tumor cells are individually scattered or loosely aggregated in the polymorphic, heterogeneous immune- inflammatory cell background. The large pleomorphic tumor cells are CD20-/+, CD3-, and CD30+. Synchronously detected masses in the spleen and liver show the same features. For this case, the predominant tumor location was the colon wall.
25 Supplementary Fig.A.8.
Supplementary Fig.A.8. Pictures of a 6mm single polyp arising in the descending colon. In this case, expansile proliferation of monotonous lymphocytes with clear cytoplasm, so called centrocyte-like or marginal zone cells, is noted. In the immunohistochemistry, diffuse, solid, dense expression of CD20 and thick expansion of Bcl2+ marginal zone B cells were observed. CD3 and
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CD10 (and Bcl6) could additionally help to discriminate the Bcl2+ CD20+ marginal zone B cell expansion with follicular colonization by tumor cells. The Ki-67 index is low in the expanded marginal B cell zone, suggesting the low grade B cell trait. CD5 or CD43 is expressed only in the CD3+ T cell zones, not the CD20+ B cell zone. Although grouped CD20+ B cells are infiltrated into the glands, so called lymphoepithelial lesions, this feature might not be diagnostic in colon sites. This case was diagnosed as extranodal marginal zone B cell lymphoma of the MALT type following additional immunoglobulin gene rearrangement tests.
27 Supplementary Fig.A.9.
Supplementary Fig.A.9. Pictures of a true lymphoepithelial lesion (LEL) in cases of extranodal marginal zone B cell lymphoma and mucosa-associated lymphoid tissue of the stomach (A–C).
Three or more neoplastic B cells, so-called centrocyte-like cells (CCLs) or monocytoid/marginal zone cells (MZCs) were infiltrating into glands, and the affected glands show epithelial
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degeneration and the destruction or distortion of glandular structures (arrows, A–C). LEL-like features of physiologic MALT (D; arrows) should not be considered as a true LEL.
A case of gastric MALT lymphoma (E–H). In the low magnification field, diffuse, expansile infiltrates of lymphocytes (E) are noted. On higher magnification, diffuse, monotonous proliferation of small to medium-sized CCLs or MZCs is noted (F). CCL or MZC tumor cells infiltrate glands, and the degenerated glandular epithelial cells reveal distorted glandular structures.
All those features could be defined as true LELs (G). The formation of a reactive lymphoid follicle is noted in an adjacent mucosa area in another gastric biopsy piece of the same case (H; arrows).
Application of CD20 and cytokeratin immunohistochemistry for MALT lymphoma (I–L). CD20 immunohistochemistry revealed diffuse proliferation and glandular infiltration of B cells (I and J).
In cases showing definite features of MALT lymphoma, CD20 might be enough to define the diffuse proliferation of B cells and LELs by neoplastic B cells (J; arrows). On occasion, however, cytokeratin might give additional help in finding lymphoepithelial lesions (K and L; arrows).
In this MALT lymphoma case (M and N), a reactive lymphoid follicle is partly formed (M; arrow);
however, CD20+ tumor B cells deeply expand into the lamina propria (M) and infiltrate the glandular epithelium, forming a LEL (N; arrows) away from the lymphoid follicles.
This MALT lymphoma case (O–R) was accompanied by plasma cell infiltration/plasmacytoid differentiation. CD20+ tumor B cells (O) were mixed with an upper layer of CD138+ plasma cells (P). Kappa and lambda light chain restriction analysis by RNA in situ hybridization (Q and R) revealed the distinctive predominance of kappa light chain-positive plasma cells over lambda light chain-positive plasma cells. Plasma cell infiltration/plasmacytoid differentiations occasionally
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contain Dutcher bodies (arrows) in the form of intranuclear eosinophilic inclusions (S), suggesting that the plasma cells are monoclonal.
Scattered large cells of centroblasts or immunoblasts (arrows) can be accepted when they occupy less than 10% of the tumor cells and don’t form sheets or large clusters (T). Reactive germinal centers could be misdiagnosed as sheets of large cells in small biopsy samples (U; arrows). The germinal centers are composed of large CD20+ B cells (V) showing Bcl6 (and CD10) expression (W). To define them as reactive germinal centers and not transformed large B cell lymphoma arising from MALT lymphoma, it might be helpful to compare Ki-67 (X) and the Bcl6 area (Y).
If a large B cell-like component expressed Bcl6, that area is likely a reactive germinal center in gastric lesions that are otherwise compatible with MALT lymphoma or reactive gastritis lesions.
If diffuse proliferation of large pleomorphic cells accompanies MALT lymphoma, transformation into diffuse large B cell lymphoma might be indicated (Z; left side).
30 Supplementary Fig.A.10.
Supplementary Fig.A.10. Representative features of MALT lymphoma. The perifollicular marginal zone has expanded and infiltrated into the lamina propria away from the follicles (A).
31
Centrocyte-like, monocytoid/marginal zone B cells show small irregular nuclei and a distinct rim of clear cytoplasm (B). Groups of three or more neoplastic B cells have infiltrated the gastric gland epithelium in a so called lymphoepithelial lesion, which also includes features of eosinophilic change to the epithelial cells and the destruction of the gland architecture (C). Plasma cells/plasmacytoid differentiation have a lymphoplasmacytic appearance with occasional Dutcher bodies (D). Diffuse proliferation and infiltration into the glandular epithelium by CD20+ B cells could be highlighted (E). Lymphoepithelial lesions were highlighted in cytokeratin immunohistochemistry (F).
32 Supplementary Fig.A.11.
Supplementary Fig.A.11. Post-therapy grading of MALT lymphoma according to the GELA (Groupe d’Etude des Lymphomes de l’Adult) system.9
A case showing no change after treatment. Dense, diffuse proliferation of tumor cells without stromal change (A and B).
A case of responding residual disease (C) showing nodular lymphocytic aggregates (right side of the figure) that extend around glands, forming LELs and focally empty lamina propria.
A case of probable minimal residual disease (D) showing lymphoid aggregates and stromal change, but not LELs.
33 Supplementary Fig.A.12.
Supplementary Fig.A.12. A case of MALT lymphoma arising in the small intestine (A–D).
Expansile, destructive lesions (A and B) are noted via the solid proliferation of tumor cells with CCL or MZC features (C). Follicular colonization by tumor cells is also noted (B; arrows). The infiltration of tumor cells into the overlying mucosal epithelium is noted, although it might not be diagnostic for MALT lymphoma of the small intestine (D; arrows).
A case of colon MALT lymphoma (E–H). The macroscopically small superficial lesion, diffuse lymphocyte proliferation of CCL or MZC features (F), and CD20 expression (G) suggest neoplastic B cell expression. Through further immunophenotyping and immunoglobulin gene rearrangement PCR assays using Biomed-2 IGH (H) and IGK multiplex primers, MALT lymphoma was confirmed. Abbreviations: M, ladder marker; N, negative control; P, positive control; S, patient sample
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