Haematologica 2002; 87:(12)ECR40
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Diffuse large B-cell lymphoma, anaplastic variant. Report on a problematic case primarily arising in the stomach
Stefano Aldo Pileri,* Stefano Ascani,* Pier Luigi Zinzani,* Gianluca Gaidano,° Milena Piccioli,* Maura Rossi,* Pier Paolo Piccaluga,* Teresa Marafioti,* Alessandro Pileri Jr.,* Roberta Pacini,# Brunangelo Falini,# Elena Sabattini*
*Chair of Pathologic Anatomy, Clinical and Pathology Lymphoma Units, Institute of
Haematology and Clinical Oncology "L. and A. Seràgnoli", Bologna University, Bologna, Italy; °Department of Medical Sciences - Division of Internal Medicine &endash; Haematology Unit, University of Western Piedmont "A. Avogadro", Novara, Italy; #Chair of Haematology and Laboratory of Haematopathology, Institute of Hematology, Perugia University, Italy.
Correspondence: Professor Stefano A. Pileri, MD, Cattedra di Anatomia Patologica, Servizio di Ematopatologia, Istituto di Ematologia ed Oncologia Medica "L. e A. Seràgnoli", Università di Bologna, Policlinico S. Orsola, Via Massarenti 9, 40138 Bologna, Italy. Phone: international +39.051.6363044. Fax: international +39.051.6363606. E-mail:pileri@almadns.unibo.it

Anaplastic large cell lymphoma (ALCL) was originally described as an aggressive neoplasm, consisting of bizarre lymphoid elements, expressing the CD30 molecule and bearing a T-, B-, null- or even hybrid-cell phenotype.1 The REAL Classification limited the term ALCL to tumors of T/null-cell derivation:2 this assumption has been maintained in the recent WHO Classification.3 Molecular and phenotypic studies, however, have revealed that even this more restrictive definition of the process does not apply to a uniform lymphoma category. In fact, about 60% of ALCL2,3 carry the t(2;5)(p23;q35) translocation or its variants and over-express the anaplastic large cell lymphoma kinase (ALK-protein), while the remaining ones do not.4-9 The exclusion of large B-cell lymphomas with anaplastic morphology from the ALCL category was based on: a) the lack of ALK-protein expression, b) possible derivation from a follicular lymphoma, c) occurrence of IgVH gene somatic mutations, and d) clinical behavior like that of diffuse large B-cell lymphoma (DLBCL)8,10,11 The authors report on a tumor, which according to current criteria could be diagnosed as T/null-cell ALK- ALCL, but at closer phenotypic and molecular examination showed unequivocal B-cell derivation.
In July 2000, the patient-a 70-year-old man-referred to another Hospital because of persistent epigastric pain and dyspepsia. He had been followed for chronic gastritis since 1989 and had undergone several endoscopic examinations and courses of antibiotic therapy because of proven Helicobacter pylori infection, one of which six months before admission to the Hospital. At endoscopy, a wide ulcerated lesion of the antrum measuring 8.5 cm across was detected. A total-body computed axial tomography (TB-CAT) displayed infiltration of the entire gastric wall, along with enlargement of the peri- and retro-gastric lymph nodes. A Jamshidi needle biopsy was unremarkable. In August 2000, the patient underwent sub-total gastrectomy with regional lymphadenectomy. The samples obtained from the surgical specimen were sent in consultation to the Lymphoma Unit of the Institute of Haematology of Bologna University. Following the diagnosis (see below), 6 cycles of VAD were administered. In April 2001, at completion of therapy, TB-CAT and endoscopic examination of the gastric stump did not reveal residual disease. At the last follow-up in September 2002, the patient was alive, well and in complete remission. Material and Methods. Multiple samples were taken from the peri-gastric lymph nodes and stomach (corresponding to the ulcerated lesion, the distal resection margin, which showed slightly swollen mucosa, and apparently normal gastric body mucosa at distance from the ulcer). They were fixed in 10% buffered formalin for 24 hours, automatically processed and paraffin-embedded according to scheduled procedures.12,13. Three micron-thick sections were cut and stained with hematoxylin and eosin (H&E) and Giemsa. Further sections were immunostained on a TechMate 500 machine by applying: previously described antigen retrieval methods,12 the panel of antibodies listed in Table 1 and either the alkaline phosphatase anti-alkaline phosphate complexes technique (APAAP) or the EnVisionTM Plus method.13 In situ hybridization (ISH) for Epstein Barr Virus (EBV) was carried out by EBER 1&2 probes.14 Micro-dissection was performed from sections corresponding to the gastric ulcerated lesion and the distal resection margin, according to previous experience.15,16 The dissected cells were pulled together, by maintaining the separation between the ones corresponding to the ulcerated lesion and those obtained from the resection margin. The DNA from each pool was extracted and purified using a commercial kit (QIAamp DNA mini Kit, QIAGEN, Italy) according to the manufacturer's instructions.17 IgVH gene rearrangements were amplified with two sets of six VH gene family-specific primers and a JH primer in separate reactions for each VH primer, as described.18,19 The VH primers used in this study hybridize to sequences in the framework region (FR) 1 or 2 of the respective VH families. PCR was performed for 45 cycles with an annealing temperature of 60°C. PCR products were directly sequenced using a commercially available kit (ThermoSequenase, Amersham Life Sciences) as previously reported.17 Sequences were matched with the V-BASE sequence directory (MRC Centre for Protein Engineering, Cambridge, UK) using MacVector 6.0.1 software (Oxford Molecular Group PLC, Oxford, UK) for comparison of the rearranged IgV genes to the most homologous germ line sequences. TCR a gene study was performed according to the protocol reported by Benhattar et al.20 TCR a gene analysis was carried out according to McCarthy et al.21 by employing the D2 and J2 primer sets.
Results. The ulcerated lesion was sustained by a vaguely nodular growth consisting of highly proliferating, cohesive large cells, at times with Reed-Sternberg-like features (Figures 1 and 2). These cells showed highly polymorphic, often horse-shoe or kidney shaped nuclei, prominent inclusion-like nucleoli, and a wide rim of cytoplasm, greyish-violet at Giemsa (Figures 2 and 3). Some collagen bands partly circumscribing the nodules were found (Figure 1). A similar population was detected in the resected lymph nodes, which showed prominent diffusion through residual sinuses (Figure 3).
The distal resection margin showed a second neoplastic population, which consisted of centrocyte-like elements, infiltrating the mucosa and sub-mucosa and producing typical lympho-epithelial lesions (Figure 4). The amount of blasts was exceedingly low, as was the mitotic activity. Occasional residual germinal centers were comprised within the neoplastic population (Figure 4). Micro-foci of similar tumoral involvement were detected in the samples from the apparently normal gastric body mucosa.
Retrospective examination of an endoscopic biopsy taken in 1999 showed foci of the small cell tumor along with Helicobacter pylori infection. No signs of the latter were detected in the surgical specimen.
The immunohistochemical and ISH findings observed in the two lymphomatous components are summarized in Table 1. At a preliminary analysis, they suggested a T/null-cell derivation of the anaplastic tumor (Figures 5-8). However, at a further investigation, performed following the results of molecular analyses (see below), large anaplastic cells displayed clear-cut expression of the PAX-5 gene product/B-cell Specific Activator Protein (BSAP) (Figure 9).
The TCRgamma and delta genes turned out to be in germline configuration in the samples taken from both the ulcerated lesion and small cell neoplastic population. By contrast, IgVH gene status revealed a clear-cut monoclonal band on both sides, although the rearranged genes used different VH families (VH2 the anaplastic population and VH6 the marginal zone one) and displayed completely unrelated sequences (Figures 10 and 11).
According to the above mentioned results, a diagnosis of DLBCL, anaplastic variant associated with an unrelated gastric MALT lymphoma was made.2,3
Discussion. The present case underlines the difficulties encountered in studying ALK- large cell lymphomas with anaplastic morphology as well as the need to apply all tools available for their classification. In particular, the observed tumor was consistent with the diagnosis of ALCL in terms of cell morphology and pattern of growth, which included intra-sinusoidal spread in the lymph nodes.1-3,7,8 Based on the results provided by the antibodies commonly used for lymphoma characterization,2,3 at a preliminary analysis a T/null-cell derivation might be easily sustained. Neoplastic cells expressed CD30 and partly CD45, in the absence of CD15, thus ruling out type II nodular sclerosis Hodgkin's lymphoma, a possibility to be taken into consideration because of the vague nodular growth pattern and the occurrence of neoplastic elements with Reed-Sternberg-like features.2,3,7,8 The stains for CD20, CD79a and Ig were all reliably negative, as assessed by the internal controls. CD2 showed a clear-cut membrane-bound positivity at least in a proportion of the neoplastic cells. CD3 was partly positive, although the results appeared somewhat ambiguous: in fact, the staining was on one hand intra-cytoplasmic, as observed in activated T-lymphocytes and tumors derived from them,7,8 but on the other appeared faint and with a gradient from the periphery to the central part of the cytoplasm, a finding suggesting passive absorption more than CD3 production.8 The lack of other T-cell markers did not contrast with the supposed T-cell derivation, as did not the positivities recorded for the transcription factors Oct-2 and BOB.1, which are quite characteristic of B-lymphocytes and B-cell lymphomas.9 In fact, ALCL commonly shows defective T-cell antigen expression.2,3,7,8 and can indeed present Oct-2 and BOB.1, as recently documented by Marafioti et al.22 However, molecular biology did not support the T-cell origin of the process, since both TCR a and d genes were in germ line configuration. Else, it suggested a B-cell derivation by showing monoclonal rearrangement of IgVH gene. Although discrepancies between phenotype and genotype have been reported in ALCL,23 the hypothesis of a B-cell origin was definitely proven by the immunohistochemical detection of the PAX-5 gene product/BSAP.9 The latter is a transcription factor, which plays a key role in B-lymphoid development and is strictly B-cell specific, being expressed in all steps of B-cell maturation (with the exception of plasma cells) and many B-cell lymphomas, but not in T-lymphocytes and T-cell tumors.9 Thus, following careful phenotypic and molecular evaluation our case was not classified as ALCL, but was diagnosed as DLBCL, anaplastic variant. In this context, the misleading CD2 positivity can be explained on the basis of a recent observation of Daibata et al., who found the molecule in question to be expressed in a pyothorax-associated B-cell lymphoma.24 These authors argued that the tumour they observed expressed CD2 because of EBV infection, in line with the knowledge that a proportion of activated normal B-cells can carry CD2 in the presence of mitogens.25 In our case, CD2 positivity occurred in a neoplastic lymphoid population bearing activated phenotype, as shown by CD30 expression7,8 but not EBV infection.
Another interesting feature of the present case is the association with gastric MALT lymphoma.2,3 On the basis of the proven B-cell derivation of both neoplasms, one might argue that the anaplastic DLBCL represented the progression of a pre-existing indolent lymphoid tumor. However, molecular analysis performed by micro-dissection PCR and gene sequencing showed that the two populations stemmed from unrelated clones. Thus, the detection of the two neoplasms seems more likely to correspond to a fortuitous event. Remarkably, the patient had a previous history of long-standing Helicobacter pylori-positive chronic gastritis, a condition which is known to facilitate the onset of malignant lymphoma due to the high oxidative levels caused by persistent infection.26 It is worthy of note that in our case the MALT lymphoma did not respond to antibiotic therapy. This is more often observed in cases which carry t(11;18) and are characterized by dissemination of the process without progression to DLBCL.27 Thus, on one hand it is not surprising that the two neoplasms stemmed from different clones, on the other the onset of the anaplastic tumor might have been favored by reduced local immune surveillance due to both the state of chronic inflammation and the partial involvement by MALT lymphoma.
More in general, the present case shows some similarities with another one recently reported by van den Berg et al.28 These authors simultaneously detected B-cell chronic lymphocytic leukemia, ALK-negative ALCL and cHL in the same lymph node. Although the three neoplasms appeared clonally related, conversely to our case, it is quite interesting to note that the anaplastic large cell population was originally interpreted as of T/null-cell derivation because of the lack of CD20 and the stain for CD45R0. Molecular analysis, however, led to the diagnosis of B-cell tumor because of the detection of clonal IgVH gene rearrangement in the absence of TCR products.
Based on the experience gained in the present case, one should be aware that the application of a limited panel of antibodies can be insufficient for the correct interpretation of ALK- anaplastic large cell lymphoid tumors. In particular, the lack of CD20 and CD79a does not definitively exclude the diagnosis of DLBCL, anaplastic variant and more refined immunohistochemical and molecular tests are required in cases with ambiguous or apparently null phenotype. This also prompts investigation on how heterogeneous might actually be the category of ALK- ALCLs, as defined by the REAL/WHO Classification,2,3 and whether these neoplasms should be definitively separated form the ALK+ forms and reclassified as morphologic variants of already existing B- and T-cell categories.  

References

  1. Stein H, Mason DY, Gerdes J, O'Connor N, Wainscoat J, Pallesen G, et al. The expression of the Hodgkin's disease associated antigen Ki-1 in reactive and neoplastic lymphoid tissue: evidence that Reed-Sternberg cells and histiocytic malignancies are derived from activated lymphoid cells. Blood 1985;66:848-58.
  2. Harris NL, Jaffe ES, Stein H, Banks PM, Chan JK, Cleary ML, et al. A revised European-American classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group. Blood 1994;84:1361-92.
  3. Jaffe ES, Harris NL, Stein H, Vardiman JW. Tumours of haematopoietic and lymphoid tissues. Lyon: IARC Press, International Agency for Research on Cancer 2001;230-5.
  4. Falini B, Bigerna B, Fizzotti M, Pulford K, Pileri SA, Delsol G, et al. ALK expression defines a distinct group of lymphomas ("ALK lymphomas") with a wide morphologic spectrum. Am J Pathol 1998;153:875-86.
  5. Benharroch D, Meguerian-Bedoyan Z, Lamant L, Amin C, Brugieres L, Terrier-Lacombe MJ, et al. Morphologic and phenotypic features of lymphomas expressing ALK protein. Blood 1998;91:2076-84.
  6. Falini B, Pileri S, Zinzani P-L, Carbone A, Zagonel V, Wolf-Peeters C, et al. ALK+ lymphoma: clinico-pathological findings and outcome. Blood 1999;93:2697-706.
  7. Stein H, Foss H-D, Dürkop H, Marafioti T, Delsol G, Pulford K, et al. CD30-positive anaplastic large cell lymphoma: a review of its pathological, genetic and clinical features. Blood 2000;96:3681-95.
  8. Falini B. Anaplastic large cell lymphoma: pathological, molecular and clinical features. Br J Haematol 2001;114:741-60.
  9. Falini B, Mason DY. Proteins encoded by genes involved in chromosomal alterations in lymphoma and leukemia: clinical value of their detection by immunocytochemistry. Blood 2002;99:409-26.
  10. Haralambieva E, Pulford KA, Lamant L, Pileri S, Roncador G, Gatter KC, et al. Anaplastic large-cell lymphomas of B-cell phenotype are anaplastic lymphoma kinase (ALK) negative and belong to the spectrum of diffuse large B-cell lymphomas. Br J Haematol 2000;109:584-91.
  11. Pileri S, Bocchia M, Baroni CD, Martelli M, Falini B, Sabattini E, et al. Anaplastic large cell lymphoma (CD30+/KI-1+): results of the prospective clinico-pathologic study of 69 cases. Br J Haematol 1994;86:513-23.
  12. Pileri SA, Roncador G, Ceccarelli C, Piccioli M, Briskomatis A, Sabattini E, et al. Antigen retrieval techniques in immunohistochemistry: comparison of different methods. J Pathol 1997;183:116-23.
  13. Sabattini E, Bisgaard K, Ascani S, Poggi S, Piccioli M, Ceccarelli C, et al. Envision Plus: a new immunohistochemical method of choice for diagnostics and research. Critical comparison with the APAAP, ChemMate, CSA, LABC, and SABC techniques. J Clin Pathol 1998;51:506-11.
  14. Pileri SA, Zinzani PL, Ascani S, Orcioni GF, Gamberi B, Piccioli M, et al. Diffuse large B-cell lymphoma with primary retroperitoneal presentation: clinico-pathologic study of nine cases. Ann Oncol 2001;12:1445-53.
  15. Kuppers R, Rajewsky K, Zhao M, Simons G, Laumann R, Fischer R, et al. Hodgkin disease: Hodgkin and Reed-Sternberg cells picked from histological sections show clonal immunoglobulin gene rearrangements and appear to be derived from B cells at various stages of development. Proc Natl Acad Sci USA 1994;91:10962-6.
  16. Marafioti T, Hummel M, Anagnostopoulos I, Foss HD, Falini B, Delsol G, et al. Origin of nodular lymphocyte predominant Hogkin's disease from a clonal expansion of highly mutated germinal center B cells. New Engl J Med 1997;337:453-8.
  17. Capello D, Vitolo U, Pasqualucci L, Quattrone S, Migliaretti G, Fassone L, et al. Distribution and pattern of BCL-6 mutations throughout the spectrum of B-cell neoplasia. Blood 2000;95:651-9.
  18. Kuppers R, Rajewsky K, Hansmann M-L. Diffuse large cell lymphomas are derived from mature B cells carrying V region genes with a high load of somatic mutation and evidence of selection for antibody expression. Eur J Immunol 1997;27:1398-405.
  19. Fais F, Gaidano G, Capello D, Gloghini A, Ghiotto F, Roncella S, et al. Immunoglobulin V region gene use and structure suggest antigen selection in AIDS-related primary effusion lymphoma. Leukemia 1999;13:1093-9.
  20. Benhattar J, Delacretaz F, Martin P, Chaubert P, Costa J. Improved polymerase chain reaction detection of clonal T-cell lymphoid neoplasms. Diagn Mol Pathol 1995;4:108-12.
  21. McCarthy KP, Sloane JP, Kabarowski JHS, Matutes E, Wiedermann LM. The rapid detection of clonal T-cell proliferations in patients with lymphoid disorders. Am J Pathol 1991;138:821-8.
  22. Marafioti T, Ascani S, Pulford K, Poggi S, Jones M, Sabattini E, et al. Expression of B lymphocyte-associated transcription factors in human T cell neoplasms. Am J Pathol, sumitted.
  23. Herbst H, Tippelmann G, Anagnostopoulos I, Gerdes J, Schwarting R, Boehm T, et al. Immunoglobulin and T cell receptor gene rearrangements in Hodgkin's disease and Ki-1-positive anaplastic large cell lymphoma: Dissociation between phenotype and genotype. Leuk Res 1989;13:103-16.
  24. Daibata M, Taguchi T, Nemoto Y, Saito T, Machida H, Imai S, et al. Epstein-Barr virus (EBV)-positive pyothorax-associated lymphoma (PAL): chromosomal integration of EBV in a novel CD2-positive PAL B-cell line. Br J Haematol 2002;117:546-57.
  25. Mills KHM, Worman CP, Cawley JC. Normal human B cells express endogenous sheep erythrocyte (E) receptor after appropriate in vitro culture. Eur J Immunol 1983;13:379-82.
  26. Pileri SA, Ascani S, Sabattini E, Fraternali-Orcioni G, Poggi S, Piccioli M, et al. The pathologist's view point: Part I - indolent lymphomas. Haematologica 2000;85:1268-84.
  27. Liu H, Ye H, Ruskone-Fourmestraux A, De Jong D, Pileri S, Thiede C, et al. t(11;18) is a marker for all stage gastric MALT lymphomas that will not respond to H. pylori eradication. Gastroenterology 2002;122:1286-94.
  28. Van den Berg A, Maggio E, Rust R, Kooistra K, Diepstra A, Poppema S. Clonal relation in a case of CLL, ALCL, and Hodgkin composite lymphoma. Blood 2002; 100:1425-9.