Gela histological scoring system for post-treatment biopsies of patients with gastric MALT lymphoma is feasible and reliable in routine practice

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1 research paper Gela histological scoring system for post-treatment biopsies of patients with gastric MALT lymphoma is feasible and reliable in routine practice Christiane Copie-Bergman, 1,2,3 Andrew C. Wotherspoon, 4 Carlo Capella, 5 Teresio Motta, 6 Ennio Pedrinis, 7 Stefano A. Pileri, 8 Francesco Bertoni, 9 Annarita Conconi, 10 Emanuele Zucca, 9 Maurilio Ponzoni 11 * and Andrés J. M. Ferreri 12 * 1 AP-HP, Groupe Henri Mondor-Albert Chenevier, Département de Pathologie, 2 Université Paris-Est Créteil, Faculté de médecine, 3 INSERM, Créteil, France, 4 Department of Histopathology, The Royal Marsden Hospital, London, UK, 5 Department of Anatomic Pathology, Ospedale di Circolo and University of Insubria, Varese, 6 Unita Operativa di Anatomia Patologica, Ospedale Bolognini di Seriate, Seriate, Italy, 7 Istituto Cantonale di Patologia, Locarno, Switzerland, 8 Chair of Pathology and Unit of Haematopathology, Department of Haematology and Oncological Sciences L and A. Seràgnoli, Bologna University, Bologna, Italy, 9 Oncology Institute of Southern Switzerland (IOSI), Bellinzona, Switzerland, 10 AOU Maggiore della Carità, Università del Piemonte Orientale Amedeo Avogadro, Novara, 11 Pathology Unit, Unit of Lymphoid Malignancies, San Raffaele Scientific Institute, and 12 Unit of Lymphoid Malignancies, Department of Onco-Hematology, San Raffaele Scientific Institute, Milano, Italy Summary The International Extranodal Lymphoma Study Group (IELSG) promoted this study to determine the inter-observer agreement in the application of the Groupe d Etude des Lymphomes de l Adulte (GELA) histological scoring system for evaluating residual disease in post-treatment gastric biopsies of patients with gastric Mucosa-Associated Lymphoid Tissue (MALT) lymphoma (GML). Twenty-one patients with Helicobacter pylori -associated GML and treated with anti-h. pylori therapies were considered. A total of 154 biopsy sets from follow-up endoscopic procedures after H. pylori eradication were examined independently by seven pathologists from four European countries, following histological criteria suggested by the GELA scoring system. The overall concordance rate was 83% with a kappa value of 064, indicating a significant agreement among the seven observers. Most non-concordant responses clustered across the border of complete remission (CR) and probable minimal residual disease (pmrd), a distinction that does not imply critical clinical impact. Accordingly, when the analysis considered CR/pMRD as a single entity, the responses showed an overall concordance rate of 89% with kappa value of 083, thus indicating a high degree of inter-observer agreement. This study provides additional validation of the GELA histological grading system. This scheme can therefore be recommended in routine practice and deserves to be used in prospective clinical trials. Keywords: gastric MALT lymphoma, inter-observer agreement, histological scoring system, response criteria for lymphoma. Received 08 May 2012; accepted for publication 31 July 2012 Correspondence: Dr. Christiane Copie-Bergman, Département de Pathologie, Hôpital Henri Mondor, 51 av du Maréchal de Lattre de Tassigny, Créteil, France. christiane.copie@hmn.aphp.fr *Maurilio Ponzoni and Andrés J. M. Ferreri are considered equally as senior authors. Primary gastric marginal zone lymphoma of Mucosa- Associated lymphoid Tissue (MALT) is a low-grade B-cell malignancy with an indolent clinical course. Ninety per cent of primary gastric MALT lymphomas (GML) are associated with H. pylori infection and the causal role of H. pylori in the development of the disease is now well established ª 2012 Blackwell Publishing Ltd First published online 9 October 2012 doi: /bjh.12078

2 C. Copie-Bergman et al (Du & Isaacson, 2002). The histological diagnostic features of MALT lymphoma rely on centrocytic-like B-cell neoplastic population involving the lamina propria, forming characteristic lymphoepithelial lesions (LELs), and associated with reactive lymphoid follicles (Isaacson et al, 2008). Tumour cells display CD20 +, CD5, CD10, BCL2 +, CD23, cyclin D1- immunophenotype. LELs may be identified with the use of cytokeratin immunostaining. Eradication of H. pylori with antibiotics induces complete histological remission in about 70% of GML cases and time to achieve remission may last up to 24 months (Wotherspoon et al, 1993; Fischbach et al, 2004). Following H. pylori eradication, the above-mentioned histological findings undergo substantial changes. In responding patients, tumour cells disappear progressively from the upper part of gastric mucosa, leaving an empty lamina propria that is characterized by loose fibrosis and gland depletion. LELs disappear or become increasingly more rare. If present, residual disease may show ill-defined nodular or diffuse lymphoid infiltrates at the basis of the lamina propria and is associated with fibrosis. Eventually, this lymphoid infiltrate may disappear but, more frequently, residual lymphoid aggregates persist. The interpretation of residual lymphoid infiltrates in follow-up gastric biopsies of GML represents a diagnostic challenge for pathologists. Crush artefacts are strengthened by the surrounding fibrosis and immunostaining has usually a lower diagnostic usefulness, since the lymphoid aggregates contain many reactive T cells and a minority of CD20 + B cells. Taken together, these features confirm that histological remission can be difficult to assess in GML. Although a molecular approach to overcome these problems is technically feasible, the actual clinical implication of molecular minimal residual disease is controversial (Savio et al, 1996; Thiede et al, 2001; Bertoni et al, 2002; de Mascarel et al, 2005; Wündisch et al, 2005; Montalban et al, 2005; Salar et al, 2005; Streubel et al, 2006; Hummel et al, 2006). Histological evaluation of gastric biopsies remains the method of choice for the evaluation of response to treatment in GML. Variable assessment systems have been used in the past to this end. Wotherspoon et al (1993) proposed a histological grading system that was initially designated to express the degree of confidence of a diagnosis of MALT lymphoma on gastric biopsies. This scheme was primarily designed for initial diagnosis, not for the evaluation of post-treatment gastric biopsies. Many investigators have found the application of the Wotherspoon score on follow-up biopsies difficult, as confirmed by a low degree of inter-observer reproducibility. Other groups adopted different criteria of partial and complete remission, but none of these systems have been universally adopted (Neubauer et al, 1997). In order to standardize the histological criteria used to evaluate lymphoma response to therapy, pathologists from the Goupe d Etude des Lymphomes de l Adulte (GELA) established an ad hoc histological grading system, named the GELA score (Copie-Bergman et al, 2003). A pilot test of this grading, in a small series of patients and on a limited amount of biopsy sets, proved to be promisingly efficient (Copie-Bergman & Wotherspoon, 2008). The need of a more general consensus on the evaluation of post-treatment GML biopsies prompted the International Extranodal Lymphoma Study Group (IELSG) to promote the present study, aimed at the evaluation of the inter-observer agreement within a group of seven pathologists from four European countries with the opportunity to study a larger series of patients with GML characterized by several follow-up gastric biopsies taken at different time-points with an accurate clinical follow-up. Materials and methods Patients Twenty-one patients with H. pylori -associated GML were considered, including 16 males and 5 females, with a median age of 65 years (range years). Diagnosis of GML was made on upper endoscopic examination and on histological analysis of gastric biopsies according to the criteria defined by Isaacson et al (2008): presence of a diffuse infiltrate of centrocyte-like CD20 + CD5 B-cells in the lamina propria with prominent LELs. Presence of H. pylori was assessed on modified Giemsa-stained sections. These patients were treated with conventional H. pylori -eradicating antibiotic therapy within a previously published randomized clinical trial (Hancock et al, 2009). Follow-up endoscopic procedures were performed to assess lymphoma response to H. pylori -eradication therapy. The mean of follow-up endoscopies from each patient was 7 (range 3 11). From each procedure a mean of two biopsies sets (range 1 4) was obtained. The median follow-up after GML diagnosis was 56 months (range months). This study complied fully with all provisions of the Declaration of Helsinki and was conducted in accordance with Good Clinical Practice rules. All patients gave written informed consent to participate and to provide tissue material for biological studies. Histological evaluation In order to study the inter-observer agreement in the diagnosis of residual disease in GML, seven pathologists from France, Italy, Switzerland, and UK reviewed haematoxylineosin stained sections of formalin-fixed paraffin-embedded gastric biopsies from a total of 154 follow-up endoscopic procedures. Each biopsy was evaluated independently by pathologists blinded to clinical data, following criteria proposed by the GELA scoring system (Copie-Bergman et al, 2003). This histological grading system of post-treatment gastric biopsies is based only on histopathological features, independently of immunostaining or molecular studies (Table I). Three essential histological parameters are evaluated: the 48 ª 2012 Blackwell Publishing Ltd

3 Gastric MALT lymphoma: GELA Histological Response Criteria Table I. GELA histological grading system for post-treatment evaluation of gastric MALT lymphoma. Score Lymphoid infiltrate LEL Stromal changes Complete Histological remission (CR) Probable minimal residual disease (pmrd) Responding residual disease (rrd) Absent or scattered plasma cells and small lymphoid cells in the LP Aggregates of lymphoid cells or lymphoid nodules in the LP/MM and/or SM Dense, diffuse or nodular, extending around glands in the LP Absent Absent Focal LEL or absent No change (NC) Dense diffuse or nodular Present may be absent Normal or empty LP and/or fibrosis Empty LP and/or fibrosis Focal empty LP and/or fibrosis No changes MM, muscularis mucosa; LP, lamina propria; SM, submucosa; LEL, lymphoepithelial lesions. Reproduced from Gut, Copie-Bergman, C., Gaulard, P., Lavergne-Slove, A., Brousse, N., Fléjou, J.F., Dordonne, K., de Mascarel, A., Wotherspoon, A.C., 52:1656, 2003 with permission from BMJ Publishing Group Ltd. neoplastic centrocyte-like (CCL) infiltrate, the presence of LELs, and the occurrence of stromal changes. Briefly, the morphological features observed in post-treatment gastric biopsies are summarized in four categories: 1 Complete response (CR) is defined as total disappearance of the CCL component and LELs and the presence of scattered plasma cells or small lymphoid cells with interstitial distribution, without the formation of lymphoid aggregates. Stromal changes including empty lamina propria and/or loose fibrosis may also occur. No need for additional treatment. 2 Probable minimal residual disease (pmrd) is defined as persistence of small lymphoid aggregates or lymphoid nodules in the basal lamina propria and/or submucosa, associated with stromal changes. LELs are absent. It is not possible to assess the actual significance of these lymphoid aggregates by morphology or immunohistochemistry alone, but it has been established that these nodules very frequently harbour cells displaying the same clonal immunoglobulin gene rearrangement as the lymphoma cells at diagnosis, consistent with the presence of a small number of residual neoplastic cells. However, given that no adverse prognostic significance has been demonstrated in such instances, these features should be considered as a state of remission because they are not associated with active disease (Savio et al, 1996; Thiede et al, 2001; Bertoni et al, 2002; de Mascarel et al, 2005; Wündisch et al, 2005; Montalban et al, 2005; Salar et al, 2005; Streubel et al, 2006; Hummel et al, 2006; Ruskoné-Fourmestraux et al, 2011). No need for additional treatment. 3 Responding residual disease (rrd) is defined by persistence of decreased dense nodular or diffuse CCL infiltrate, extending around glands; LELs may be absent. Stromal changes are a prominent feature of this state and are indicative of an ongoing remission process. These features indicate a partial and ongoing response. Management should be individually tailored; however, additional treatment can be postponed until further endoscopic assessment in the case of absence of unfavourable endoscopic features or clinical evidence of tumour progression. 4 No change (NC) is defined by the persistence of virtually unmodified CCL neoplastic cells infiltrate, with or without LELs; stromal changes are absent. Oncological treatment should be proposed when NC is associated with persisting macroscopic lesions or evidence of lymphoma dissemination. Conversely, oncological treatment can be postponed in cases where only microscopic infiltration is present. Management should be individually tailored when NC features persist for up to 24 months after Helicobacter pylori eradication. An important recommendation states that, in order to offer the most accurate information to the clinician, each follow-up gastric biopsy should be scored in comparison to the previous gastric biopsy by the same pathologist. According to this histological scoring system, criteria for treatment response in GML can be defined as follow (Ruskoné-Fourmestraux et al, 2011): 1 Complete remission (CR) is defined as normalization of endoscopic findings and negative histology (CR or pmrd) in two subsequent follow-up investigations. 2 Partial remission (PR) is defined as normalization or reduction of macroscopic findings, histological signs of lymphoma regression (rrd) and no signs of progressive disease. 3 Stable disease (SD) is characterized by unmodified gastroscopic findings and/or unmodified histology (NC). 4 Progressive disease (PD) is defined by worsening of macroscopic findings or dissemination of gastric MALT ª 2012 Blackwell Publishing Ltd 49

4 C. Copie-Bergman et al lymphoma or transformation into diffuse large B-cell lymphoma. 5 Relapse is defined as the re-occurrence of histologically-confirmed lymphoma after a CR was previously documented. Statistical analysis Inter-observer agreement was evaluated using the weighted kappa test of Cohen (Cohen, 1960). Kappa Cohen was interpreted as currently: a score <02 was considered as slight ; between 02 and 04 was fair ; between 04 and 06 was moderate ; between 06 and 08 was substantial ; and a score >08 was considered as almost perfect (Cohen, 1960). Results Patient follow-up Helicobacter pylori eradication was achieved in all patients. Twenty patients were in histological remission (CR/pMRD) and one had persistent disease corresponding to unmodified histology NC according to the GELA assessment system. Evaluation of inter-observer agreement One hundred and fifty-four samples from 21 patients were analysed. Every sample was reviewed by seven independent expert pathologists, resulting in 1046 assessable data; 32 data were not available for analysis (Table S1). The overall concordance rate was 83% with a kappa value of 064, indicating a significant agreement among the seven observers. The median concordance rate was 80% or more in all patients but six. Concordance rate was 80% in 97 (63%) samples and <70% in 27 (18%) samples. Among the latter, relevant discordances were essentially observed in distinguishing CR from pmrd in 15 samples, pmrd from rrd in seven, and rrd from NC in three. In two samples, pathologists had some difficulties in distinguishing between pmrd, rrd and NC. In these particular cases, this discrepancies may be explained by the fact that the score NC = no change refers to the presence of lymphoma and not to the comparison with the previous score in the preceding biopsy. Distinguishing CR from pmrd displays a low impact on therapeutic decision, because both are assimilated to histological remission and no further therapy is needed. Accordingly, when the analysis was repeated considering CR/pMRD as a single entity, results showed an overall concordance rate of 89% with kappa value of 083, indicating almost perfect agreement among observers. The median concordance rate was 100% in all patients but three. Concordances were 100% in 116 (75%) samples, between 80% and 90% in 13 (8%) samples, between 70 and 79% in 13 (8%) and lower than 70% in 12 (8%) samples. Among these 12 discordant cases, difficulties were observed between CR/pMRD and rrd in eight samples, between rrd and NC in two samples and among CR/pMRD, rrd and NC in two samples. Discussion The development of a standardized method to evaluate the lymphoma response to treatment is a prerequisite for the clinical management of patients, for the comparison among trials and for testing new therapeutic schemes. The need for standardized response criteria for definition of response in lymphomas has been highlighted by recently introduced guidelines incorporating PET, immunohistochemistry and flow cytometry (Cheson et al, 2007). Herein, through a large series of gastric biopsies evaluated by a multi-institutional, international group of pathologists and supplied by a long clinical follow-up, we provide evidence that GELA histological score is a feasible and reliable tool in the evaluation of response to treatment in GML. Equally important, because pmrd is clinically assimilated to histological remission and therefore does not require further treatment, we pooled CR and pmrd under a single variable during statistical analysis, obtaining the result of an almost perfect agreement among observers. The GELA histological grading system, proposed in 2003 (Copie-Bergman et al, 2003), was based on the evaluation of a series of 10 patients and analysis of 45 sets of gastric biopsies of GML patients enrolled in the GELA trial, with a median follow-up of 19 months (Hancock et al, 2009). The inter-observer agreement among the seven pathologists in that study was good and showed a 084 weighted kappa value, but this histological grading required additional validation on a larger series of GML patients. In the present study, we analysed 1046 data from 154 sets of gastric biopsies from 21 H. pylori -positive GML patients treated with H. pylori eradicating therapy with a median follow-up of 56 months. Interestingly, only two pathologists of the present panel were part of the review panel in the original GELA scoring paper. The inter-observer agreement reached was 83% with a kappa value of 064, thus indicating a significant agreement among the seven examinators. Significantly, the choice of considering as a whole entity both CR and pmrd categories raised the overall concordance rate to 89%, with a kappa value of 083. The context of GML greatly differs from other types of non-hodgkin lymphoma, also in terms of clinical management, which is specific to this disease. In fact, GML is a very indolent lymphoma that can be cured in 70% of the cases by antibiotics alone (Wotherspoon et al, 1993; Fischbach et al, 2004). Time to remission may last take from several months up to two years and most patients have a favourable clinical course without additional oncological treatment. On this basis, a watch and wait strategy has been suggested as the approach of choice in patients with minimal persisting lymphoma infiltrates, providing that regular endoscopic 50 ª 2012 Blackwell Publishing Ltd

5 Gastric MALT lymphoma: GELA Histological Response Criteria control is performed (Fischbach et al, 2004). Patients who do not respond to H. pylori eradication or who are H. pylori negative at diagnosis are referred for chemotherapy and/or immunotherapy, or radiation treatment (Ruskoné-Fourmestraux et al, 2011). Notwithstanding these reassuring characteristics, a longterm follow-up study has shown that transient histological recurrence or late relapse may occur and life-long repeated gastric biopsies are recommended in the follow-up procedure (Raderer et al, 2005). A basic question involves the choice of the best method for monitoring GML. To this aim, evaluation of lymphoma response to treatment is presently based on endoscopic features and, essentially, on histological assessment of gastric biopsies, as lymphoma may persist despite normalization of endoscopic findings. Molecular evidence of monoclonality detected by polymerase chain reaction (PCR) and/or molecular monitoring of BIRC3-MALT1 fusion transcripts detected by reverse transcription PCR in t(11;18)- positive GML proved to be sensitive techniques (Savio et al, 1996; Thiede et al, 2001; Bertoni et al, 2002; de Mascarel et al, 2005; Wündisch et al, 2005; Montalban et al, 2005; Salar et al, 2005; Streubel et al, 2006). However, persistence of molecular disease in the absence of histological disease is frequently observed in the context of GML and the significance of this persistent clone is still unclear. Molecular results should be therefore interpreted only in the context of histology and molecular monitoring of residual disease in posttreatment gastric biopsies is presently not recommended in routine practice (Ruskoné-Fourmestraux et al, 2011). In the context of the central role played by histopathological examination, the interpretation of residual lymphoid infiltrates can be difficult. Review of the literature shows that the histological criteria used for assessing lymphoma remission are either poorly defined or variable among different series and hamper a reliable comparison of the clinical trials, especially when testing new therapeutic schemes. This heterogeneity is also semantic, as different terms have been used to define residual disease like minimal histological residuals (Fischbach et al, 2004) or partial histological regression (Neubauer et al, 1997) and may introduce some degree of confusion for pathologists and clinicians, with obvious implications on the correct management of GML; for instance, this uncertainty could lead to overtreatment of a significant fraction of patients (Fischbach et al, 2004). Some important histopathological considerations must be briefly added. Above all, some confusion could rise in the definition of NC ( no change ). It must be emphasized that NC refers to the lymphoma status itself and not necessarily in comparison with the immediately earlier biopsy. On the other hand, in case of lymphoma relapse, the score NC is recommended and means presence of overt lymphoma. To circumvent this ambiguity, we suggest that pathologists should report NC, presence of overt lymphoma. Even more important, most diagnostic difficulties were encountered in differentiating between the phases of histological remission (CR/pMRD) and responding residual disease (rrd). The strict application of features of CR/pMRD and rrd according to the GELA criteria should improve inter-observer agreement. CR/pMRD status is very similar to chronic gastritis and the density of lymphoid aggregates is low. The rrd score aims to certify the clear evidence of residual lymphomatous infiltrate and, at the same time, indicate an ongoing remission process, characterized by loose fibrosis and rare, if any, LELs. In conclusion, this study showed an excellent inter-observer agreement and provides additional validation of the GELA histological grading system. Our figures underscore the importance of this grading scheme also in patients with long followup. Importantly, these lymphoma response criteria also apply to follow-up gastric biopsies of patients who do not respond to H. pylori eradication and who receive immunotherapy and/or chemotherapy, or radiation. This scheme accurately assesses the response to treatment in GML and its use in routine practice and future clinical trials is strongly encouraged, as will result in the worldwide use of an harmonized language in reporting and monitoring GML. Acknowledgements We are indebted to Cristina Morinini for secretarial assistance and excellent organization of the histology review sessions of this study. The authors are thankful to Dr. Roberto Crocchiolo (Unit of Lymphoid Malignancies of the San Raffaele Scientific institute, Milan, Italy) for database management and technical support during statistical analysis. Funding This work was supported by Oncosuisse grant OCS ; Fondazione per la Ricerca e la Cura sui Linfomi (Lugano, Switzerland). Author contributions Conception and design: C Copie-Bergman; AC Wotherspoon; E Pedrinis; SA Pileri; E Zucca; M Ponzoni; AJM Ferreri; Provision of study materials or patients : C Copie-Bergman; AC Wotherspoon; C Capella, T Motta, E Pedrinis; SA Pileri; F Bertoni; A Conconi; E Zucca; M Ponzoni; AJM Ferreri; the IELSG clinicians and Pathology network. Collection and assembly of data: C Copie-Bergman; AC Wotherspoon; E Pedrinis; E Zucca; M Ponzoni; AJM Ferreri; Data analysis and interpretation: C Copie-Bergman; AC Wotherspoon; E Zucca; M Ponzoni; AJM Ferreri; Manuscript writing : C Copie-Bergman; AC Wotherspoon; E Zucca; M Ponzoni; AJM Ferreri; Final approval of manuscript : C Copie-Bergman; AC Wotherspoon; C Capella, T Motta, E Pedrinis; SA Pileri; F Bertoni; A Conconi; E Zucca; M Ponzoni; AJM Ferreri; Competing interests: there is no conflict of interest in this report; this report is not currently under consideration at any other ª 2012 Blackwell Publishing Ltd 51

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