published_or_final_version / Medicine / Master / Doctor of Medicine

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published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Pathology / Master / Master of Philosophy

Thesis (PhD)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: BACKGROUND Lymphomas arise in cells of the immune system. They vary widely in cytomorphology, immunophenotype and clinical course as well as response to treatment - all of which are factors that determine prognosis. The substantial geographical differences that exist for Hodgkin and other lymphoproliferative disorders have been previously reported from the Lymphoma Reclassification Project. OBJECTIVE This investigation was in two parts. In the first we tested the hypothesis that, using comparable treatment regimens, outcome from a private academic centre in the Western Cape region of South Africa would be similar to that from the first world. To this end a series of 512 individuals were analysed. In the second tissue samples from these patients where the most common subtype is diffuse large B-cell lymphoma (n=93) randomly receiving either standard combination chemotherapy in the form of the CHOP regimen or the identical program with rituximab which is an anti-CD20 monoclonal antibody were further investigated in cooperation with the University of Nebraska Medical Centre using an immunohistochemistry based method, also known as tissue microarray. PATIENTS AND METHODS In the first cohort consecutive and comprehensive records of patients diagnosed with lymphoma aged 14 years and older seen between October 1998 in July 2006, were scrutinized. After exclusion for a variety of reasons 398 cases suitable for further definitive study remained. In the second group tissue samples from a total of 149 biopsy proven de novo diffuse large Bcell variants could be further evaluated. After additional refinement a total of 93 remained that met all the entry criteria for the study in which 48 received chemotherapy alone and the remaining 45 chemo-immunotherapy. Demographic features were well matched. The initial stratification of these 93 cases, based on phenotyping with immunohistochemistry employing a tissue microarray method, yielded two populations depending on the criteria used. Each of these primary subdivisions was further evaluated for expression of BCL2 and LMO2 both of which are recognised to predict response. Finally, for each variable, clinical characteristics and survival outcome were compared between chemotherapy as a single modality or the same regimen combined with rituximab. STATISTICAL ANALYSIS Overall and event-free survival were calculated as the years from diagnosis to death, loss to follow-up, first progression or relapse respectively. The Kaplan Meier method was used to estimate distribution and the log rank test to compare survival between groups. Patient characteristics for each cohort specified were tested with Chi-squared or Fisher's exact test for small samples. RESULTS In the first part of this study all 398 cases were retrospectively analysed and showed a similar treatment outcome with regards to overall and event-free survival at 36 months when compared to first world reference centers. Adverse factors identified were similar to published experience comprising constitutional symptoms, prior treatment with chemotherapy, intermediate or high-risk scores as defined by the International Prognostic Index, histologic grading and certain anatomical sites of primary tumour. In contrast gender, staging by Rye or Rai classification, retroviral infection and prior treatment with radiotherapy were without effect. In the second part tissue samples from 93 de novo DLBCL, subtyped using an investigational immunohistochemical based Tissue Micro Array (TMA) were contrasted to the approximately corresponding categories as defined either by Hans and associates using a three marker panel into germinal or non-germinal centre subtypes or by Choi and colleagues with two additional antibodies into germinal centre or activated B-cells. Not surprisingly when compared to DNA-based gene expression profiling, as a reference point, concordance was different being 86 as opposed to 93% for the respective algorithms. The rationale for this exercise was to determine relative cost-effectiveness in an affordable but accurate technology that may be applicable to under resourced areas of the globe. Additionally the prognostic marker expression for BCL2 and LMO2 was investigated with regards to treatment outcome. Using the investigational tissue microarray approach the addition of rituximab to standard chemotherapy group did not show any significant improvement on 5 years overall (63% vs 59%, p 0.68) or event-free survival (42% vs 39%, p 0.94). Similarly no differences were evident in subtype analysis. Interestingly however, when segregated on the Choi criteria, cytotoxic drugs alone showed a non-significant trend in improved survival (74% vs 55%, p 0.32) as well as event-free survival (44% vs 40%, p 0.42) for the germinal centre as opposed to the activated B-cell subtype. Nevertheless not even a small difference could be demonstrated in the presence of rituximab. According to Choi, both regimens (chemotherapy with or without the addition of rituximab) revealed similar results to the Hans algorithm on 5 years OS as well as 3 year EFS when comparing GCB versus non-GCB subgroups. BCL2 and LMO2 marker expression of the respective immunohistochemical (IHC) subtype, despite small sample size, revealed the following. Analysis by Choi criteria on survival for BCL2, no matter for which subsets (GCB or ABC) or treatment modality (chemotherapy with or without the addition of rituximab) showed no difference in 5 years OS or EFS. However, in contrast, a significant difference for better EFS (p=0.0015) in the BCL2 positive group of the ABC subgroups subtypes treated with rituximab containing chemotherapy. These results must be interpreted with care due to the very low sample size and the follow up time of only 9 months. For LMO2 similar results on survival outcome were seen thus showing no difference in 5 years OS or EFS – regardless of subtype or treatment modality. Also here, this was contrasted by better EFS (p=0.039) in the LMO2 positive group of ABC subtypes when treated with the rituximab containing regimen. Again the reservation about small numbers and the 14 months observation period apply. DISCUSSION AND CONCLUSION Most of the studies examining the proportions of subtypes in large series of DLBCL patients have been predominantly carried out on Western populations. While 50% of patients in North America or Europe express GCB phenotypes, this is only 31% in their Asian counterparts. Thus far, no study has been published on lymphoma subtypes in South African populations using a Tissue Micro Array (TMA) method. Despite certain limitations of this study, due to a variety of reasons such as loss of analysable data, variability of representation of the South African population as a whole or low sample size leading to a potential source of error, these results confirm that lymphoproliferative disorders are heterogeneous. Neverless this group can be treated successfully if exact staging, classification and risk assessment defines the holistic management plan – no matter if in a developed or under resourced setting. With the introduction of sophisticated methods such as gene expression profiling (GEP), diffuse large B-cell lymphoma (DLBCL) can be classified into the prognostically favourable germinal center B-cell–like (GCB) and the more aggressive activated B-cell–like (ABC) subtypes. Against the background of resource restriction we therefore used an immunohistochemical (IHC) stain method to analyse formalin-fixed, paraffin-embedded tissue samples. Here the algorithm by Choi et al., originally described by Hans et al., showing a high concordance rate, was comparable to the GEP classification. The use of the IHC based TMA methodology was shown to be a simple, cost effective and a robust alternative to GEP which is currently regarded as the gold standard for the classification in lymphomas. It provides a useful prognostic tool in stratifying DLBCL or other entities in future, even when frozen tissue samples are not available for GEP analysis. With the current budgetary limitations in public hospitals chemotherapy protocols for lymphoproliferative disorders exclude agents such as rituximab. Local therapeutic drug committees consider the approximately 15% overall survival benefit seen at 5 years for DLBCL when rituximab is added to combination chemotherapy as too marginal for justifying the arising additional expenses. Accordingly, demonstration that a specific molecular subtype accounts for superior outcome when using these regimens is needed and would provide convincing evidence for the use of this monoclonal antibody in a resource constrained setting. / AFRIKAANSE OPSOMMING: AGTERGROND Limfome ontstaan vanuit selle van die immuunsisteem. Hulle toon ‘n groot verskil met betrekking tot sitomorfologie, immunofenotipe, kliniese verloop, asook respons op behandeling – almal faktore wat prognose bepaal. Die Limfoom Herklassifikasieprojek het getoon dat daar substansiële geografiese verskille bestaan vir Hodgkin se limfoom en ander limfoproliferatiewe toestande. Huidige navorsing oor hierdie maligniteite skep die indruk dat hulle skaars is in Afrika, met die inligting wat wel beskikbaar is hoofsaaklik gebaseer op gevallestudies deur klinici en patoloë. DOEL Hierdie studie bestaan uit twee dele. In die eerste toets ons die hipotese dat die uitkomste vir die behandeling van limfoom in ‘n privaat akademiese instansie geleë in die Wes-Kaap, Suid- Afrika, dieselfde is as wat in die res van die wêreld gesien word indien soortgelyke behandeling toedgedien word.`n Reeks van 512 pasiënte is in die analise gebruik. Vir die tweede hipotese het`n kohort van die mees algemene subtipe, naamlik diffuse groot B-sellimfoom (DLBCL) (n = 93), lukraak òf die standaardkombinasie-chemoterapie in die vorm van CHOP òf `n identiese program met rituximab, `n anti-CD20 monoklonale teenliggaam, gekry. Die teenliggaam is in samewerking met die Universiteit van Nebraska se Mediese Sentrum getoets met behulp van `n immunohistochemies-gebaseerde metode, ook bekend as weefsel mikro-skikking (tissue microarray or TMA). PASIËNTE EN METODE Vir die eerste kohort is die opeenvolgende en volledige rekords van pasiënte wat ouer as 14 was en tussen Oktober 1998 en Julie 2006 in `n privaat-gebaseerde akademiese instansie gediagnoseer is, noukeurig ondersoek. Na sekere uitsluitingskriteria toegepas is, was daar 398 gevalle wat geskik was vir verdere analise. In die tweede deel van die navorsing kon 149 pasiënte wat nuut met diffuse groot B-sel-limfoom (DLBCL) gediagnoseer is, verder geëvalueer word. Drie-en-neëntig van hierdie 149 pasiënte het aan die kriteria vir insluiting voldoen; 48 het slegs die standaard chemoterapie (CHOP) ontvang en die oorblywende 45 het chemo-immunoterapie (chemoterapie plus rituximab (R-CHOP) ontvang. Die demografiese eienskappe van beide groepe het goed vergelyk. Vanweë die kriteria wat gebruik is, het die aanvanklike stratifikasie van 93 gevalle, wat deur middel van die TMA-metode op fenotipering met immunohistochemie gebaseer was, twee populasies gelewer. Hierdie primêre subdivisies is verder geëvalueer vir uitdrukking van BCL2 en LMO2, beide erkende voorspellers van respons. Laastens is kliniese eienskappe en oorlewingsuitkoms ná behandeling met chemoterapie as `n enkel modaliteit of dieselfde chemoterapie met rituximab, vir elke veranderlike met mekaar vergelyk. STATISTIESE ANALISE Algehele en insident-vrye oorlewing is bereken as die jare vanaf diagnose tot sterfte, verdwyning van pasiënte tydens opvolg, eerste progressie of terugval van die toestand. Die Kaplan Meier-metode is gebruik om distribusie te bepaal en die log rank-toets om oorlewing tussen die groepe te vergelyk. Pasiëntkenmerke vir elke gespesifiseerde kohort is met die Chikwadraattoets of Fisher se eksakte toets in die geval van kleiner groepe getoets. RESULTATE In die eerste gedeelte van die studie is al 398 gevalle terugwerkend geanaliseer en daar is gevind dat die behandelingsuitkoms met betrekking tot totale oorlewing en insident-vrye oorlewing teen 36 maande vergelykbaar was met uitkomste in eerstewêreldsentrums. Nadelige faktore met betrekking tot oorlewing was soortgelyk aan reeds gepubliseerde data en het die volgende ingesluit: gestelsimptome, voorafgaande behandeling met chemoterapie, intermediêre of hoë-risiko tellings soos deur die Internasionale Prognostiese Indeks bepaal, histologiese gradering en sekere anatomiese setels van primêre tumor. In teenstelling met internasionale ondervinding het geslag, steiering volgens Rye- of Rai-klassifikasie, retrovirale status en vorige behandeling met radioterapie geen invloed gehad nie. In die tweede gedeelte is weefsel van 93 nuut-gediagnoseerde DLBCL-pasiënte wat deur middel van die TMA-metode subtipeer is, vergelyk met naastenby ooreenstemmende kategorieë soos deur Hans et al. met `n drie-merkerpaneel in kiem- en nie-kiemsentrumsubtipes, of deur Choi et al. met twee bykomende teenliggame in kiemsentrum of geaktiveerde B-selle gedefinieer. Met die DNA-gebaseerde geen-uitdrukkingsprofiel as `n verwysingspunt, was die ooreenstemming tussen die onderskeie algoritmes na verwagting verskillend, met 86% teenoor 93%. Die onderliggende rasionaal was om die relatiewe lonendheid te bepaal van `n bekostigbare maar akkurate tegnologie, wat moontlik in gebiede met onvoldoende hulpbronne toegepas kon word Verder is die prognostiese merkeruitdrukking vir BCL2 en LMO2 ook met die oog op die uitkomste van behandeling ondersoek. In vergelyking met standaard chemoterapie, het die gebruik van die TMA-tegniek met toevoeging van rituximab geen noemenswaardige verbetering in die algehele 5-jaar oorlewing (63% vs 59%, p = 0.68) óf insident-vrye oorlewing (42% vs 39%, p = 0.94) getoon nie. Insgelyks was daar geen duidelike verskille in subtipe-analise nie. Dit is egter interessant dat, met die toepassing van die Choi-kriteria, sitotoksiese middels op hul eie, in teenstelling met die geaktiveerde B-sel-subtipe, nie ‘n statisties belangrike neiging tot beter oorlewing (74% vs 55%, p = 0.32) of insident-vrye oorlewing (44% vs 40%, p = 0.42) vir die kiemsentrumsubtipe (GCB) getoon het nie. Dit was selfs nie eers moontlik om ‘n klein verskil in die teenwoordigheid van rituximab te demonstreer nie. Volgens Choi, het chemoterapie (met óf sonder die toevoeging van rituximab) by vergelyking van kiemsentrumsubtipes met nie-kiemsentrumsubtipes soortgelyke resultate gelewer as die Hans algoritme na 5-jaar oorlewing, sowel as 3-jaar insident-vrye oorlewing. Ten spyte van ’n klein steekproef het verdere subtipe-analise van BCL2- en LMO2- merkeruitdrukking van die onderskeie immunohistochemiese (IHC) subtipes die volgende aan die lig gebring: Analise met gebruik van die Choi-kriteria vir subtipe-analise vir BCL2- uitdrukking het, ongeag die subtipe (GCB of ABC) en die behandelingsmodaliteit (chemoterapie met of sonder toevoeging van rituximab), geen verskil getoon in 5-jaar oorlewing en insident-vrye oorlewing nie. Daar was egter `n noemenswaardige verskil ten opsigte van beter insident-vrye oorlewing (p = 0.0015) in die BCL2-positiewe groep van die ABC-subtipes wat met rituximab-bevattende chemoterapie behandel is. Hierdie resultate moet egter met sorg geïnterpreteer word weens die klein steekproef en kort opvolgperiode van slegs nege maande. Soortgelyke resultate met betrekking tot die uitkoms vir oorlewing is vir LMO2 gelewer, naamlik geen verskil ten opsigte van 5-jaar oorlewing of insident-vrye oorlewing ongeag die subtipe of behandelingsmodaliteit. Hier ook was daar egter beter insident-vrye oorlewing (p = 0.039) in die LMO2-positiewe groep van die ABC-subtipe wanneer rituximab-bevattende chemoterapie toegedien is. Weereens moet die resultate met sorg interpreteer word weens die klein steekproef en die kort opvolgperiode van 14 maande. BESPREKING EN GEVOLGTREKKING Die meeste studies wat reeds die verhoudings van subtipes in groot getalle DLBCL-pasiënte ondersoek het, is onder westerse populasies uitgevoer en daar bestaan onsekerheid oor of dieselfde verhoudings in Afrika van toepassing is. Terwyl 50% van alle DLBCL-pasiënte in Noord-Amerika of Europa die GCB-fenotipe uitdruk, kom dit in slegs 31% van hierdie pasiënte in Asië tot uitdrukking. Geen studie is tot dusver met behulp van die TMA-metode onder limfoomsubtipes in populasies in Afrika onderneem nie, veral nie onder pasiënte met verswakte immuunstelsels nie. Ten spyte van sekere beperkings van hierdie studie, waarvoor daar verskeie redes is, soos die verlies van analiseerbare data, die wisselende verteenwoordiging van die Suid-Afrikaanse bevolking as ‘n geheel en die klein steekproef, wat vergissing in die hand kan werk, bevestig die resultate dat limfoproliferatiewe toestande heterogeen is. Desnieteenstaande kan hierdie groep met sukses behandel word indien ‘n holistiese bestuurplan presies volgens stadiums, klassifikasie en risiko-assessering uitgevoer word – ongeag of dit in `n ontwikkelde gebied of een met min hulpbronne plaasvind. Met die beskikbaarheid van ingewikkelde metodes soos geen-ekspressie profielskepping (GEP), kan DLBCL in prognosties-voordelige kiemsentrum B-sel-agtige (GCB) en die meer aggressiewe geaktiveerde B-sel-subtipes geklassifiseer word. Teen die agtergrond van beperkte hulpbronne, is immunohistochemiese (IHC) kleuringsmetodes gebruik om - weefselmonsters wat in formalien gefikseer en in paraffien ingebed was, te analiseer. Hier was die algoritme wat oorspronklik deur Hans et al. beskryf is en deur Choi et al. verder ontwikkel is, en hoë ooreenstemming toon, vergelykbaar met die GEP-klassifikasie. Die gebruik van die IHC-gebaseerde metodologie is as ‘n eenvoudige, effektief lonende en kragtige alternatief tot GEP, wat tans as die goudstandaard vir klassifikasie van limfoom beskou word, bewys. Dit verskaf ‘n nuttige prognose-hulpmiddel vir die stratifisering van DLBCL of ander entiteite in die toekoms, selfs wanneer gevriesde weefselmonsters nie vir GEP-analise beskikbaar is nie. Onder die huidige begrotingsbeperkings in staatshospitale is middels soos rituximab by die protokolle vir die behandeling van limfoproliferatiewe toestande uitgesluit. Plaaslike terapeutiese middel-komitees beskou die nagenoeg 15% algehele oorlewingsvoordeel teen vyf jaar, wat vir DLBCL moontlik is met die toevoeging van rituximab by kombinasiechemoterapie, as te randstandig om die bykomende onkoste te regverdig. Daarvolgens is dit noodsaaklik om te demonstreer dat `n spesifieke molekulêre subtipe tot ’n beter uitkoms lei wanneer hierdie metode gebruik word en dat dit oortuigende bewyse sal lewer vir die gebruik van hierdie monoklonale teenliggaam in `n omgewing met beperkte hulpbronne.