IMPACT OF BCR-ABL1 MONITORING AMONG PATIENTS WITH BCR- ABL1-POSITIVE B-ACUTE LYMPHOBLASTIC LEUKEMIA Chong Siew Lian Thesis submitted in partial fulfilment for the degree of MASTER OF MEDICAL SCIENCE UNIVERSITY SAINS ISLAM MALAYSIA December 2021 i AUTHOR DECLARATION I declare that this dissertation is entirely my own original work. I declare that, except where fully referenced direct quotations have been included, no aspect of this dissertation has been copied from any other source. I declare that all other works cited in this dissertation have been appropriately referenced. Date: 1st December 2021 Signature: Name: Chong Siew Lian Matric No: 3192577 Address: 1, Jalan Aman Bayan 2, Bandar Tropicana Aman, 42500 Telok Panglima Garang, Selangor, Malaysia. ACKNOWLEDGEMENTS I would like to express my deep gratitude to Professor Madya Dr. Asral Wirda Ahmad Asnawi, my research supervisor, for her kindness, a very patient guidance, an enthusiastic encouragement, and useful critiques she gave of this research work. I would like to offer my special thanks to Dr Tan Sen Mui for her early advice on project writing guidelines. Advice given has been a great help in getting a head start on my final year project. I am particularly grateful for the assistance given by deans of faculties and heads of programme, from USIM. I wish to acknowledge the help provided by all laboratory staff of Haematology Department Ampang Hospital Malaysia on assistance with my project sampling and data issues. Finally, I wish to thank my husband and children for their understanding, unfailing support, and encouragement throughout my studies. iii ABSTRAK Leukemia limfoblastik akut atau Acute lymphoblastic leukaemia (ALL) dengan BCR- ABL1 membawa kepada risiko yang tinggi untuk relaps serta prognosis yang teruk. Akses kepada perencat tyrosine kinase sebagai salah satu strategi rawatan telah mengubah stratifikasi risiko untuk pesakit ALL yang mempunyai gen tertaup ini. Memandangkan BCR-ABL1 dikuantifikasi secara rutin untuk pemantauan penyakit mieloproliferatif, penggabungan kuantifikasinya melalui reaksi rantai polymerase pada masa nyata boleh dijadikan sebagai parameter yang boleh dipercayai sebagai pemantau sisa penyakit atau measurable residual disease (MRD) untuk ALL yang mempunyai BCR-ABL1. Sama ada ini telah menggantikan faktor risiko konvensional dalam menentukan pesakit mana yang memerlukan transplan atau tidak masih belum ditentukan. Kajian ini adalah bertujuan untuk menentukan kesan pemantauan BCR- ABL1 pada pesakit ALL dengan BCR-ABL1 terhadap keputusan mereka selepas pemindahan sel stem alogenik. Kami menganalisa hasil kelangsungan hidup pesakit ini secara retrospektif berdasarkan kuantifikasi BCR-ABL1 pada tiga titik-masa; titik-masa pertama pada akhir induksi, titik-masa kedua pada minggu ke-16 selepas konsolidasi, dan titik-masa ketiga pada akhir rawatan pesakit yang sama ada layak untuk transplan atau tidak layak untuk transplan. Dari tahun 2006 hingga 2018, sejumlah 96 pesakit dewasa yang baru didiagnosis dengan Leukemia Limfoblastik Akut dengan BCR-ABL1 telah dirawat dengan kemoterapi dan perencat tyrosine kinase. Tiga puluh lapan (41.3%) mencapai remisi keseluruhan dan 33 pesakit telah menjalani pemindahan sel stem alogenik. Data kami menunjukkan bahawa pemantauan sisa penyakit melalui tindak balas rantai polymerase kuantitatif pada masa nyata yang dilakukan sebelum transplan iv menunjukkan hubung kait kelangsungan hidup yang tertinggi dalam pesakit ALL dengan BCR-ABL1 terutamanya bagi mereka yang menjalani transplan sel stem alogenik. Pesakit yang mempunyai MRD negatif pra-transplan, mempunyai kelangsungan hidup yang lebih tinggi berbanding mereka yang MRD positif dan menunjukkan hasil jangka panjang yang cemerlang selepas transplan sel stem alogenik. Dengan kemunculan perencat/inhibitor tyrosine kinase dan penggabungan pemantauan penyakit sisa yang boleh diukur dengan ketat, stratifikasi risiko untuk ALL dengan BCR-ABL1 telah berubah dengan ketara. v ABSTRACT Acute lymphoblastic leukaemia (ALL) with BCR-ABL1 confers a high risk of relapse and a poor prognosis. Access to the tyrosine kinase inhibitor as part of the treatment strategy has changed the risk stratification for ALL patients harbouring this fusion gene. As BCR-ABL1 is routinely quantified for the monitoring of its myeloproliferative counterpart, incorporation of its quantification by real-time polymerase chain reaction could be a reliable parameter for the monitoring of measurable residual disease (MRD) for ALL with BCR-ABL1. Whether this has replaced conventional risk factors in deciding whether patients would need a transplant or not is yet to be determined. This study aimed to determine the impact of BCR-ABL1 monitoring in patients with ALL with BCR-ABL1 on their outcome after allogeneic stem cell transplantation. We retrospectively analysed the survival outcome of these patients based on the quantification of BCR-ABL1 at three time-points; time-point 1 at the end of induction, time point 2 at post-consolidation week 16, and time point 3 at the end of treatment for patients who were transplant-eligible or nontransplant eligible. From 2006 to 2018, a total of 96 adult patients newly diagnosed with acute lymphoblastic leukemia with BCR-ABL1 were treated with chemotherapy and a tyrosine kinase inhibitor. Thirty- eight (41.3%) achieved overall remission; 33 patients underwent an allogeneic stem cell transplant. Our data showed that residual disease monitoring by real-time quantitative polymerase chain reaction performed prior to transplantation showed the highest survival correlation in ALL with patients with BCR-ABL1, especially for those who underwent allogeneic stem cell transplantation. Patients with MRD negative before transplantation had better survival compared to those who were MRD positive and showed excellent long-term outcomes after allogeneic stem cell transplantation. vi With the emergence of tyrosine kinase inhibitors and the incorporation of stringent, measurable residual disease monitoring, risk stratification for ALL with BCR-ABL1 has changed significantly. iiv ُملَخَّص ى . أدّ س وإنذار سّي خطورة عالية للنّك 1LBA-RCB جين ) مع LLA الدم الليمفاوي الحاد (ابيضاض يحمل جميع المرضى ل تصنيف الخطورةة العلاج إلى تغيير الوصول إلى مثبطات التيروزين كيناز كجزء من استراتيجيّ . المندمجهذا الجين لديهمذين الّ قيمته اعتماد، فإن نقوياثر كت من هما يقابل مراقبةجل بشكل روتيني لأ 1LBA-RCB ة كميّ قياس بما أنّه يتم ّ الحد ّلرصد ا ً موثوق معياراً أن يكون تفاعل البوليميراز المتسلسل في الوقت الفعلي يمكن المحدّدة كميّا ًبواسطة .1LBA-RCB جين ) مع LLA الليمفاوي الحاد (لابيضاض الدّم ) DRM ( الأدنى من المرض المتبّقي تحديد المرضى ديّة في التّقليعوامل الخطر استخدام ينوب عن استخدام هذه القيمة قد تحديد ما إذا كان بعدلم يتم ّ ى في المرض 1LBA-RCB راسة إلى تحديد تأثير مراقبة ة زرع أم لا. هدفت هذه الدّ الذين يحتاجون إلى عمليّ ة المثليّة.عيّ ى نتائجهم بعد زرع الخلايا الجذعل 1LBA-RCB جين ولديهم LLAالذين يعانون من زمنية؛في ثلاث نقاط 1LBA-RCB على القياس الكمي لـ اعتماداً رجعّياً هؤلاء المرضى ياققمنا بتحليل نتائج ب في نهاية 3قطة الزمنية والن مابعد الاندماج،61الأسبوع في 2، النقطة الزمنية تّحفيزفي نهاية ال 1النقطة الزمنية راعة. لين للز ّراعة أو غير مؤه ّلين للز ّكانوا مؤه ّ للمرضى سواءالعلاج -RCB ع ممفاوي الحاد بسرطان الدم اللّ ا ً حديثم شخَّص مريض بالغ 69 علاج 8102و 6002 تّم بين عامي ّ فيا ً تعا منهم) ٪3.14ثمانية وثلاثون (حقّق ز. االتيروزين كين اتطمثبّ العلاج بعلاج الكيميائي وبال 1LBA الحد ّالأدنى راقبة . أظهرت بياناتنا أن مالمثلّيةة لعملية زرع الخلايا الجذعيّ منهم مريض 33خضع ؛ ا ً إجماليّ جراؤه قبل الّزرع ي في الوقت الفعلي الذي تم إالمتسلسل الكم ّ وليميرازعن طريق تفاعل الب المتبقّي من المرض ولئك الذين خضعوا لعملية أةً خاص ّ 1LBA-RCB ن لديهم جينالّذي LLA لدى مرضى مع البقياأعلى ارتباط يملك ة المثليّة.زرع الخلايا الجذعيّ ،إيجابية DRM مقارنة مع أولئك الذين كانت نسبة بقيا أعلىسلبيّة قبل الّزرع ب DRM ذين لديهم المرضى الّ يتمتّع LLA تصنيف خطورةر تغيّ لقد مثليّة. أظهروا نتائج ممتازة على المدى الطويل بعد زرع الخلايا الجذعية اللقد و iiiv للحدّ مراقبة صارمة إجراء بشكل كبير مع ظهور مثبطات التيروزين كيناز و 1LBA-RCBالمترافق مع جين .الأدنى المتبّقي من المرض ix TABLES OF CONTENTS TABLE OF CONTENTS AUTHOR DECLARATION .................................................................................... i ACKNOWLEDGEMENTS ..................................................................................... ii ABSTRAK .............................................................................................................. iii ABSTRACT ............................................................................................................. v صََّخلُم ........................................................................................................................ vii TABLES OF CONTENTS ..................................................................................... ix LIST OF TABLES ................................................................................................. xii LIST OF FIGURES .............................................................................................. xiii LIST OF APPENDICES ...................................................................................... xiv LIST OF ABBREVIATIONS ................................................................................ xv CHAPTER 1 .......................................................................................................... 17 INTRODUCTION ................................................................................................. 17 1.1 Introduction .................................................................................................. 17 1.2 Background of the Study ............................................................................. 18 1.3 Problem Statement ....................................................................................... 21 1.4 Research Questions ...................................................................................... 22 1.5 Objectives of the Study ................................................................................ 22 1.5.1 General Objective .................................................................................. 22 1.5.2 Specific Objectives ................................................................................. 22 1.6 Significance of The Study ............................................................................. 23 1.7 Scope of the Study ........................................................................................ 24 1.8 Research Conceptual Framework ............................................................... 24 1.9 Operational Definitions ................................................................................ 26 1.9.1 BCR-ABL1 Molecular Monitoring ........................................................ 26 1.9.2 Complete Haematological Response (CHR) ......................................... 26 1.9.3 Complete Molecular Response (CMR).................................................. 27 1.9.4 Major Molecular Response (MMR) ...................................................... 27 1.9.5 First Complete Remission (CR1) ........................................................... 27 1.9.6 Engraftment ........................................................................................... 27 1.9.7 Disease Relapse ...................................................................................... 28 1.9.8 Disease-free Survival (DFS) ................................................................... 28 1.9.9 Overall Survival (OS) ............................................................................ 28 CHAPTER 2 .......................................................................................................... 29 LITERATURE REVIEW...................................................................................... 29 x 2.1 Overview of Acute Lymphoblastic Leukaemia ........................................... 29 2.2 BCR-ABL1 in B-Acute Lymphoblastic Leukaemia ..................................... 31 2.3 Minimal Residual Disease Monitoring in B-ALL with BCR-ABL1 ............ 35 2.4 Management of B-ALL with BCR-ABL1 ..................................................... 36 2.5 Gap Analysis ................................................................................................. 41 CHAPTER 3 .......................................................................................................... 43 METHODOLOGY ................................................................................................ 43 2.1 Introduction .................................................................................................. 43 2.2 Research Design ........................................................................................... 43 2.3 Molecular Monitoring Strategy ................................................................... 44 2.4 Study Location.............................................................................................. 45 2.5 Population and Sampling ............................................................................. 45 2.5.1 Inclusion Criteria ................................................................................... 45 2.5.2 Exclusion Criteria .................................................................................. 45 2.6 Research Instruments .................................................................................. 45 2.6.1 Hospital Information System (HIS) ...................................................... 45 2.6.2 Laboratory Results ................................................................................ 46 2.6.3 Data Collection ....................................................................................... 47 2.7 Statistical Analysis........................................................................................ 47 CHAPTER 4 .......................................................................................................... 49 FINDINGS ............................................................................................................. 49 4.1 Characteristics of Patients with ALL with BCR-ABL1 in Hospital Ampang ............................................................................................................................ 49 4.2 Treatment Modalities and BCR-ABL1 Monitoring..................................... 49 4.3 Outcomes of BCR-ABL1 Positive B-ALL Patients ...................................... 51 4.4 Characteristics of Transplanted Patients with B-ALL with BCR-ABL1 .... 53 4.5 Transplant Modalities and Outcome of Allogeneic Stem Cell Transplant 55 4.7 Patients Achieving CMR Using BCR-ABL1 Transcript Levels at Different Time Points ......................................................................................................... 59 CHAPTER 5 .......................................................................................................... 67 DISCUSSION, RECOMMENDATIONS AND CONCLUSION ......................... 67 5.1 Introduction .................................................................................................. 67 5.2 Summary and Discussion of Findings ......................................................... 67 5.3 Implications of The Study ............................................................................ 70 5.4 Recommendations of The Study .................................................................. 71 5.5 Conclusion .................................................................................................... 71 APPENDIX ............................................................................................................ 72 xi REFERENCES ...................................................................................................... 75 xii LIST OF TABLES Table 1: Chemotherapy regimen for B-ALL with BCR-ABL1 at Hospital Ampang .. 37 Table 2: Long-term results of TKI-based clinical trials for adult Ph+ (patients in complete hematologic remission), with an emphasis on allogeneic stem cell transplantation. ........................................................................................................ 38 Table 3: Baseline patient characteristics ................................................................... 50 Table 4: Treatment modalities and molecular monitoring of BCR-ABL1 .................. 51 Table 5: Characteristics of Characteristics of transplanted patients ........................... 54 Table 6: Transplant modalities and outcomes in patients with B-ALL with BCR-ABL1 ................................................................................................................................ 56 Table 7: Univariable analysis of factors predictive of OS ......................................... 61 Table 8: Multivariable analysis of factors predictive of OS and DFS in transplant patients. ................................................................................................................... 62 xiii LIST OF FIGURES Figure 1: Number of stem cell transplants performed from 1999 to 2019. ................ 25 Figure 2: Conceptual framework of the research ...................................................... 26 Figure 3: Schematic representation of the structure of the breakpoint cluster region (BCR) proto-oncogene tyrosine protein kinase (ABL1) gene and protein. ................. 33 Figure 4: Transplantation outcome, OS according to BCR-ABL1 at certain time points. (A) Post-induction MRD (B) Post-consolidation week 16 (C) End of treatment (D) The overall cohort .......................................................................................................... 64 Figure 5: Transplantation outcome, DFS according to BCR-ABL1 at certain time points. (A) Post-induction MRD (B) Post-consolidation week 16 (C) End of treatment (D) The overall cohort .......................................................................................................... 66 xiv LIST OF APPENDICES Appendix 1: NMRR registration and MREC approval Ministry of Health of Malaysia ................................................................................................................................ 72 Appendix 2: Article published in Blood Res 2021; 56(3): 175-183. Published online: 30 September 2021 DOI: https://doi.org/10.5045/br.2021.2021045.......................... 74 xv LIST OF ABBREVIATIONS aGVHD Acute graft-versus-host disease AML Acute myeloid leukaemia AYA Adolescent and young adult B-ALL B-Acute Lymphoblastic Leukaemia BCR-ABL1 Breakpoint Cluster Region-Abelson 1 fusion protein BFM Berlin-Frankfurt-Munich protocol cGVHD Chronic GVHD CLAEG Cladribine, VP16, AraC, GCSF, methotrexate and dexamethasone regime CML Chronic myeloid leukaemia CML-N Neutrophilic-chronic myeloid leukaemia CMR Complete molecular response CMV Cytomegalovirus CNS Central nervous system CR1 First complete remission DFS Disease-free survival FISH Fluorescence in-situ hybridization FLAG-IDA Fludarabine, AraC, Idarubicin, GCSF, methotrexate, dexamethasone regime GMALL 07/2003 vincristine, daunorubicin, dexamethasone, L-asparaginase, methotrexate, rituximab, GCSF regime GVHD Graft-versus-host disease. HSCT Haemopoetic stem cell transplant Hyper-CVAD VP16, GCSF, rituximab dexamethasone, vindesine, methotrexate, folinic acid, AraC, regime IS International system of units LC Light cycle MMR Major molecular response MRD Measurable residual disease xvi MSD Matched sibling donor MUD Matched unrelated donor OS Overall survival RCPA Royal College of Pathologists of Australasia RIC Reduced intensity conditioning SCT Stem cell transplant TBI Total body irradiation TCR T-cell receptor TKI tyrosine kinase inhibitor TP Time-point TP1 Time-point 1, post-induction TP2 Time-point 2, post-consolidation or week 16 TP3 Time-point 3, end-of-treatment Ph Philadelphia chromosome qRT-PCR Quantitative real-time polymerase chain reaction UKALL International ALL Trial UKNEQAS United Kingdom National External Quality Assessment Service VDJ Variability Diversity Joining WCC White cell count WHO World Health Organization