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行政院國家科學委員會補助專題研究計畫成果報告
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兒童急性淋巴性白血病殘留癌細胞之研究----聯合交替使用聚合
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鏈反應與流式細胞儀
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※ Minimal Residual Disease of Childhood Acute Lymphoblastic Leukemia (II)
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-- Combined Application of Polymerase Chain Reaction and Flow Cytometry
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計畫類別：
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個別型計畫
□整合型計畫
計畫編號：NSC 89－2314－B－006－159
執行期間：89 年 8 月 1 日至 90 年 7 月 31 日
計畫主持人：陳建旭
計畫參與人員：邱元佑、鄭兆能
小兒部 國立成功大學醫學院附設醫院
本成果報告包括以下應繳交之附件：
□赴國外出差或研習心得報告一份
□赴大陸地區出差或研習心得報告一份
□出席國際學術會議心得報告及發表之論文各一份
□國際合作研究計畫國外研究報告書一份
執行單位：
小兒部
國立成功大學醫學院附設醫院
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行政院國家科學委員會專題研究計畫成果報告
計劃名稱：兒童急性淋巴性白血病殘留癌細胞之研究----聯合交替使用聚合脢鏈反應與
流式細胞儀
Minimal Residual Disease of Childhood Acute Lymphoblastic Leukemia (II) -- Combined
Application of Polymerase Chain Reaction and Flow Cytometry
計畫編號：NSC 89-2314-B006-159
執行期限：89 年 08 月 01 日至 90 年 07 月 31 日
主持人：陳建旭 小兒部 國立成功大學醫學院附設醫院
一、中文摘要 雖然現代抗癌藥物以及輔助治療之進步，已經 大幅提昇了兒童白血病的治療成果，但是，仍然有 20~25%左右的病童會復發。偵測殘留癌細胞(MRD) 是提高成功率的方法之一，但是以傳統光學顯微鏡 檢查之方法，靈敏不夠，須要利用更靈敏的方法， 才能偵測出殘留之癌細胞。在緩解後之 MRD 已經 被公認是一個獨立的預後因子。甚至緩解後 MRD 之消長變化，也漸漸被認為與預後相當有關。本計 劃之前身，我們已經採用了半定量聚合脢鏈反應 （PCR）之方法，來偵測殘留癌細胞的多寡。由於 偵測率偏低(46.7%)，本年度，我們加上流式細胞 儀之方法，與 PCR 方法交替使用，期望能提高偵 測率。本年度我們前瞻性地總共收集了 16 位急性 淋巴性白血病的病童，先用流式細胞儀之方法，其 中有 7 位能找到合適的抗体組合，及 2 位有適當之 PCR 引子，作為偵測 MRD 之用(56.2%)，其餘 2 位為 polyclonal 或 multiple bands 無法分離者，因重 組之核酸甘太少或引子功能不足以區別癌細胞與 正常細胞者有 5 位。緩解之後總共有 14 個病童之 骨髓檢体進行系列 MRD 之分析，其中一位在緩解 誘導完成之後仍然可測得 MRD (0.364%)，所幸在 之後的追蹤檢驗，MRD 逐漸降低到 0.01%以下（第 16 週為 0.143%，32 週則<0.01%）。此病童在臨床 上尚無復發之跡象，目前仍密切追蹤中。而其他所 有病人都沒有臨床之復發。流式細胞儀合併 PCR 來偵測殘留癌細胞是一種可行的方法。它的敏感性 可以高達 10-4~10-5。本實驗已經明顯提高了有效之 偵測率，若能增加個案數，相信必能更加提高偵測 率，可含蓋更多的病童。由於本實驗之個案數目有 限，以及追蹤時間仍稍短，目前我們還無法下一個 定論。但是，我們推論，系列的追蹤 MRD，比單 一定點 MRD 之偵測，更能顯示出治療預後。假若 系列追蹤之 MRD 數值一直降低，可推測其預後也 將不差。我們需要更多的個案以及較長的追蹤期。 隨著以往二個計劃的累積，我們已經持續追蹤接近 17 位個案。對預計的成果，將指日可待。 關鍵詞：殘留癌細胞、流式細胞儀、聚合脢鏈反應、 急性淋巴性白血病 Abstr actAlthough the treatment result of childhood
lymphocytic leukemia (ALL) has much improvement in the past decades, there still has 20-25% patients will eventually experience relapses. The level of minimal residual disease (MRD) during clinical remission is one of the most powerful prognostic factors. The most useful methods to monitor the MRD are polymerase chain reaction (PCR) amplifications of antigen-receptor genes and flow cytometric identification of leukemic-associated immunophenotypes. We prospectively use both methods alternatively to follow the fate of the leukemic clone. MRD was examined at time of complete remission and different time-point of treatment protocols. We have enrolled 16 newly diagnosed childhood ALL patients in the past one year. Total 9 patients had individualized markers for their subsequent bone marrow MRD monitoring (56.2%). 7 of these 9 patients had suitable individualized leukemic-associated MoAb combinations and 2 had successful clone-specific primers for MRD monitoring. Fourteen follow-up bone marrow samples from these 9 ALL cases were analyzed for MRD assays. We identified the levels of MRD greater than 0.01% in two samples of a patient after the remission induction (week 11 and 16) and which decreased gradually to less than 0.01% 4 months later (week 32). There was no clinical relapse in all cases during the period of follow-up. In conclusion, alternating with semi-quantitative PCR and flow cytometry methods is a feasible and powerful tool in MRD monitoring and can increase the detection rate up to 56.2% in our study. However, with the limited sample size and follow-up duration, our current study is hard to draw a final conclusion and answer those questions prompted in our proposal. Large-scale study and long-term follow-up is warranted to answer those questions.
Keywor ds: minimal residual disease, flow cytometry,
polymerase chain reaction, childhood acute lymphoblastic leukemia
Intr oduction
With the advent of contemporary multidrug chemotherapy and supportive care, the survival of childhood acute lymphocytic leukemia (ALL) has been much improved1. However, at least 20-25% of
those who are regarded as standard risk will still experience relapses. A number of biological and clinical parameters have been used for prediction of these relapses, but none is completely reliable2. Sequential monitoring of minimal residual disease (MRD) of bone marrow is thought to be one of the most promising parameter for prediction of relapse. Among the various available techniques for MRD detection in ALL, flow cytometric detection of aberrant immunophenotypes and polymerase chain reaction (PCR) amplification of clone-specific antigen-receptor genes rearrangement have been considered as the most sensitive and reproducible methods3-6. Several retrospective and prospective studies have shown that detection of MRD with these methods during clinical remission appears to be an independent prognostic factor for treatment outcome. However, neither can be applied to all patients7.
There are several consensus about MRD in the treatment of childhood ALL. Absence of MRD after remission induction is independently to be a factor of good prognosis8;9. A steady absence of MRD throughout the follow-up duration is associated with a favorable prognosis but persistence of MRD in the course of treatment generally will lead to a dismal relapse outcome10-12. Recently, some studies have shown that the trend rather than the levels of MRD are more significantly related to the treatment outcome8;11;13. These findings provide us a strong rationale to use the sequential MRD levels as not only for monitoring but for a criteria for risk re-classification in the treatment of childhood ALL. So, we conducted a prospective study to sequentially monitor the MRD in the course of treatment in childhood ALL. To increase the applicability in diagnostic patients, we use semiquantitative PCR and flow cytometry methods alternatively.
Mater ials and Methods
All patients who were newly diagnosed as ALL according FAB criteria from august 1, 2000 to July 31, 2001 and sticked to the treatment protocols of TPOG were enrolled in this prospective study. Bone marrow (BM) samples were collected at diagnosis, the end of remission, before consolidation, before reinduction, one year, two year and the end of therapy, according to the protocol used. Mononuclear cells (MNC) were separated by centrifugation by a density gradient. All samples were processed within 4 hours. Cells were maintained in RPMI-1640 medium with 10% fetal calf serum, 2mM L-glutamine and antibiotics.
Flow Cytometr y Leukemia-associated
immunophenotypes were detected by multiparameter flow cytometry, with various combinations of monoclonal antibodies conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein (PerCP), and phycoerythrin cyanin (PC). Matched nonreactive fluorochrome-conjugated antibodies served as
controls. The staining procedure has been described elsewhere14. For each case, marker combinations with the sensitivity of one leukemic cell per 104 normal nucleated bone marrow cells or greater were selected at diagnosis and then applied for subsequent MRD monitoring. We used a FACSort flow cytometer equipped with CellQuest software (Becton Dickinson, San Jose, CA). The staining procedure used for MRD detection has been described. For intracellular staining, cells were permeabilized with 8E solution during the cell-labeling procedure. In all samples, we acquired data from all mononuclear cells in each test tube (more than 1 x 105).
PCR amplification of Immunoglobin Heavy Chain genes Those who failed to obtain a suitable
combination of immunophenotypic markers proceeded to semiquantitative PCR assay. DNA samples were prepared by using QIAamp blood kit. IgH gene rearrangements present at diagnosis were identified with the consensus V-region primer FR1C or FR2B, accompany with a universal JH primer15-17.
Reaction mixture of 20 ul contained 1xPCR buffer, 100 ng of genomic DNA, 1.5mM MgCl2, 200 uM dNTPs, 0.25 uM primers and 1 unit of AmpliTaq Gold (PE Applied Biosystems). The typical amplification conditions consisted of an initial incubation for 10 minutes at 95C, then 45 cycles of 30 sec at 94C and 60 sec at 60C, followed by a final incubation of 5 min at 72 C. PCR products were purified and sequencing. The VH, DH and JH regions
were identified by comparison with sequences in GeneBank using GCG software. For analysis of remission samples, primers complementary to N-D-N and D-N-J nucleotides of the leukemic IgH gene rearrangement were synthesized. A semi-nested PCR assay was used to detect a single copy of the leukemic gene rearrangement among 105 normal genomes. All PCR products were analyzed by electrophoresis on a 3% agarose gel. Specificity was confirmed by using pooled peripheral-blood MNC DNA and sterile water as negative controls. The sensitivity of each assay was established by making serial 10-fold dilutions of leukemic DNA with DNA prepared from pooled PB MNC until no PCR product was observed. MRD was quantified by limiting-dilution analysis using 10 replicates and Poisson statistics18;19.
Results
Total 16 cases of childhood ALL were eligibly enrolled in this study including 7 boys and 9 girls; 14 B-lineage ALL and 2 T-cell ALL. 8 patients were stratified to standard risk group, 4 to high-risk group and 3 to very-high-risk group and 1 to L3 protocol according to the criteria of TPOG guideline. 13 diagnostic samples were examined for MRD markers and seven patients had useful leukemic-associated immunophenotypic markers, which can effectively differentiate leukemic cells from normal BM cells (53.8%). The remaining 9 diagnostic samples were sent for IgH gene rearrangement sequencing. Only 2
5 patients had successful clone-specific primers, which were suitable for quantification study (22.2%). The reasons for failure were polyclonal amplified Ig heavy-chain genes or more than two amplified Ig heavy-chain genes that could not be readily separated for sequencing in 4 diagnostic samples (33.3%); inability to design a primer owing to too short CDR3 sequence or lack of specificity or sensitivity of the primers in 4 patients (44.4%). Total 9 out of 16 patients had either leukemic-associated immunophenotypic markers or clone-specific primer markers for subsequent MRD analysis (56.2%).
Fourteen follow-up bone marrow samples from these 9 patients were analyzed for MRD. We identified the levels of MRD greater than 0.01% in two samples of the same patient after remission induction (week 11 and week 16, respectively). The percentage of blasts in both samples was all less than 5% by light microscope examinations. Fortunately, the level of MRD decreased from 0.364% to 0.143% and <0.01% in the week 16 and week 32, respectively. No clinical relapse was noted in all 9 patients studied. But, the prognosis of those whose MRD levels converted from positive to negative after remission induction seem to have a relative good outcome.
Discussion
Alternating with flow cytometry and PCR techniques for detection of MRD is feasible and can detect leukemic cells down to 10-5 level. The advantage of the three-colored flow cytometry assay is it’s a fast, mature technique and accurate in cell quantitation14. The weakness is expensive, skillful, instrument-dependent and immunotyping switch. On the contrary, semiquantitative PCR amplification of IgH gene rearrangement technique is cheaper but low applicability, laborious and time-consuming. With alternating application of both techniques, we have increased the detection rate up to 56.2% in our series. We found that MRD detected during the early clinical course can become undetectable at later time point and that this conversion is associated with a relatively good clinical outcome. These observations also prompt the importance of sequential monitoring of MRD in childhood ALL. Sequential monitoring of MRD can provide a measure of drug sensitivity, indicating the number of leukemic cells remained to be eradicated, and enables early talor of treatment strategy.
A major limitation of MRD assay was its lack of applicability in a substantial proportion of newly diagnosed cases, especially for cases with B-cell phenotype. Our previous study has shown that with solely semiquantitative PCR technique, only half of the patients had suitable individualized primers for MRD monitoring. Currently, with alternating application of both three-colored flow cytometry and semiquantitative PCR assay, we have been improving this limitation as what was reported by others20;21. We estimate that the suitable MRD markers could
ultimately be quantifiable in 70-80% of patients by such approach.
One of the most difficulties in leukemia treatment is to distinguish patients who need more intensive therapy from those who only need less intensive therapy. Sequential MRD monitoring can provide direct measurements of leukemic cell responses to chemotherapy. The MRD at the end of induction remission can be used for risk re-stratification and protocol re-selection. We suggest that those cases with MRD positive at the end of remission induction should be closely monitored for changes in MRD. In our current study, with the limited sample size and follow-up duration, it is too early to say whether it is significant in the prediction of clinical relapse after the remission induction, MRD is protocol-dependent or which time point is critical in clinical course. More cases and longer follow-up duration are needed to answer these questions.
Refer ences
1. Pui CH, Evans WE: Acute lymphoblastic leukemia. N.Engl.J.Med. 339: 605-15, 1998
2. Campana D, Pui CH: Detection of minimal residual disease in acute leukemia: methodologic advances and clinical significance. Blood 85: 1416-34, 1995
3. Farahat N, Morilla A, Owusu-Ankomah K et al.: Detection of minimal residual disease in B-lineage acute lymphoblastic leukaemia by quantitative flow cytometry. British Journal of Haematology 101: 158-64, 1998
4. van Dongen JJ, Macintyre EA, Gabert JA et al.: Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease. Report of the BIOMED-1 Concerted Action: investigation of minimal residual disease in acute leukemia. Leukemia 13: 1901-28, 1999
5. Foroni L, Harrison CJ, Hoffbrand AV, Potter MN: Investigation of minimal residual disease in childhood and adult acute lymphoblastic leukaemia by molecular analysis. British Journal of Haematology 105: 7-24, 1999
6. van Dongen JJ, Breit TM, Adriaansen HJ, Beishuizen A, Hooijkaas H: Detection of minimal residual disease in acute leukemia by immunological marker analysis and polymerase chain reaction. Leukemia 6 Suppl 1: 47-59, 1992 7. Cave H, van der Werff ten Bosch, Suciu S et al.:
Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia. European Organization for Research and Treatment of Cancer--Childhood Leukemia Cooperative Group. New England Journal of Medicine 339: 591-8, 1998
8. van DJ, Seriu T, Panzer-Grumayer ER et al.: Prognostic value of minimal residual disease in
acute lymphoblastic leukaemia in childhood. Lancet 352: 1731-8, 1998
9. Steenbergen EJ, Verhagen OJ, van LE et al.: Prolonged persistence of PCR-detectable minimal residual disease after diagnosis or first relapse predicts poor outcome in childhood B- precursor acute lymphoblastic leukemia. Leukemia 9: 1726-34, 1995
10. Ciudad J, San Miguel JF, Lopez-Berges MC et al.: Prognostic value of immunophenotypic detection of minimal residual disease in acute lymphoblastic leukemia. Journal of Clinical Oncology 16: 3774-81, 1998
11. Coustan-Smith E, Behm FG, Sanchez J et al.: Immunological detection of minimal residual disease in children with acute lymphoblastic leukaemia. Lancet 351: 550-4, 1998
12. Gruhn B, Hongeng S, Yi H et al.: Minimal residual disease after intensive induction therapy in childhood acute lymphoblastic leukemia predicts outcome. Leukemia 12: 675-81, 1998
13. Coustan-Smith E, Sancho J, Hancock ML et al.: Clinical importance of minimal residual disease in childhood acute lymphoblastic leukemia. Blood 96: 2691-6, 2000
14. Campana D, Coustan-Smith E: Detection of minimal residual disease in acute leukemia by flow cytometry. Cytometry 38: 139-52, 1999
15. Aubin J, Davi F, Nguyen-Salomon F et al.: Description of a novel FR1 IgH PCR strategy and its comparison with three other strategies for the detection of clonality in B cell malignancies. Leukemia 9: 471-9, 1995
16. Ramasamy I, Brisco M, Morley A: Improved PCR method for detecting monoclonal immunoglobulin heavy chain rearrangement in B cell neoplasms. Journal of Clinical Pathology 45: 770-5, 1992 17. Deane M, Norton JD: Immunoglobulin heavy
chain variable region family usage is independent of tumor cell phenotype in human B lineage leukemias. Eur.J.Immunol. 20: 2209-17, 1990 18. Ouspenskaia MV, Johnston DA, Roberts WM,
Estrov Z, Zipf TF: Accurate quantitation of residual B-precursor acute lymphoblastic leukemia by limiting dilution and a PCR-based detection system: a description of the method and the principles involved. Leukemia 9: 321-8, 1995 19. Sykes PJ, Neoh SH, Brisco MJ, Hughes E, Condon
J, Morley AA: Quantitation of targets for PCR by use of limiting dilution. Biotechniques 13: 444-9, 1992
20. Neale GA, Coustan-Smith E, Pan Q et al.: Tandem application of flow cytometry and polymerase chain reaction for comprehensive detection of minimal residual disease in childhood acute lymphoblastic leukemia. Leukemia 13: 1221-6, 1999
21. Pui CH, Campana D: New definition of remission in childhood acute lymphoblastic leukemia. Leukemia 14: 783-5, 2000 【計畫成果自評】 本研究和原提計劃內容相符，本實驗連接上一 年度之研究計劃，加入了流式細胞儀之檢驗方法， 果然偵測率提高甚多（56.2%），而且比率還在增 高中，雖然只是短短一年，但是我們證實了 Flow Cytometry 加上 PCR 是一個可行而且簡單的方法， 可以用來偵測孩童急性淋巴性白血病之殘留癌細 胞。我們的結論是以流式細胞儀偵測優先，以 PCR 為輔，然而仍有一些病例無法含括在內，殊為可 惜，實在有引進新一代四色流式細胞儀之偵測方法 之必要。至於 MRD 與臨床之關係，因本研究個案 數尚在累積之中，且目前無人有臨床上之復發發 生，不過從其中一位起初 MRD 陽性之個案，後來 追蹤之後慢慢消失，而病人也沒症狀發生，隱約可 見系列追蹤 MRD 的變化，在臨床上的重要性。最 後，本 MRD 之研究須要更多的個案，以及時間的 追蹤，才可以得到答案，嘉惠病童以及論文發表。
