兒童急性淋巴性白血病殘留癌細胞在週邊血與骨髓液之比較

行政院國家科學委員會專題研究計畫 成果報告

兒童急性淋巴性白血病殘留癌細胞在週邊血與骨髓液之比

較

中 華 民 國 92 年 10 月 30 日

行政院國家科學委員會補助專題研究計畫成果報告

兒童急性淋巴性白血病殘留癌細胞在週邊血與骨髓液之比較

Prospective Comparison of Minimal Residual Disease in Peripheral Blood

and Bone Marrow in Childhood Acute Lymphoblastic Leukemia

計畫類別：

;

個別型計畫 □整合型計畫

計畫編號：NSC 91-2314-B-006-071-

執行期間：91 年 8 月 1 日至 92 年 7 月 31 日

計畫主持人：陳建旭

計畫參與人員：蕭志誠*、孫孝芳**、鄭兆能

小兒部 國立成功大學醫學院附設醫院

血液腫瘤部 高雄長庚兒童醫院*

臨床醫學研究所 國立成功大學醫學院**

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

中 華 民 國 90 年 10 月 31 日

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在週邊血與骨髓液之比較

Prospective Comparison of Minimal Residual Disease in Peripheral Blood and Bone Marrow in Childhood Acute Lymphoblastic Leukemia

計畫編號：91-2314-B-006-071- 執行期限：91 年 08 月 01 日至 92 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 25~30%左右的病童會 復發。在白血病的治療期間，系列地偵測 病患體內微量殘留癌細胞(MRD)之多寡， 已經被公認是一個獨立的預後因子。但 是，目前之偵測是需要施行骨髓穿刺檢 查，以便取得骨髓檢體，這會造成病童之 不舒服，從而限制了更密集偵測之可能。 所以，我們提出微量殘留癌細胞，是否可 以使用週邊血液來取代骨髓檢體之假說。 我們使用三色流式細胞儀之方法，其靈敏 度可以達到一萬分之一。我們前瞻性地總 共收集了 19 位新診斷急性淋巴性白血病 的病童，這些都是有合適的抗体組合，可 偵測其微量殘留癌細胞者。10 位女生與 9 位男生。18 位 B 細胞系列，1 位 T 細胞系 列。11 位是屬於標準群，5 位危險群和 3 位最高危險群。追蹤之平均時間是 12.1 個 月。19 位之中有 10 位之骨髓檢體，在誘 導期治療之後，仍然可以偵測到微量殘留 癌細胞之存在，這都是屬於 B 細胞系列之 患者。而從週邊血檢體，只有 6 位可偵測 到。這 6 位之骨髓，也都可以偵測到微量 殘留癌細胞之存在。有 4 位是只有骨髓有 微量殘留癌細胞，但是週邊血卻是偵測不 到的。這二群之骨髓微量殘留癌細胞之數 值，並沒有統計學上之差異。骨髓與周邊 血之微量殘留癌細胞之數值變異頗大。週 邊血之數值，一般是遠比骨髓要低，最大 者，可有 300 倍之差別存在。由於本實驗 之個案數目相當有限，目前我們還無法下 一個最後之定論。但是，我們推論，B 細 胞系列的微量殘留癌細胞追蹤，目前仍無 細胞之存在，或許可能代表疾病之侵略性 較強。因為 T 細胞細之個案數只有 1 例， 我們還不能下任何結論。我們需要更多的 個案以及較長的追蹤期，才能證明用週邊 血取代骨髓（B 以及 T 細胞系列），來偵 測兒童及性淋巴性白血病之微量殘留癌細 胞追蹤之可行性。 關鍵詞：急性淋巴性白血病、微量殘留癌 細胞、流式細胞儀、週邊血液 Abstract

The level of minimal residual disease (MRD) during clinical remission is one of the most powerful prognostic factors in childhood acute lymphoblastic leukemia (ALL). In this prospective study, we evaluate whether the bone marrow sampling can be replaced by peripheral blood sampling. We used flow cytometry technique to compare MRD measurements in 19 pairs of bone marrow and peripheral blood samples collected at the end of remission induction therapy in 19 newly diagnosed ALL children. MRD was detected in both marrow and peripheral blood samples in 6 pairs, and in marrow but not in peripheral blood in 4 pairs. In B-lineage ALL, 6 out of 10 positive marrow samples had a corresponding positive peripheral blood samples. The MRD levels in bone marrow and peripheral blood varied significantly with MRD in bone marrow being up to 300 times higher than in the corresponding peripheral blood samples. The MRD levels did not have a significant difference between group of double-positive pairs and group of positive MRD in bone

cannot be replaced by peripheral blood for MRD analysis in childhood B-lineage ALL. Larger scale studies are warranted to answer its usefulness in childhood T-ALL.

Keywords: Childhood acute lymphoblastic

leukemia, Minimal residual disease, flow cytometry. Peripheral blood

Introduction

Monitoring of minimal residual disease (MRD) has become one of the strongest prognostic factors about childhood acute lymphocytic leukemia (ALL)1. Among the various available techniques for MRD detection in ALL, flow cytometric detection of aberrant immunophenotypes has been considered as the most sensitive and reproducible methods2-5. Sequential monitoring of MRD during treatment can be used to tailor the intensity of the therapy during treatment. However, the frequency of monitoring MRD is limited by the discomfort accompanied with the procedure of bone marrow aspirate in children. A few investigators have recognized the potential of using peripheral blood instead of bone marrow for MRD assays.

It was noted that the detection of MRD in peripheral blood samples paralleled those in bone marrow samples, but the levels in peripheral blood were much lower than those measured in paired bone marrow samples6. But, only a few studies have simultaneously examined paired peripheral blood and bone marrow samples using flow cytometry method, which can identify one leukemic cell out of 104 normal nucleated cells. So, we prompt a hypothesis that MRD studies using peripheral blood samples could also be helpful and informative as those in bone marrow. So, we conducted a prospective study to compare the MRD results by flow cytometry in paired peripheral blood and bone marrow samples at the end of remission induction of childhood ALL.

Materials and Methods

Patients

All patients who were newly diagnosed as ALL according FAB criteria from June 1, 2002 to August 31, 2003, stuck to the treatment protocols of TPOG and had useful MRD markers were enrolled in this prospective study. Bone marrow and peripheral blood samples were collected at diagnosis, the end of remission according to the protocol used. Mononuclear cells (MNC) were separated by centrifugation by a density gradient. Cells were collected in preservative-free heparin. All samples were processed within 4 hours.

Flow Cytometry

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 5.1 (PC5). Matched nonreactive fluorochrome-conjugated antibodies served as controls. For each case, marker combinations with the sensitivity of one leukemic cell per 104 normal nucleated bone marrow or peripheral blood cells or greater were selected at diagnosis and then applied for subsequent MRD monitoring. We used a 3-color FACSort flow cytometer equipped with CellQuest software (Becton Dickinson, San Jose, CA). The staining procedure used for MRD detection has been described elsewhere 7. 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 ). Detective MRD was defined as 0.01% or more cells expressing a leukemic-associated immunophenotype among mononuclear cells in the sample.

Exact X2 analysis and Fisher exact test were used to compare the difference in the distribution of presenting features at the end of remission induction therapy according to the presence of MRD in the peripheral blood. The cut-off date for follow-up observation was on October 15, 2003.

Results

Total 19 cases of newly diagnosed childhood ALL were eligibly enrolled in this study including 10 boys and 9 girls; 18 B-lineage ALL and 1 T-cell ALL. 11 patients were stratified to standard risk group, 5 to high-risk group and 3 to very-high-risk group protocol according to the criteria of TPOG guideline. The median age of diagnosis was 4.5 years with a range of 1.1 to 12.7 years. The median follow-up duration was 12.1 months (ranging, 1.9 to 16.2 months). 2 patients had their initial white blood cell count more than 100,000/ul at diagnosis and 2 between 50,000/ul and 100,000/ul and 15 were less than 50,000/ul.

10 out of 19 follow-up bone marrow samples were noted to have detectable MRD (52.6%), while only 6 out of 19 peripheral blood samples were MRD negative (31.6%). Of the corresponding 10 peripheral blood samples, 6 (60%) contained 0.01% or more cells with the leukemic phenotype. But, the remaining 4 patients who had detectable residual disease in bone marrow samples were MRD negative in peripheral blood samples. Levels of residual disease in bone marrow ranged from 0.03% to 22.47% (median 1.875%). In the peripheral blood, the MRD levels ranged from 0.02% to 2.09% (median 0.04%). 9 bone marrow samples were MRD-negative and all their paired peripheral blood samples were MRD-negative too.

The presence of MRD in the blood was generally associated with higher levels of MRD in bone marrow. The 6 patients who

0.041%-22.47%), while the 4 patients who had only MRD in the marrow had a median of 0.34% MRD (range, 0.027%-0.597%). The levels of bone marrow MRD to peripheral blood MRD had a median of 82.27 fold high in bone marrow (range, 2.28-317).

Discussion

B-lineage ALL often had no detectable MRD in the peripheral blood, despite their presence in the bone marrow. Our result is consistent with previous studies of MRD with peripheral blood6;9;10. Brisco et al claimed that the levels of MRD in blood were lower than those in bone marrow by a factor of 10 with the method of polymerase chain reaction amplification of immunoglobulin gene6. Velden VHJ et al found that the MRD levels in paired bone marrow and peripheral blood were very comparable and strongly correlated9.

In B-lineage ALL, the MRD levels in bone marrow and peripheral blood samples varied significantly with MRD levels in bone marrow being up to 300 times higher than in the corresponding peripheral blood. The MRD levels in bone marrow did not have a significant difference between samples of double-positive pairs and samples with positive MRD in bone marrow but negative in peripheral blood. Our study only had one case with T-ALL and it showed paralleled result in the bone marrow and peripheral blood (both undetected). But, in other’s studies, the MRD from the bone marrow samples might be replaced by the peripheral blood sampling for MRD monitoring in T-ALL 9;10. We need larger scale study to prove its usefulness in childhood T-ALL.

In conclusion, previous studies, taken together with our small-scale study results, indicate that for B-lineage ALL, levels of MRD in bone marrow are usually significantly higher than those in the

aggressive nature of the disease. Although the peripheral blood might not be used to monitor MRD in patients with B-lineage ALL, they might provide important information of relapse. Bone marrow sampling so far cannot be replaced by peripheral blood for MRD analysis in childhood B-lineage ALL. Larger scale studies are necessary to evaluate the usefulness of using peripheral blood to monitor the MRD in childhood ALL.

References

1. Pui CH, Campana D, Evans WE: Childhood acute lymphoblastic leukaemia--current status and future perspectives. Lancet Oncology 2: 597-607, 2001

2. Campana D, Pui CH: Detection of minimal residual disease in acute leukemia: methodologic advances and clinical significance. Blood 85: 1416-1434, 1995 3. 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

4. Campana D, Coustan-Smith E: Detection of minimal residual disease in acute leukemia by flow cytometry. Cytometry 38: 139-152, 1999

5. Coustan-Smith E, Sancho J, Hancock ML, Boyett JM, Behm FG, Raimondi SC, Sandlund JT, Rivera GK, Rubnitz JE, Ribeiro RC, Pui CH, Campana D: Clinical importance of minimal residual disease in childhood acute lymphoblastic leukemia. Blood 96: 2691-2696, 2000

6. Brisco MJ, Sykes PJ, Hughes E, Dolman G, Neoh SH, Peng LM, Toogood I, Morley AA: Monitoring minimal residual disease in peripheral blood in B-lineage acute lymphoblastic leukaemia. British Journal of Haematology 99: 314-319, 1997

7. Campana D, Coustan-Smith E: Detection of minimal residual disease in acute leukemia by flow cytometry. Cytometry 38: 139-152, 1999

8. Sievers EL, Radich JP: Detection of minimal residual disease in acute leukemia. Current Opinion in Hematology 7: 212-216, 2000

9. van dV, V, Jacobs DC, Wijkhuijs AJ, Comans-Bitter WM, Willemse MJ, Hahlen K, Kamps WA, van Wering ER, van Dongen JJ: Minimal residual disease levels in bone marrow and peripheral blood are comparable in children with T cell acute lymphoblastic leukemia (ALL), but not in precursor-B-ALL. Leukemia 16: 1432-1436, 2002

10. Coustan-Smith E, Sancho J, Hancock ML, Razzouk BI, Ribeiro RC, Rivera GK, Rubnitz JE, Sandlund JT, Pui CH, Campana D: Use of peripheral blood instead of bone marrow to monitor residual disease in children with acute lymphoblastic leukemia. Blood 100: 2399-2402, 2002 【計畫成果自評】 本研究和原提計劃內容相符，進度洽 當。本實驗使用流式細胞儀之檢驗方法， 方便又快速，加上之前歷年之計畫，本實 驗室已建立了良好、穩定之基礎。雖然只 是短短之一年多，我們已經收集了 19 例新 診斷個案。我們的初步結論是：用流式細 胞儀的方法，在 B 細胞系列，週邊血並無 法取代骨髓液，來偵測微量殘留癌細胞。 在 T 細胞系列方面，因為個案數只有一 位，所以無法下任何結論。最後，本研究 尚須要更多的個案，以及時間的追蹤，才 可以得到最後之答案，嘉惠病童，以及論 文發表。