Treatment of Leukemia in Patients with t(8;21), t(15;17) or t(9;22)

107

An unrelated Clone of 20q Deletion Following Successful

Treatment of Leukemia in Patients with t(8;21), t(15;17) or t(9;22)

Chorong Hahm

, Yeung Chul Mun

, Chu Myong Seong

, Wha Soon Chung

, and Jungwon Huh

Department of Laboratory Medicine

, Department of Internal Medicine

,

Ewha Womans University School of Medicine, Seoul, Korea

t(8;21), t(15;17) 또는 t(9;22)를 동반한 백혈병 환자에서 성공적인 치료 후에 무관성 클론으로 발생한 20번 염색체 장완 결손

함초롱

․문영철

․성주명

․정화순

․허정원

이화여자대학교 의학전문대학원 진단검사의학과

, 내과

접수일: 12 / 7 / 30

최종재심접수일: 12 / 10 / 23 게재승인일: 12 / 10 / 29 교신저자：허정원

우)158-710 서울시양천구안양천로1071, 이화여자대학교 의과대학 부속 목동병원 진단검사의학과

전화 : 02)2650-5287 FAX : 02)2650-5091

E-mail : [email protected]

Cases of clonal cytogenetic abnormalities in Philadelphia-negative cells during the treatment of Philadelphia-positive CML have been previously reported. However, clonal abnormalities unrelated to the original t(8;21) or t(15;17) karyotype are not common. Deletion of 20q (del(20q)) is one of the most common recurrent cytogenetic abnormalities in myeloid neoplasms. Here we describe 3 patients with t(8;21), t(15;17), or t(9;22) who developed unrelated del(20q) after successful treatment of leukemia.

We retrospectively reviewed the cytogenetic results of 23 AML patients with t(8;21)(q22;q22), 28 AML patients with t(15;17)(q22;q12), and 47 CML patients with t(9;22)(q34;q11.2). We identified 3 patients with del(20q) as the only clonal aberration unrelated to the primary karyotype when they achieved complete morphologic and cytogenetic remission. The latency period between diagnosis and emergence of del(20q) was 1, 114, and 35 months for the 3 patients, respectively. There was no evidence of therapy-related MDS/AML during the follow-up period. In 1 AML patient with t(8;21), relapse occurred in a t(8;21)(q22;q22) clone and the del(20q) clones were lost. The clinical significance of del(20q) as an unrelated clonal aberration is unknown, but our study suggests that del(20q) does not cause therapy-related MDS/AML or indicate disease progression.

Key Words: Chromosome deletion, Chromosome 20, Unrelated, t(15;17), t(8;21), t(9;22)

INTRODUCTION

Deletion of chromosome 20q, del(20q), is one of the most common recurrent cytogenetic ab- normalities in a variety of myeloid neoplasms such as MDS, AML, and myeloproliferative neoplasm [1].

The del(20q) cytogenetic abnormality may confer a proliferative advantage on the myeloid cells through

deletion of a tumor suppressor gene, but the underlying mechanism of action by which del(20q) affects hematopoietic stem cells remains unresolved [1].

In MDS, del(20q) alone is associated with good prognosis with regard to survival and potential for AML evolution, as defined by the International Prognostic Scoring System (IPSS) for MDS prog-

J Lab Med Qual Assur 2012 ; 34：107-11

nosis. However, in de novo acute leukemia, poor response to treatment and reduced survival are seen [1]. In myeloproliferative disorders, the presence of del(20q) does not appear to adversely affect survival, although it is considered a potential cytogenetic marker for clonal evolution in chronic myelogenous leukemia (CML) [2].

The occurrence of clonal abnormalities unrelated to the original primary karyotype is rare. Some cases with clonal cytogenetic abnormalities in the Philadelphia chromosome (Ph)-negative cells during treatment of Ph-positive CML have been reported [2-4]. Del(20q) has been reported in the Ph-negative cells of CML patients with cytogenetic and morphologic remission [2]. On the other hand, unrelated clonal abnormalities following remission in patients with t(8;21) or t(15;17) is a rare occurrence whose clinical significance remains to be clarified.

Here, we describe and study 3 patients who acquired del(20q) as a clonal aberration unrelated to the primary karyotype after successful treatment.

CASE REPORTS

We retrospectively reviewed the cytogenetic data of 23 AML patients with t(8;21)(q22;q22), 28 AML patients with t(15;17)(q22;q12), and 47 CML patients with t(9;22) between 1996 and 2011 and identified 3 cases with a del(20q) locus as the only clonal aberration unrelated to the primary karyotype during follow-up. Conventional meta- phase cytogenetic analysis (MC) was performed on unstimulated 24- and 48-hour cultures using fresh bone marrow aspirates obtained at diagnosis. When possible, at least 20 metaphases per sample were analyzed and karyotypes were described according to the International System for Human Cytogenetic Nomenclature (2009) [5].

Fluorescence in situ hybridization (FISH) was performed using commercially available probes (Abbott/Vysis, Downers Grove, IL, USA): BCR/

ABL1 dual color, dual fusion translocation probe;

RUNX1/RUNX1T1 dual color, dual fusion translocation probe; PML/RARA dual color, dual fusion translocation probe; D20S108 (20q12) Spectrum Orange probe. At least 200 interphase cells were scored for each probe by 2 independent

experienced examiners.

Quantitative real‐time reverse transcriptase- PCR for RUNX/RUNXT1, PML-RARA, and BCR- ABL1 translocation was performed using a commercially available kit (FusionQuant kit;

Ipsogen, Marseilles, France).

Detailed follow-up data of each patient is shown in Table 1.

Case 1

Case 1 was of an 18-year-old male patient diagnosed with AML with t(8;21)(q22;q22) who received standard induction chemotherapy (dauno- rubicin 60 mg/m

× 3 days and Ara-C 200 mg/m

× 7 days). This patient achieved complete response (CR). At diagnosis, MC showed t(8;21) in 95% of metaphase cells and FISH analysis with the D20S108 probe yielded equivocal results: del(20q) was detected in 2.3% of interphase cells (cutoff:

2.5%). However, 1 month later, t(8;21) was not detected on MC, but del(20)(q13.1) was seen in 30% of the metaphase cells. Quantitative PCR with RUNX1/RUNX1T1 showed positive results and FISH analysis with the D20S108 probe confirmed the presence of del(20q) in 4% of the interphase cells. Two cycles of consolidation chemotherapy were administered followed by autologous peripheral blood stem cell transplantation. A relapse of the t(8;21)(q22;q22) clone was seen after 9 months of the initial diagnosis. However, the del(20q) clones disappeared prior to the relapse. The patient then received re‐induction chemotherapy with the FLAG regimen (fludarabine [30 mg/m

× 5 days], high‐

dose cytarabine [2 g/m

× 5 days], and granulocyte colony-stimulating factor) followed by allogeneic peripheral blood stem cell transplantation. At the last follow‐up visit (14 months), another relapse was seen with the clonal evolution of the t(8;21)(q22;q22) cell line with del(5q) and del(7q), and the patient finally died because of complications from severe pneumonia.

Case 2

Case 2 was of a 66-year-old male patient diagnosed as having AML with t(15;17)(q22;q12).

The patient was treated with tretinoin (45 mg/m

Table 1. Characteristics of patients with a deletion of chromosome 20q as a clone unrelated to their primary karyotype following successful treatment

No. Case Age Sex Month Bone marrow

study Cytogenetics FISH PCR

1 18 M 000 AML 46,XY,t(8;21)(q22;q22)[19]/46,XY

[1]

RUNX1/RUNX1T1 : 95%

del(20q): 2.3%

(cutoff: 2.5%) 001 Hypercellular

marrow 46,XY,del(20)(q13.1)[6]/46,XY[14] del(20q): 4% RUNX1/RUNX1T1 : Positive (3.97)*

006 Hypocellular

marrow 46,XY[20] del(20q): 0% RUNX1/RUNX1T1 :

Positive (1.20)*

009 Relapse 46,XY,t(8;21)(q22;q22)[16]/46,XY

[4] RUNX1/RUNX1T1 :

Positive (146.9)*

010 Hypocellular

marrow 46,XY,t(8;21)(q22;q22)[1]/46,XY

[19] RUNX1/RUNX1T1

: 2.8% RUNX1/RUNX1T1 :

Positive (11.7)*

012 Hypocellular

marrow 46,XY[20] RUNX1/RUNX1T1

: 0% RUNX1/RUNX1T1 : Positive (0.12)*

014 Relapse 46,XY,del(5)(q31~q32),

t(8;21)(q22;q22)[6]/46,idem,del(7) (q22)[6]/46,XY[8]

2 66 M 000 AML 46,XY,t(15;17)(q22;q12-21)[7]

/47,XY,idem,+mar[13]

002 Hematologic

remission 46,XY[20]

021 Hematologic

remission 46,XY[14]

033 Hematologic

remission 46,XY[20] PML/RARA : Negative

065 Hematologic

remission 46,XY[20] PML/RARA : Negative

077 Hematologic

remission 46,XY[20] PML/RARA : Negative

114 Hematologic

remission 46,XY,del(20)(q12)[8]/46,XY[12] PML/RARA : Negative

120 Hematologic

remission 46,XY,del(20)(q12)[11]/46,XY[9] del(20q): 42% PML/RARA : Negative

3 34 F 000 CML,

chronic phase 46,XX,t(9;22)(q34;q11.2)[25] BCR/ABL1 : 98.5% BCR/ABL1 : Positive b3a2

008 Hematologic

response 46,XX[20]

022 Hematologic

response 46,XX[20]

035 Hematologic

response 46,XX,del(20)(q11.2)[11]/46,XX[9] BCR/ABL1 : Negative

* The quantity of RUNX1/RUNX1T1 rearrangement is expressed as a normalized ratio of RUNX1/RUNX1T1/ABL1.

daily) combined with the standard induction chemotherapy, which comprised daunorubicin (60 mg/m

× 3 days) and cytarabine (200 mg/m

× 7 days). Three cycles of consolidation chemotherapy were also administered, and the patient maintained

a continued CR. However, del(20q) was detected in

40% of the metaphase cells at 114 months after

diagnosis. The t(15;17) was not detected on MC,

and the results of the quantitative PCR for the

PML-RARA rearrangement were negative. Further,

a bone marrow analysis did not show evidence of relapse. At the last follow-up visit (120 months), del(20q) was still seen in 55% of the metaphase cells and was confirmed by FISH analysis in 42% of the interphase cells. Complete blood cell counts (CBC) were as follows: Hb, 12.1 g/dL; platelet count, 107,000/µL; and leukocyte count, 3,490/µL (63% segmented neutrophils, 25% lymphocytes, 10% monocytes, and 2% atypical lymphocytes).

Case 3

Our case 3 patient was a 35-year-old female patient diagnosed as having CML with t(9;22)(q34;q11.2). The patient received 400 mg imatinib treatment and showed a continued com- plete cytogenetic response. Thirty-five months later, 55% of the metaphase cells on MC contained a del(20q) in the Ph chromosome-negative cells. The bone marrow samples at this juncture did not show evidence of AML or MDS. The BCR/ABL1 transcript was also not detected on PCR and CBC were as follows: Hb, 11.2 g/dL; platelet count, 250,000/µL;

and leukocyte count, 4,360/µL (62% segmented neutrophils, 30% lymphocytes, 4% monocytes, and 4% eosinophils).

DISCUSSION

In this study, we described 3 patients with AML or CML of t(8;21), t(15;17), or t(9;22) origin who acquired an unrelated del(20q) clonal abnormality after successful treatment of leukemia. The del(20q) was not associated with relapse of the original neoplasm or to therapy-related MDS/AML in any of these patients. Although there have been a few reports of del(20q) as an unrelated clonal ab- normality in patients with t(15;17) or t(9;22) during treatment, to our knowledge, this is the first report of on the occurrence of a clonal del(20q) after remission in an AML patient with t(8;21) [2-4, 6-8].

One study showed that 10% of the leukemias of t(15;17) origin developed a variety of unrelated clonal abnormalities including del(20q) [8], whereas 3.4% of the patients developed unrelated clonal abnormalities in the Ph-negative cells of Ph-positive CML patients during imatinib treatment [4]. In our

study, the incidence of the unrelated del(20q) clone was 4.3% (1/23) in AML patients with t(8;21)(q22;q22), 3.6% (1/28) in AML patients with t(15;17)(q22;q12), and 2.1% (1/47) in CML patients.

Previous reports have also found other chromosomal abnormalities of unrelated origin such as trisomy 8, monosomy 7, del(7q), del(5q), and less commonly, del(20q) in patients with t(9;22) or t(15;17) [2-4,6-8]. With regard to clinical sig- nificance, it is unlikely that an unrelated del(20q) clone after successful treatment implies the development of therapy-related MDS/AML or relapse of original disease [2-4,6-8]. During follow- up, the del(20q) karyotype was seen to spon- taneously revert to a normal karyotype [8] or the patient remained in CR without dysplasia in bone marrow cells [7]. Similar to these studies, there was no evidence of therapy-related MDS/AML in our study. Although relapse occurred in 1 AML patient with t(8;21), del(20q) clones were lost when the original t(8;21)(q22;q22) karyotype re-appeared.

The median latency period between diagnosis of AML with t(15;17) and emergence of the unrelated clone was reported to be 28–36 months [6,8]. In the present study, the interval between diagnosis of AML with t(15;17) and appearance of del(20q) clones was 114 months, whereas in a patient with t(8;21), the unrelated del(20q) clone was observed in 30% of metaphase cells within 1 month of the diagnosis. As for CML, a previous study reported del(20q) in Ph-negative cells in 50% of the patients after cytogenetic response and, less commonly, in 20% of patients before the response or in 30% of the patients at the time of the response [2]. The latency period between diagnosis and emergence of 20q deletion was 4 to 38 months [2]. In this case, the latency period between diagnosis and the emergence of del(20q) was 1, 114, and 35 months for AML with t(8;21)(q22;q22), AML with (15;17)(q22;q12), and CML with t(9;22)(q34;

q11.2), respectively.

Why these unrelated clonal abnormalities are

observed in patients during CR is a question that

remains to be answered. One theory is that an

initial “hit” to the hematopoietic stem cells might

have given rise to several abnormal clones, of

which, 1 may have dominated while the others remained undetectable. Once the dominant leuke- mia clone had been controlled or eradicated by treatment, the other clones may have become

“unmasked” and may have expanded, thus becoming detectable at the cytogenetic level [7]. This has been specifically suggested in the case of CML where imatinib treatment induced or favored the emergence of Ph‐negative clones via the involve- ment of the DNA damage repair pathways [2]. In our study, del(20q) clones appeared after imatinib therapy. It was also suggested that in CML, genomic instability leads to the production of multiple abnormal clones, including the clone with t(9;22) and other Ph-negative clones [2]. In other words, the imatinib-induced cytogenetic response could eliminate the proliferative advantage of the t(9;22) and unmask the Ph-negative clones. The pathogenesis and clinical significance of the unrelated clone to the original karyotype during drug response remains to be clarified.

In summary, our study shows that the del(20q) chromosomal aberration, unrelated to the original karyotype, could be observed following successful treatment of leukemia in patients with t(8;21), t(15;17) or t(9;22). The prognostic significance of del(20q) as an unrelated clone is unknown, but this study suggests that del(20q) does not seem to cause therapy-related MDS/AML nor does it serve as an indicator of disease progression.

요 약

필라델피아 염색체 양성 만성골수성백혈병의 치료 기간 동 안 필라델피아 염색체 음성인 세포에서 클론성의 세포유전학 적인 이상이 발생한다는 몇몇 보고가 있다. 그러나 t(8;21) 또는 t(15;17)의 염색체 이상을 가진 경우 무관성으로 클론 성 이상이 발생하는 경우는 흔하지 않다. 20번 염색체 장완 의 결손은 골수성 종양에서 가장 흔하게 관찰되는 세포유전 학적 염색체 이상이다. 저자들은 t(8;21), t(15;17) 또는 t(9;22) 염색체 이상을 보이는 백혈병의 성공적인 치료 후에 무관성 클론으로 20번 염색체 장완의 결손이 발생한 3명의 환자에 대해 기술하고자 한다. 저자들은 23명의 t(8;21) (q22;q22) 염색체 이상을 가진 급성골수성백혈병 환자와 28 명의 t(15;17)(q22;q12) 염색체 이상을 가진 급성골수 성백혈병 환자와 47명의 t(9;22) 염색체 이상을 가진 만성 골수성백혈병 환자들의 세포유전학적 결과를 후향적으로 검

토하였다. 3명의 환자에서 형태학적, 세포유전학적 완전 관 해 후에 20번 염색체 장완의 결손이 발생했으며, 진단을 받 고 20번 염색체 장완의 결손이 나타나기까지 잠복기는 각각 1, 114, 35개월이었다. 마지막 추적 관찰까지 치료와 연관 된 골수이형성증후군이나 급성골수성백혈병이 나타난 경우는 없었으며, t(8;21)를 보인 1명의 급성골수성백혈병 환자에 서만 20번 염색체 장완의 결손을 보이는 클론이 사라진 후 t(8;21) 염색체 이상이 다시 관찰되면서 재발이 발생하였다.

20번 염색체 장완의 결실을 보이는 무관성으로 발생한 클론 의 임상적 의의는 아직 확실하지 않으나, 이번 연구를 통해서 20번 염색체 장완의 결실은 치료와 연관된 골수이형성증후군 이나 급성골수성백혈병을 유발하지 않으며, 질병의 진행과는 무관한 것으로 생각된다.

REFERENCES

1. Huret JL. Atlas of genetics and cytogenetics in oncology and h a em ato lo g y . h ttp ://atlasg en eticso n co lo g y .o rg /A n o m a lies/

tri8ID1017.html (Accessed December 22, 2011).

2. Sun J, Yin CC, Cui W, Chen SS, Medeiros LJ, Lu G.

Chromosome 20q deletion: a recurrent cytogenetic abnormality in patients with chronic myelogenous leukemia in remission. Am J Clin Pathol 2011;135:391-7.

3. Kim M, Lee S, Jung CK, Lim J, Cho SG, Kim DW, et al.

Transient trisomy 8 abnormality in Philadelphia ‐negative cells during imatinib mesylate treatment of chronic myelogenous leukemia. Int J Lab Hematol 2008;30:508-2.

4. Terre C, Eclache V, Rousselot P, Imbert M, Charrin C, Gervais C, et al. Report of 34 patients with clonal chromosomal abnormalities in Philadelphia ‐negative cells during imatinib treatment of Philadelphia ‐positive chronic myeloid leukemia.

Leukemia 2004;18:1340-6.

5. Shaffer LG, Slovak ML, et al. eds. ISCN (2009): An International System for Human Cytogenetic Nomenclature.

Basel: Karger, 2009.

6. Mallo M, Salido M, Espinet B, Cervera J, Canellas A, Pajuelo JC, et al. Could ATRA/Idarubicin treatment of acute promyelocytic leukemia induce the appearance of new clonal cytogenetic abnormalities in patients in complete remission?

Leuk Res 2007;1315-7.

7. Lin Y, Bruyère H, Horsman DE, Pantzar T, Barnett MJ, Hogge DE, et al. Philadelphia ‐negative clonal hematopoiesis following imatinib therapy in patients with chronic myeloid leukemia: a report of nine cases and analysis of predictive factors. Cancer Genet Cytogenet 2006;170:16-23.

8. Batzios C, Hayes LA, He SZ, Quach H, McQuilten ZK, W all

M, et al. Secondary clonal cytogenetic abnormalities following

successful treatment of acute promyelocytic leukemia. Am J

Hematol 2009;84:715-9.