Molecular remission in Philadelphia-positive adult acute lymphoblastic leukaemia rapidly induced by conventional-dose chemotherapy. JAE Irving1, R Finney2, ...
Bone Marrow Transplantation, (1998) 21, 323–325 1998 Stockton Press All rights reserved 0268–3369/98 $12.00
Case report Molecular remission in Philadelphia-positive adult acute lymphoblastic leukaemia rapidly induced by conventional-dose chemotherapy JAE Irving1, R Finney2, A Lennard1 and SJ Proctor1 1
Department of Haematology, Royal Victoria Infirmary, Newcastle upon Tyne; and 2Department of Haematology, North Tees General Hospital, Stockton-on-Tees, Cleveland, UK
Summary:
Case report
This report describes a patient presenting with Ph-positive ALL. RT-PCR analysis of diagnostic marrow revealed the presence of three bcr/abl transcripts; the ALL type e1a2 along with the CML types, b2a2 and b3a2. After initial induction therapy, bcr/abl transcripts were only detectable after two rounds of PCR but after MIDAC consolidation, remission samples were tworound negative. The relationship between the unusual molecular biological profile of this leukaemia and the rapid attainment of molecular remission is discussed. Keywords: Ph+ ALL; molecular remission; bcr/abl transcripts; RT-PCR
A white male aged 43 years presented with a haemoglobin of 7.2 g, white count 14.9 × 109/l, platelets 51 × 109/l. Morphologically 75% of the peripheral blood cells were lymphoblasts. They expressed CD34, CD19, CD10 and HLADR. Cytogenetic analysis demonstrated the Philadelphia chromosome. He began treatment on the induction protocol shown in Figure 1. During the first month he was given the induction schedule according to the Regional Protocol (NEALL III)5 utilising vincristine, cytosine arabinoside, prednisolone and idarubicin with intrathecal methotrexate. At the end of this initial sequence the patient was in morphological remission and proceeded to consolidation with the use of mitozantrone and high-dose cytosine. During this period it was noted that he had an HLA-identical family donor. Following this consolidation period it was noted that haematological remission was sustained and molecular remission had been achieved. Since the patient had an identical sibling donor, it was not necessary to proceed to the experimental auto-transplant sequence associated with stem cell harvest so further consolidation therapy using a modified FLAG regimen was given (Figure 1). Following this consolidation, 6 months after presentation, the patient underwent allogeneic bone marrow transplant from his brother utilising high-dose melphalan and total body irradiation as pre-conditioning. The patient suffered quite severe graft-versus-host disease (GVHD) (clinical grade III) and had a number of infective complications but after 12 months the GVHD had become quiescent and the patient was in sustained haematological and molecular remission.
It is now well recognised that adult acute lymphoblastic leukaemia (ALL) is a heterogenous group of diseases biologically and clinically.1 The different molecular genetic defects are indicators of the nature of the disease in a given individual and provide information of prognostic significance.2 Ph-positive ALL carries the worst clinical prognosis independent of age and the suggestion has been made that this occurs due to involvement of the stem cell compartment.3,4 We believe that entities which involve the stem cell compartment may not usually be chemo-curable because of the capacity of cells to stay in Go, thus avoiding the effects of combination chemotherapy. In such diseases, including Ph-positive ALL, allogeneic transplant from a matched sibling donor or from a matched unrelated donor are the only treatments that prolonged event-free survival associated with molecular remission. Our group has recently begun a pilot study of a new protocol aimed at early peripheral stem cell harvesting, providing material for double autologous transplantation as an alternative therapy for those who do not have a donor. In this report we describe the second patient entering the pilot protocol, whose disease had an interesting molecular biological profile and showed a complete molecular remission on the induction chemotherapy.
Correspondence: JAE Irving, Department of Haematology, Royal Victoria Infirmary, Newcastle upon Tyne, NE1 4LP, UK Received 1 July 1997; accepted 25 September 1997
Molecular investigations Diagnostic bone marrow aspirates were analysed by reverse transcriptase multiplex PCR for bcr/abl gene products.6 Three transcripts were expressed: the ALL type e1a2 along with the CML types, b2a2 and b3a2 (Figure 2a), the latter being the predominant form. This was confirmed by PCR with separate amplimers for major and minor transcripts (Figure 2b). In our experience of using one-round PCR methods to detect bcr/abl transcripts in a panel of adult ALL, 14 of 77 patients were PCR positive: nine expressed e1a2 transcripts, four expressed b2a2 or b3a2 transcripts,
Molecular remission in Ph+ ALL JAE Irving et al
324
Week
Month I 1
Prednisolone – oral 60 mg
Vincristine – i.v. 1.4 mg/m2 (max 2 mg)
2 Oral
3
Month II 4
Month III
MIDAC Cytosine 1 g twice a day/days 1–4 Mitozantrone 10 mg/m2/days 1–4
FLAG G-CSF 300 µ g i.v. over 30 min Days 1–5 (incl) Fludarabine 30 mg/m2/days 1–5 over 30 min then 4 h later Cytosine arabinoside 2 g/m2 over 4 h
21 day recovery period
21 day recovery period
Idarubicin 12 mg/m2 Cytosine arabinoside – i.v. 100 mg/m2 Methotrexate (10 mg) Cytogenetics
Ph+ve
bcr/ abl PCR
Figure 1 The induction chemotherapy consisted of NE ALL III therapy.5 Consolidation in month II was MIDAC following which molecular remission was achieved and sustained after FLAG chemotherapy in Month III. Subsequently, Month IV was treatment-free and during Month V allogeneic transplant was performed utilising melphalan 3 mg/kg × 1 and TBI 1200 cGy (six fractions) pre-conditioning. PCR was negative from the end of Month II and remains negative 12 months post-transplant. (d) 1-round positive; (g) 1-round negative, 2-round positive; (s) 2-round negative.
but only one patient, described in this report, expressed all three forms (unpublished observations). Minimal residual disease was assessed in remission samples initially by the one-round multiplex PCR and if this proved negative was then further analysed by a tworound nested PCR method.7 Positive controls for one- and two-round PCR were 1% K562 or 1% SD-1 cells and 1 in 105 K562 or 1 in 104 SD-1 cells, respectively, in a background of MOLT-4 cells, and were present at each stage of the processing. Appropriate negative controls were used in all PCR assays and expression of bcr and abl genes controlled for RNA and cDNA integrity in patient samples as described in the original methods. For aspirates taken after the initial remission induction sequence bcr/abl transcripts were not detectable after one round of PCR. However, after nested PCR both major transcripts were still in evidence. Further samples taken after consolidation therapy with MIDAC and FLAG were molecularly negative for all bcr/abl transcripts after nested PCR. Samples taken post-allogeneic transplant have remained negative.
Discussion The outcome of Philadelphia-positive acute lymphoblastic leukaemia on conventional chemotherapy is very poor. For those patients who achieve haematological remission, minimal residual disease is usually detectable by PCR. In our experience, bcr/abl transcripts are invariably detected after only one round of PCR and molecular remission is achieved only after BMT. Surprisingly, the patient presented here attained one-round negativity after initial induction treatment and became two-round negative after MIDAC consolidation chemotherapy. Although PCR negativity has been documented after conventional chemotherapy,8,9 to our knowledge our case is unique in the speed at which
molecular remission was achieved, ie within 8 weeks of diagnosis. Co-expression of major and minor transcripts may be explained by the presence of a second Philadelphia translocation with a breakpoint in the mBCR but which is cytogenetically cryptic or masked. Sample availability prohibited further characterisation by Southern blotting or FISH. However, the most likely explanation is the presence of a single breakpoint downstream of the b3 exon with the three transcripts arising by alternative splicing of the primary transcript. In the largest series of ALL examined, only one out of 90 bcr/abl-positive samples expressed all three bcr/abl transcripts.10 However, it has been recently demonstrated that most p210 ALL and indeed CML express minor transcripts to a varying degree, but are more usually detected after two rounds of PCR.11,12 The significance of more abundant expression of minor transcripts in p210 ALL is not known. It is possible that in this instance, achieving molecular remission may be related more to the unusual biological features of this form of leukaemia rather than the particular chemotherapy used. Patients who can achieve molecular remission on standard chemotherapy may provide complete Ph-negative CD34+ progenitors and in the absence of a donor may perform well following autologous transplant (marrow or PBSC). It now appears that the Ph-positive ALL is itself a heterogenous disorder and outcome varies according to additional cytogenetic abnormalities present at diagnosis.13 The present case represents a Ph-positive ALL variant which may have the potential for chemo-curability. This may reflect biological disease variation rather than treatment strategy but detailed analyses of populations of such cases is warranted when assessing the impact of new therapies.
Molecular remission in Ph+ ALL JAE Irving et al
References
a 1
2
3
4
5
6
Ela2 b3a2 b2a2
b 1
2
3
4
5
6
7
8
Ela2 b3a2 b2a2
Figure 2 (a) Analysis of bcr/abl mRNA transcripts after multiplex PCR with amplimers for bcr, major bcr/abl and minor bcr/abl transcripts. Lane 1, dilute positive control (1% K562); lane 2, patient diagnostic sample; lane 3, dilute positive control (1% SD-1); lane 4, negative control; lane 5, base pair markers (rBR322 AluI); lane 6, base pair markers (fX174 HaeIII). (b) Analysis of bcr/abl mRNA transcripts after PCR with amplimers for major bcr/abl transcripts (lanes 2–4) and minor bcr/abl transcripts (lanes 6–8). Lane 1, base pair markers (rBR322 AluI); lane 2, dilute positive control (1% K562); lane 3, patient diagnostic sample; lane 4, negative control; lane 5, base pair markers (fX174 HaeIII); lane 6, dilute positive control (1% SD-1); lane 7, patient diagnostic sample; lane 8, negative control.
Acknowledgements We would like to thank J Wilkinson for expert secretarial assistance. JAE Irving is supported by the Tyneside Leukaemia Research Fund.
1 Proctor SJ. Acute lymphoblastic leukaemia in adults: the case for a strategic shift in study approach. Br J Haematol 1994; 88: 229–233. 2 Secker-Walker LM, Prentice HG, Durrant J et al. Cytogenetics adds independent prognostic information in adults with acute lymphoblastic leukaemia on MRC trial UKALL XA. Br J Haematol 1997; 96: 601–610. 3 Jasmin C, Charpentier A, Marion S, Proctor SJ. Concepts of remission, curability and lineage involvement in relationship ` to the problems of minimal residual leukaemia. In: Bailliere’s Clin Haematol 1991; 4: 599–607. 4 Greaves MF. Stem cell origins of leukaemia and curability. Br J Cancer 1993; 67: 413–423. 5 Proctor SJ, Hamilton PJ, Taylor P et al. A comparative study of combination chemotherapy versus marrow transplant in first remission in adult acute lymphoblastic leukaemia. Br J Haematol 1988; 69: 35–39. 6 Cross NCP, Melo JV, Feng L, Goldman JM. An optimized multiplex polymerase chain reaction (PCR) for detection of bcr/abl fusion mRNAs in haematological disorders. Leukemia 1994; 8: 186–189. 7 Cross NCP, Feng L, Bungey J, Goldman JM. Minimal residual disease after bone marrow transplant for chronic myeloid leukaemia detected by the polymerase chain reaction. Leuk Lymphoma 1993; 11 (Suppl. 1): 39–43. 8 Mitterbauer G, Fodinger M, Scherrer R et al. PCR-monitoring of minimal residual leukaemia after conventional chemotherapy and bone marrow transplantation in BCR-ABL-positive acute lymphoblastic leukaemia. Br J Haematol 1995; 89: 937–941. 9 Preudhomme C, Henic N, Cazin B et al. Good correlation between RT-PCR analysis and relapse in Philadelphia (Ph-1)positive acute lymphoblastic leukaemia (ALL). Leukemia 1997; 11: 294–298. 10 Maurer J, Janssen JWG, Thiel E et al. Detection of chimeric BCR-ABL genes in acute lymphoblastic leukaemia by the polymerase chain reaction. Lancet 1991; 337: 1055–1058. 11 Van Rhee F, Hochhaus A, Feng L et al. p190 BCR-ABL mRNA is expressed at low levels in p210-positive chronic myeloid and acute lymphoblastic leukaemias. Blood 1996; 87: 5213–5217. 12 Saglio G, Pane F, Gottardi E et al. Consistent amounts of acute leukemia-associated p190BCR/ABL transcripts are expressed by chronic myelogenous leukemia patients at diagnosis. Blood 1996; 87: 1075–1080. 13 Rieder H, Ludwig WD, Gassman W et al. Prognostic significance of additional chromosome abnormalities in adult patients with Philadelphia chromosome positive acute lymphoblastic leukaemia. Br J Haematol 1996; 95: 678–691.
325
