Blut (1990) 61:226-231

Blur

© Springer-Verlag 1990

Original article Interferon alfa-2c in chronic myelogenous leukemia (CML): Hematologic, cytogenetic and molecular-genetic response of patients with chronic phase CML previously resistant to therapy with interferon gamma F. Herrmann 1, D. Jonas I, S.G. Helfrich I, A. Lindemann I, E. Schleiermacher 2, and R. Mertelsmann 1 1 Department of Hematology and Oncology, University of Freiburg, Freiburg, Federal Republic of Germany 2 Institute for Anthropology, University of Mainz, Mainz, Federal Republic of Germany Received February 23, 1990/Accepted July 5, 1990

Summary. Alpha- and gamma-interferons have been shown to actively suppress hematopoiesis in patients in the chronic phase of chronic myelogenous leukemia in vitro and in vivo. Since both interferons act through different receptors on their hematopoietic target cells, they are expected to be capable of independently inhibiting abnormal blood cell development in patients with chronic myelogenous leukemia. We have utilized recombinant human interferon alfa-2c to treate 11 patients with Philadelphia chromosome positive chronic myelogenous leukemia in chronic phase, who were resistant to previous interferon gamma therapy. Ten of the patients were evaluable for hematologic, cytogenetic and molecular-genetic response following interferon alfa-2c therapy for 6 to 30 months. In 5 patients, IFN alfa-2c treatment failed due to lack of hematologic response. A complete hematologic or partial hematologic response was achieved in the remaining 5 patients. Three of these experienced cytogenetic improvement with reappearence of 100% diploid hematopoietic cells and disappearence of c-abl/bcr rearrangement in one patient. In two patients interferon alfa-2c did not prevent transformation of the disease into an accelerated state or blast crisis, respectively. We conclude that recombinant human interferon alfa-2c may also control hematopoiesis in interferon-gamma resistant chronic myelogenous leukemia patients, although the long-term response will need to be elucidated in further studies. Key words: CML - Phase II trial - IFN treatment

Introduction Chronic myelogenous leukemia (CML) is a leukemic disorder with a typical biphasic clinical course. The initial Offprint requests to: E Herrmann, Department of Hematology and Oncology, University of Freiburg, Hugstetter Strasse 55, W-7800 Freiburg, Federal Republic of Germany

chronic phase (CP) preceedes a rapidly evolving fatal phase referred to as blast crisis (BC). During the past decades there has been little improvement in the ability to therapeutically aker the course of the disease. The median survival time after diagnosis for untreated patients is 31 months, for those patients receiving high-dose combination chemotherapy 48-65 months, and 35-48 month for patients undergoing treatment with single agents such as hydroxyurea or busulfan [5]. Allogeneic bone marrow transplantation has become increasingly successful in the treatment of CP-CML [4], but is restricted to the availability of HLA-compatible donors and appropriate for a limited number of younger patients only. Recently, interferons (IFN) including natural IFN alpha [18], recombinant human (rhu) IFN alfa-2a [18], rh IFN alfa-2b [1, 2, 12] and rh IFN gamma [7, 10] have undergone extensive clinical trials and have produced encouraging results in the management of CML. The alfa-IFNs induced hematologic remissions in about 6 5 % - 7 0 % of early CP-CML and about 5 0 % - 7 0 % of the responders also experienced varying degrees of cytogenetic improvement [1,11, 18]. About 40% of CP-CML patients subjected to IFNgamma therapy attained a hematologic remission and a half of these showed a cytogenetic response [10]. Since alpha and gamma-IFNs exhibit synergistic antiproliferative effects [9], possibly related to their binding to distinct cell surface receptors [3], combination IFN therapy is expected to improve disease outcome. Furthermore, it seems also possible for patients who are resistant to IFN gamma to respond to IFN alpha, and vice versa. On the basis of a recent phase Ib clinical trial by the authors, utilizing IFN gamma to treat CP-CML [7], patients who responded to IFN gamma were subjected to combination therapy with IFN alpha, and those who were previously resistant to IFN gamma were treated with IFN alpha only. This report describes our experience with purified IFN alfa-2c, a recombinant human IFN alpha 2 subtype, in the treatment of previously IFN gamma resistant patients with Philadelphia chromosome (Ph') positive CPCML, evaluating toxicity, hematologic, cytogenetic and molecular-genetic changes associated with therapy.

E H e r r m a n n et al.: IFN alfa-2c in IFN g a m m a resistant CML

Materials and m e t h o d s Patients. Between December 1986 and December 1988 11 patients (six women and five men) with Ph' positive CP-CML entered the study. All patients were previously treated with subcutaneous rh IFN gamma for a median duration of 14 weeks (monthly escalating doses ranging from 0.1 to 0.5 m g / m 2 per day, 5 times weekly), but failed to show improvements in hematologic variables [7]. The median age of the patients was 45 years (range, 20-61). Bone marrow samples from all patients contained 100% Ph' positive metaphases. One patient had additional karyotype alterations. None of the patients had received antileukemic treatment for at least 4 weeks before entry into the study. Further eligibility criteria included: informed consent according to the guide-lines of the local ethics Committee, Karnofsky performance status of > 50%, life expectancy of at least 3 months, and preserved hepatic, renal and coagulation functions. The clinical characteristics of the patients enrolled in this study are detailed in Table 1. Interferon preparation. Recombinant h u m a n (rhu) IFN alfa-2c produced in Escherichia Coli by Boehringer Ingelheim, Vienna, Austria, was provided by Karl Thomae GmbH, Biberach, FRG, in the form of a sterile lyophilized powder. This material is > 98% pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contains < 0.5 ng endotoxin by the Limulus amebocyte lysate assay. The specific activity is 3.2 x 108 U/rag of protein. Study design. This was an uncontrolled open-label phase II clinical trial. The rh IFN alfa-2c (galenic code: IF-RC 1XXAAMT1B1A) was given daily by single subcutaneous injection; 5x106 U per square meter of body-surface area was the starting dose. When significant toxicity occured the dose was reduced by 50% and was reduced by a further 50% if toxicity continued. All patients who ex-

Table 1. Patient characteristics at enrollment Number of patients

11

Median age (range)

45 (20-61) years

Male/female

5/6

Median duration of disease (range)

17 (9-34) m o n t h

Median performance status (range) according to the Karnofsky index

80 (60-100) percent

Splenomegaly

10

Mean WBCs (range)

59 (15-258) × 103(ill)

Mean H b (range)

13.3 (11.3-15.1) g/d1

Mean platelet counts (range)

65.0 (19.5-123.0) X 104///1

Prior treatment IFN g a m m a

11

Other investigational drugs

4

Busulfan

3

Hydroxyurea

8

Other chemotherapy

1

Prognostic category according to Sokal et al. [15] Low risk

5

Intermediate risk

4

High risk

2

227 perienced hematologic remission continued to receive the IFN at half of the dose that they had received during the 3 m o n t h induction phase. Pretreatment evaluation of patients included physical examination, spleen and liver ultrasound, ECG, and chest X-ray, complete white blood counts (WBCs) with differential and platelet counts, neutrophil alkaline phosphatase (NAP), serum chemistry analyses including determinations of liver, renal and coagulation functions, measurement of uric acid, lactate dehydrogenase (LDH), and vitamin-B12 levels. Bone marrow aspirations were performed for morphological study, cytogenetic and molecular evaluation. WBCs, N A P and chemical analyses were repeated every other week; ECG, chest X-ray, spleen and liver ultrasound and bone marrow analyses were performed at intervals of 3 month. Physical examination, including assessment of performance status was performed weekly during the first 4 weeks and every 4 weeks thereafter.

Cytogenetic and molecular studies. Cytogenetic analysis was performed according to the Giemsa banding technique previously described [14]. At least 20 bone marrow metaphases per sample were examined. For detection of rearranged c-abl/bcr sequences, D N A (10//g) obtained from bone marrow specimens of CP-CML patients and h u m a n placenta were digested with various restriction endonucleases (Boehringer, Mannheim, FRG), electrophoresed on a 0.7% agarose gel, and hybridized to the 2.0-kb 5' bcr cDNA [6l as described [16]. Polymerase chain reaction (PCR) was essentially performed as described [8]. The cDNA was synthesized in 20//1 reaction volume using 200 U of murine leukemia virus reverse transcriptase in the presence of 20 U RNasin (Promega Biotec, Madison, WI) with 10 nmoles of each dNTP and 100 pmoles of random hexamer. After incubation for 1 h at 37°C, the reaction mixture was denatured by heating to 95°C for 5 min and immediately chilled on ice. Subsequently, the reaction mixture was diluted with 80//1 of PCR reaction buffer followed by addition of 5' and 3' primer (10 pmoles each), and 1 unit AmpliTaq rTaq D N A Polymerase (PerkinElmer, Cetus). Cycles of PCR consisted of denaturating by heating to 94°C for 30 s, annealing of primers at 55°C for 15 s and primer extension at 72°C for 30 s. This cycle was repeated 45 times by using a programmable heat block (Cetus, Emeryville, CA) with increments of the primer extension step (5 s/cycle). One tenth of each reaction mixture was run on composite gels containing 3% NuSieve (FMC Bioproducts, Rockland, ME) and 1% Agarose (BRL) and was blotted onto Zeta-Probe membranes (Bio-Rad) by alkaline transfer. The membranes were prehybrized with 0.9 M NaC1/0.05 M NaHzPO 4, pH 7.0, 5 times and were then treated with Denhardt's solution containing 2 0 0 / / g / m l denatured salmon sperm DNA, 10 mM EDTA, 1% SDS. This solution was replaced with the same containing 4 x 106 cpm of 5'-32p-labeled probing oligonucleotides, hybridizing to a ca. 200 bp product amplified from the joining region of the second c-abl exon to the third exon of the major breakpoint cluster region. After hybridization for 2 h at 60°C, blots were washed at the same temperature in 2.5 x SSC/0.1% SDS and exposed to Kodak XAR5 film with Dupont intensifying screen. Criteria for response. Hematologic and cytogenetic response was evaluated according to criteria of Talpaz et al. [18]. A complete hematologic remission (CHR) was defined as decline of peripheral white blood counts (WBC) to < 9 × 103 cells///l with normal differential, disappearence of clinical signs and symptoms of the disease including splenomegaly. A partial hematologic remission (PHR) was defined as decline of WBC by > 50% below 20x103 cells///1 a n d / o r persistence of splenomegaly. In patients who experienced less than a PHR, the treatment was considered to have failed (TF). A complete cytogenetic response (CCR) was considered to have been achieved when Ph' positive metaphases were completely eliminated. Persisting Ph' positive chromosomes in all analyzable rectaphases were defined as no cytogenetic response (NCR). Ph' suppression to levels of 5% to 34% was defined as partial cytogenetic response (PCR) and Ph' suppression to levels of 35% to 99% was considered m i n i m u m cytogenetic response (MCR).

F. Herrmann et al.: IFN alfa-2c in IFN gamma resistant CML

228

Table 2. Hematologic and cytogenetic response to IFN alfa-2c therapy

Results

Hematologic, cytogenetie, and molecular-genetic response Hematologic response Cytogenetic response O f 11 patients with C P - C M L entering this trial, ten patients were evaluable for hematologic, cytogenetic, and molecular-genetic response. One patient had to be removed from the study because of therapy-associated side effects (see below). The remaining 10 patients were treated with IFN alfa-2c for at least 6 months. After this time 4 patients had experienced C H R . Three of these belonged to the low-risk group according to the criteria of Sokal et al. [15]. A P H R was recorded in one additional patient. Five patients failed to respond to IFN alfa-2c (Table 2) and were therefore excluded from the study. They also failed to show any cytogenetic improvement at this time. Therapy failure was indicated by progressive leukocytosis (n = 3), and clonal evolution (n = 2). In one of the latter patients an additional Ph' chromosome and the appearence of an isochromosome 17q heralded an accelerated stage of the disease, clinically manifested by progressive splenomegaly, basophilia and anemia requiring transfusion of packed red blood cells. In the second patient blast crisis of myeloid phenotype developed. This patients showed trisomy 8. In patients who responded to IFN alfa-2c, treatment was maintained for at least 6 additional months (range, 6 - 2 4 months). Table 2 also illustrates the cytogenic response encountered in the group of patients experiencing hematologic remission. Following 12 m o n t h of treatment one patient each achieved CCR, PCR, and MCR. In two patients no cytogenetic response was recorded. In line with a previous report suggesting predominant in vivo sensitivity of megakaryopoiesis to IFN alpha inhibition [161, all 10 patients who were studied for at least 6 month, experienced normalization of previously increased platelet counts (Fig. 1). Serum levels of L D H and

120 105

CCR PCR MCR NCR

4

1

1

5

1

1

2b

a For abbreviations see "Materials and methods" b Five additional patients who failed to respond to IFN alfa-2c therapy also showed no cytogenetic improvement

vitamin-B12 that were increased in all patients (550 + 320 U/ml) and 4711 + 2836 pg/ml, respectively) returned to normal values only in the patients experiencing hematologic remission (Fig. 2). Molecular analysis to probe for rearranged c-abl/bcr sequences using a 5' bcr c D N A in the three patients who experienced cytogenetic improvement, demonstrated persistent rearrangements in the two patients with P C R and MCR. However, in the one patient with CCR, Southernblotting showed that c-abl/bcr rearrangement had disappeared 12 month after commencing IFN-alfa-2c treatment (Fig. 3 B). When investigating c-abl/bcr transcripts by means of the more sensitive P C R technique 45 cycles of amplification of the specimen of this patient yielded a strong 200 base-pair (bp) signal before IFN alfa-2c therapy. RNA from bone marrow cells of this patient 12 months after initiation of IFN alfa-2c yielded the same 200 bp PCR product, indicating fusion of the second c-abl exon to the third exon of the major breakpoint cluster region, but the signal was markedly weaker than before therapy (Fig. 3 A). This patient has now been under treatment for 30 months and is still in CCR.

10! 91

90

2" 75 ~2

7! 6~

60 45

4~

30

31

15

1!

0

Number of patients

CHRa PHR TF

P/CHR

TF

//'// //// ////

//// //// //// // // ////

////

//// //// Ii lI tIlI // // // // //// I///

5//~ ///¢/, ////

i////

P/CHR

P/CHR

[

//// ////

//// // ////// //// //// 1

//// TF

~o 2

TF

Fig. 1. White blood cell count (WBC) (1~), platelet count (ITS)and hemoglobin (Hb) value (k-'q) in IFN gamma resistant patients with CPCML undergoing therapy with rh IFN alfa-2c for at least 6 months. The values are classified according to the state of hematologic remission achieved. P/CHR indicates patients having experienced a partial or complete hematologic remission. TF indicates patients who failed to respond to IFN-alfa-2c therapy. The open columns represent post-treatment values

F. H e r r m a n n et al.: IFN alfa-2c in IFN gamma resistant CML

229 500

00o

I000 I

2

800 500

d .~

-~ eq

61)o

ee~

~oo ""

500

200

P/CttR

TF

1

fi

!

z

40o

2

P/CHR

'~

TF

3

Fig. 2. Serum LDH ( D ) and vitamin-B12 values ( ~ in the same patient population as described in the legend to Fig. 1. The open columns represent post-treatment values. The normal range in our laboratory for LDH is 100-200 U / m l and for vitamin-B12 200-1100 pg/ml

Table 3. Acute and chronic toxicity associated with IFN alfa-2c therapy Number of patients Toxicity

200 bp~

Mild/moderate

Severe

11

--

Acute Fever

A

Chills

9

--

Malaise

7

--

Arthralgias

6

1

Myalgias

5

--

Nausea/vomiting

2

--

Headache

2

1

Dry mouth

1

--

Eye discomfort

1

--

Chronic

- 5kb

Malaise

4

1

Arthralgias

4

1

Myalgias

4

--

Headache

2

--

Hair loss

2

--

Weight loss

2a

b

a Less than 10% of the total body weight b More than 10% of total body weight 1

2

3

4

co

5

6

Fig. 3. A Detection of bcr-abl-specific transcripts by PCR. R N A was prepared from PBMLs before (lane 1) and after (lane 2) treatment with IFN alfa, reverse transcribed, amplified by 45 cycles of PCR, separated on an agarose gel, transferred onto a membrane, hybridized with a bcr-abl-specific oligonucleotide that detects the third exon of bcr joined to the second exon of c-abl. Lane 3 represents a control with healthy d o n o r - d e r i v e d blood mononuclear cells. B Southern blot analysis of bcr sequence of h u m a n placenta D N A (Co) and bone marrow DNA from 3 patients before (lanes /, 3, 5) and after IFN-alfa-2c therapy (lanes 2, 4, 6). All patients had experienced cytogenetic improvement by the IFN alfa-2c treatment. DNA wad digested with Bgl II and hybridized to a Bgl I I / H i n d III 5' bcr probe that detects a 5 kb normal bcr fragment. Digests with Pvu II gave comparable results (not shown). The arrows indicate the aberrant bands.

Toxicity o f I F N alfa-2c Acute and chronic toxicities associated with IFN alfa-2c t r e a t m e n t a r e s h o w n i n T a b l e 3. O v e r a l l t o x i c i t i e s e n c o u n tered with IFN alfa-2c treatment were mild to moderate, i n c l u d i n g fever a n d c h i l l s n e v e r l a s t i n g f o r m o r e t h a n o n e week, acute and chronic myalgias, arthralgias, headache, m a l a i s e , h a i r loss a n d w e i g h t loss, t h a t d i d n o t l e a d t o t r e a t m e n t i n t e r r u p t i o n . O n l y o n e p a t i e n t s h a d severe acute arthralgias and headache, not manageable by nonsteroidal analgetics, and was therefore removed from the s t u d y . Two w e e k s a f t e r d i s c o n t i n u i n g t r e a t m e n t s t h e c o m p l a i n t s h a d d i s a p p e a r e d . A severe c h r o n i c m a l a i s e s y n -

230 drome also associated with arthralgias developed in one patient not requiring removal from study. None of the side effects coincided with lack of response. No hepatic, renal or cardiac toxicity and no alteration of coagulation variables were observed.

Discussion Interferons are members o f a family of naturally occuring polypeptides which, in addition to their well-known antiviral activity, exert substantial anticellular effects particularly on rapidly dividing ceils such as hematopoietic progenitors. Consequently, several investigators have made use of IFNs in the treatment of patients with various hematologic disorders including hairy cell leukemia [13], multiple myeloma [11] and chronic-phase CML [1,2, 12, 17-19]. Based on their antigenic and physiochemical properties, type I IFNs, including IFN alpha and IFN beta, can be distinguished from type II 1FNs (= IFN-gamma). IFN alpha represents a group of 20 highly homologous species, of which three different human IFN alpha species have been cloned: IFN alfa-2a, b, and c. IFN alfa-2a [19], and IFN alfa-2b [1, 2, 12] have already proven to be of benefit for C P - C M L patients by producing 4 0 % - 8 0 % hematologic remissions when administered as single agents. In addition, alpha-IFNs have also shown to be active in prolonging chemotherapy-induced remissions in 90% of C P - C M L patients [2]. Also promising is the observation that during IFN treatment normal metaphases lacking the Ph' chromosome abnormality have reappeared in the bone marrow of some patients [1,2, 12, 18, 19]. More recently, IFN gamma was also shown to satisfactorily reduce abnormal hematopoiesis in C P - C M L [10]. Since type I and type II IFNs act through disparate binding sites on IFN receptive cells [3], it has been suggested that patients who are resistant to IFN gamma treatment may respond to IFN alpha, and vice versa [10]. In the present study we report on 10 evaluable patients with Ph' positive C P - C M L refractory to a preceeding IFN-gamma therapy, who had secondary treatment with rhu IFN alfa-2c for 6 - 3 0 months. We demonstrate that IFN alfa-2c is as effective in the management of this particular group of C P - C M L patients as reported for IFN alfa-2a, and -2b in previously untreated patients. Five of 10 patients experienced a hematologic remission and normalization o f previously increased serum L D H and vitamin-B12 levels. All of these patients with C H R also developed a normal index of the N A P (not shown). In three of these, cytogenetic improvement was achieved. In one patient, the Ph' positive clone completely disappeared (in 48 bone marrow metaphases evaluated) following 12 months of therapy. This patient is still (30 months after commencing treatment) in CCR and has developed germline bcr configuration by Southern blot analysis. Two patients who responded initially to IFN alfa-2c have progressed from the CP to an accelerated state and BC respectively, thus showing that IFN alpha does not necessarily prevent clonal evolution. The possibility that IFN alpha will, in the long run, slow down or prevent disease progression needs therefore to be studied in prospec-

E Herrmann et al.: IFN alfa-2c in IFN gamma resistant CML tive randomized trials. Assessment of different treatment schedules (e.g. low-dose therapy or pulsatile therapy) will be required as well. A complete cytogenetic response should be the major objective of future 1FN therapy. Several concepts should be contemplated to optimize the cytogenetic response: combining synergizing IFNs (alpha and gamma), combining IFNs with other cytoreductive biologic agents (e.g. with Tumor Necrosis Factor) or combining IFNs with differentially acting chemotherapeutic agents.

Acknowledgements. This study was supported in part by the Deutsche Forschungsgemeinschaft (He 1380-2-1) and the Karl Thomae GmbH, Biberach, who provided the recombinant human IFNalfa-2c. We are grateful to C. Bartram, University of Ulm, for providing the bcr cDNA and to E. Kawasaki, Cetus Corp., Emeryville, CA, for providing the oligonucleotides for amplification and detection of bcr-abl mRNA by PCR. Ms. C. Zommernig and R. Freitas are acknowledged for their excellent secretarial help.

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