Abstract

Background: The purpose of this study was to assess the efficacy and safety of ISIS 3521, an antisense phosphorothioate oligonucleotide to protein kinase C α in patients with relapsed low-grade non-Hodgkin's lymphoma (NHL).

Patients and methods: Twenty-six patients received ISIS 3521 (2 mg/kg/day) as a continuous infusion over 21 days of each 28-day cycle.

Results: The median age of the patients was 53 years (range 37–77). Histological subtypes were low-grade follicular lymphoma (n=22) and B-cell small lymphocytic lymphoma (n=4). Twenty-one (81%) had stage III/IV disease. The median number of previous lines of chemotherapy was two (range one to six). A total of 87 cycles of ISIS 3521 were administered. Twenty-three patients were assessable for response. Three patients achieved a partial response. No complete responses were observed. Ten patients had stable disease. Grade 3–4 toxicity was as follows: neutropenia (3.8%) and thrombocytopenia (26.9%).

Conclusions: ISIS 3521 has demonstrated anti-tumour activity in patients with relapsed low-grade NHL. There may be a potential role for this agent in combination with conventional chemotherapy for advanced low-grade lymphoma, and further trials are warranted.

Introduction

Follicular lymphoma accounts for 22% of all non-Hodgkin's lymphoma (NHL) worldwide [1]. The majority of these patients have advanced disease at presentation, which is incurable. This is characterised by an indolent course, and thus median survival ranges from 5 to 10 years. In chemo-naïve patients ∼70–80% will respond to single-agent alkylating therapy, of whom 30–40% achieve a complete response [2]. The median duration of response is between 1.5 and 2 years. Combination chemotherapy, high-dose chemotherapy and monoclonal antibodies have been investigated in this setting; none of these treatments has had a positive impact on long-term survival.

Protein kinase C (PKC) is a family of serine threonine protein kinases that are involved in signal transduction pathways regulating proliferation and apoptosis [3]. There are many isoforms of this family, and the isoenzyme PKC-α is found in a number of transformed cell lines [4, 5]. It appears to play a pivotal role in tumour growth and progression in certain malignancies [4]. In vitro experiments have demonstrated that overexpression of PKC-α in the breast cancer cell line MCF-7 leads to increased anchorage-independent growth and proliferation, and a greater degree of tumorigenicity [6].

Single-stranded antisense DNA forms a covalent bond with a specific sequence of messenger RNA (mRNA) inhibiting translation and rendering the complex susceptible to degradation by the enzyme RNaseH. ISIS 3521 is a 20-base phosphorothioate antisense oligonucleotide targeted to the 3′-untranslated region of the human PKC-α mRNA. In vitro experiments in the A549 human non-small-cell lung carcinoma cell line showed that administration of ISIS 3521 caused a significant decrease in PKC-α protein levels [79].

Antitumour activity of ISIS 3521 has been demonstrated in phase I trials. Nemunaitis et al. [10] reported objective complete responses in two patients with NHL receiving ISIS 3521 three times a week as a 2-h infusion, whilst a partial response was observed by Yuen et al. [11] in a patient with ovarian cancer receiving ISIS 3521 as a continuous infusion over 21 days. The toxicity profile was acceptable with both these schedules of administration. Thrombocytopenia was the dose-limiting toxicity and was more frequent with the continuous infusion, whilst coagulation abnormalities were seen more often with the thrice-weekly regimen. The 21-day infusion regime has been adopted for further studies based on a prolonged rate of tumour exposure to the treatment and fewer hospital visits for the patient. The maximum tolerated dose of ISIS 3521 was 2 mg/kg/day by continuous infusion. This dose was recommended for phase II studies.

This study was therefore performed to evaluate the efficacy and safety of ISIS 3521 given at 2 mg/kg/day in previously treated patients with low-grade NHL.

Patients and methods

Study design

This open-label, non-randomised multicentre phase II study was conducted in 10 centres. Written informed consent was obtained from all patients. The study was approved by the local research and ethics committees at all participating centres.

Eligibility criteria were: histologically confirmed low-grade, follicular or lymphocytic non-Hodgkin's B-cell lymphoma; documented progressive disease after at least one previous line of chemotherapy; adequate bone marrow reserve (platelets >100 × 109/l, neutrophils >1.0 × 109/l), renal function (serum creatinine <140 μmol/l), hepatic function (serum bilirubin <35 μmol/l, serum aspartase transaminase and alanine transaminase <3× the upper limit of normal), activated partial thromboplastim time (APTT) within normal range and bidimensionally measurable disease; and Eastern Cooperative Oncology Group (ECOG) performance status 0–2. Patients were excluded if they had an active bleeding disorder, were on therapeutic doses of warfarin or heparin, had a history of other malignancy (apart from curatively treated superficial skin cancers, or carcinoma in situ of the uterine cervix), or if there was central nervous system involvement with lymphoma.

Therapy

ISIS 3521 (5′-GTTCTCGCTGGTGAGTTTCA-3′) 2 mg/kg/day was administered as a continuous intravenous infusion over 21 days of each 28-day cycle, followed by a 7-day treatment-free interval. The chemotherapy was infused by a portable pump via a skin-tunnelled central venous catheter. Patients could continue on therapy until there was objective evidence of disease progression or, in the event of complete response, for an additional two cycles.

Toxicity evaluation and dose modifications

Toxicity was evaluated weekly and graded according to the National Cancer Institute Common Toxicity Criteria. For non-haematological toxicity of grade 3 or 4, treatment was suspended until this had resolved to grade <1 and then treatment was reinstituted with 50% dose reduction (1 mg/kg/day). The following dose reductions were made for haematological toxicity: for grade 3 or 4 thrombocytopenia, treatment was suspended until recovery to grade 1 and then treatment was recommenced with 50% dose reduction (1 mg/kg/day); for grade 3 or 4 neutropenia, treatment was stopped until resolution to grade 2 and then restarted with 50% dose reduction (1 mg/kg/day). Any patient not meeting the above recovery criteria within 2 weeks of the last dose of study drug was withdrawn from the study.

Pre-treatment evaluation, assessment during treatment and follow-up

Patients were assessed at baseline with a full medical history and physical examination including ECOG performance status and measurement of any palpable disease, full blood count, coagulation tests, serum biochemistry including electrolytes, hepatic and renal function tests, urinalysis, bone marrow aspirate and trephine, and computed tomography (CT) scans of chest, abdomen and pelvis. Histological samples were reviewed centrally by an independent pathologist.

During the study, medical history, physical evaluation, full blood count, coagulation screen and serum chemistry were obtained every 28 days.

Response assessment

Tumour response was assessed by CT scans (according to World Health Organisation criteria), clinical measurement of palpable tumour masses and bone marrow biopsy where applicable. These tests were repeated after every second cycle of treatment.

A complete response was defined as disappearance of all radiological and clinical evidence of tumour confirmed on two observations at least 4 weeks apart. In addition, a bone marrow biopsy was required to confirm the absence of bone marrow disease and patients must have been free of lymphoma-related symptoms. A partial response was defined as a ≥50% decrease in the sum of the products of the measurable lesions confirmed on two observations at least 4 weeks apart. No increase in size in any existing lesion or the appearance of any new lesion may have occurred. Bone marrow disease may not have appeared when previously absent. Stable disease was defined as disease less than a partial response, i.e. ≤50% decrease in the overall sum of the products of the measurable lesions or the absence of progressive disease at 8 weeks. Progressive disease was defined as a ≥25% increase in the overall sum of the products of measurable lesions or the appearance of a new lesion or the development of bone marrow involvement when previously absent.

Statistics

Patient demographics and baseline characteristics were summarised. Patients who received any study drug were evaluated for toxicities. Adverse events were summarised by COSTART preferred terms and toxicity grades. Patients who completed at least one cycle of treatment were considered evaluable for efficacy. Proportions of patients experiencing complete response, partial response, stable disease and progressive disease were calculated. Median time to progression was estimated using the Kaplan–Meier method.

Results

Patient characteristics

Patient characteristics are shown in Table 1 and demographic data are summarised in Table 2. Twenty-six patients were enrolled into the study between January 1999 and April 2001. Median time from patients' diagnosis to study entry was 68 months (range 9–175). All patients had received at least one prior line of chemotherapy; the median number of previous lines of treatment was two (range one to six). Nine patients had received previous radiotherapy.

Drug administration

Twenty-six patients received the study drug. Two patients received <7 days of treatment and were not assessable for response. The median number of completed treatment cycles was two (range one to 14), with 14 patients receiving two or more cycles of treatment. A total of 87 cycles of treatment were delivered. Eighteen cycles of treatment were delayed, nine due to recovering thrombocytopenia, seven due to infections and two due to diarrhoea and vomiting. Twenty-six cycles of treatment were administered at a reduced dose due to thrombocytopenia according to the protocol.

Toxicities

All 26 patients were evaluable for toxicities. Common side effects reported were nausea, fever, headache and fatigue. The most commonly reported events are shown in Table 3. Clinically significant haematological toxicities were largely limited to thrombocytopenia. Grade 3 thrombocytopenia requiring dose reduction occurred in four patients (15.4%), and one patient was withdrawn with grade 4 thrombocytopenia. There was one case of grade 3/4 neutropenia encountered. Grade 3/4 lymphopenia was reported in 12 patients. No significant coagulation disturbance was seen. Hepatic toxicity was mild, with no grade 3 or 4 toxicity seen. In three patients, grade 1 renal toxicity (elevation of creatinine) was observed, contributed to by the co-administration of gentamicin in two cases. A further patient discontinued the study drug after developing grade 2 renal toxicity, again occurring in conjunction with the co-administration of gentamicin. During the course of the study, eight venous catheter infections required treatment with intravenous antibiotics.

The study drug was discontinued in five patients (19.2%) due to adverse events, specifically indwelling venous line infection, renal failure, sepsis, gastrointestinal bleed and exertional chest pain. The gastrointestinal bleed was upper gastrointestinal in origin, and occurred in a patient receiving concurrent non-steroidal analgesia and also warfarin therapy for a line-related venous thrombosis. At the time of the bleed the platelet count was 91 × 109/l and international normalized ratio (INR) 3.3. The patient with exertional chest pain received treatment with oral anti-anginal therapy with resolution of symptoms. Five patients withdrew from the study at their request (19.2%) and one patient was withdrawn due to toxicity (grade 4 thrombocytopenia).

Efficacy

Three patients were excluded from response assessment: one patient withdrew voluntarily from the study prior to completion of cycle 1, another patient discontinued the study prior to completion of cycle 1 because of a serious adverse event (renal dysfunction), a further patient had undergone excision of a marker lesion whilst receiving the study drug, thus response assessment was inaccurate in this case. Response data are shown in Table 4. No complete responses were seen. Amongst the patients assessed for response, three (11.5%) had a partial response as their best, 10 (38.5%) achieved stable disease as their best response and 10 (38.5%) had progressive disease. Responses were observed in patients with low-grade follicular lymphoma stage IVB, IIIA and IE. The patients were 38, 59 and 9 months from the time of initial diagnosis, and had all previously received two or more lines of treatment prior to study entry. One patient had relapsed disease following previous treatment with fludarabine, chlorambucil and rituximab; the second responding patient had relapsed disease following previous therapy with CVP (cyclophosphamide, vincristine and prednisolone), fludarabine and CHOP (cyclophosphamide, adriamycin, prednisolone and vincristine); and the third patient had disease that was refractory to both CVP and CHOP chemotherapy. The partial responses occurred at cycle 11, 5 and 2, respectively. The overall median time to progression for all evaluable patients was 2.8 months.

Discussion

Patients with advanced stage low-grade lymphoma that respond to chemotherapy will inevitably relapse over time. In one prospective study, 148 patients with newly diagnosed follicular lymphoma were evaluated over a 12-year period. The median survival of patients after their first relapse was 5 years, and the remission rates and durations of remission were similar (1 year) for the first three lines of chemotherapy but then decreased with each subsequent course of treatment [12]. This confirms that the disease becomes more resistant to chemotherapy with time, and therefore increasingly difficult to treat after multiple relapses.

Antisense oligonucleotides have been investigated in follicular lymphoma previously. Webb et al. [13] demonstrated that BCL2 antisense caused tumour regression, symptom relief and decreased numbers of circulating lymphoma cells in multiply treated patients with advanced stage low-grade lymphoma. Furthermore, this agent exhibited chemosensitising properties, as several patients treated with chemotherapy after antisense achieved a partial response. Non-antisense-mediated effects may contribute to the clinical activity seen with phosphorothioated antisense oligonucleotides. Preclinical studies have evaluated the immune stimulatory activity of oligonucleotides and have found it to be related to the presence of certain oligonucleotide sequences, in particular the presence of CG dinucleotides [14]. The oligonucleotide sequence of ISIS 3521 does not contain oligonucleotide sequences with high immunostimulatory properties, although of note the phosphorothioate backbone is known to have some immunostimulatory activity independent of the oligonucleotide sequence [15]. This study does not allow us to assess the relative contribution of the antisense and non-antisense (immunostimulatory) activity to the clinical outcomes seen.

This study has demonstrated that ISIS 3521 is a feasible treatment for patients with multiply treated low-grade NHL in terms of safety and antitumour activity. Three patients achieved a partial response and a further 10 patients with follicular NHL had stable disease. The median time to progression after treatment with ISIS 3521 was 2.8 months. Furthermore, one of the responders had refractory low-grade NHL having failed to respond to two previous lines of treatment including an anthracycline-containing regimen.

The earliest response was observed after two cycles of treatment in this study, but the other two responders had their first responses at cycles 5 and 11. The median treatment delivery for all study patients was only two cycles, suggesting that a higher response rate may have been observed if patients had received a longer treatment course.

The treatment was generally well tolerated, with no excess or unexpected toxicity. There were a number of treatment-related infections, although there was only one case of febrile neutropenia. Thrombocytopenia was the principle haematological toxicity observed, which appears to be a class effect of oligonucleotide phosphorothiates [1620], and in this study may be partly accounted for by a large proportion of patients having advanced stage disease and receiving multiple lines of previous chemotherapy. Despite 26.9% of patients experiencing grade 3–4 thrombocytopenia, there was only one case of severe haemorrhage reported and this occurred in a patient receiving concurrent warfarin medication for a Hickman line thrombosis.

Whilst ISIS 3521 has shown promising antitumour activity in this study, it may be possible to further improve these data by combining this treatment with conventional chemotherapy. This has already been evaluated in a number of other malignancies; a phase I study has recently reported encouraging antitumour activity of ISIS 3521 in combination with 5-fluorouracil and leucovorin in patients with refractory advanced cancer, predominantly of colorectal origin [21]. ISIS 3521 administered with carboplatin and paclitaxel in patients with advanced non-small-cell lung cancer has produced promising efficacy data without incurring any additional toxicity [22]. Yuen et al. [22] reported an objective response rate of 42% with this treatment, and only 17% of patients progressed during treatment. These particular data have led to a large randomised phase III study comparing this novel combination with carboplatin and paclitaxel alone in this cohort of patients.

The results achieved in this study with ISIS 3521 are comparable to those reported with antisense oligonucleotides targeting BCL2 in patients with multiply pre-treated follicular lymphoma [12, 13, 23]. ISIS 3521 required prolonged central venous access, and the earliest time to response was 2 months. Therefore, we would not recommend its use as monotherapy for these patients. Preclinical data have indicated that ISIS 3521 administered in combination with conventional chemotherapy agents produces a greater antitumour effect than observed with the individual agents alone [24]. ISIS 3521 may cause tumour cells to be more susceptible to apoptopic injury secondary to cytotoxic agents, and there may be a potential role for ISIS 3521 in combination with conventional chemotherapy for follicular lymphoma. Further trials are warranted.

Table 1.

Patient characteristics

Patient Gender Age (years) ECOG PS Stage Histology Time from diagnosis to study entry (years) Previous therapies prior to study entry
 
    Time from last therapy to study entry (months) 
       Radiotherapy Alkylating agent Anthracycline/ anthracenedione Purine analogue Rituximab  
65 IIIA Follicular 5.3    31.3 
67 IIIA Follicular 9.4    4.4 
46 IIIB Follicular 2.7    16.8 
4a 37 IVB Follicular 3.2   15.3 
75 IVB Follicular 10.7    3.8 
60 IIIE Follicular 14.6    1.4 
53 IIA Follicular 5.5    26.8 
44 IVA Follicular 1.8    13.6 
45 IVB Follicular 7.1     0.4 
10 46 IIA Follicular 3.3    23.3 
11 69 IIIA Small  lymphocytic 4.6     29.1 
12 46 IIIA Follicular 1.0     5.7 
13 46 IVB Follicular 0.8    6.6 
14 57 IVB Follicular 5.7   1.0 
15a 53 IIIA Follicular 4.9   16.9 
16 53 IVA Follicular 10.4    49.8 
17 51 IVA Follicular 3.2  31.4 
18 69 IIA Follicular 12.4  16.4 
19 57 IVA Follicular 2.6   6..3 
20 69 IIA Follicular 5.2   17.7 
21 45 IIIA Follicular 7.3    22.7 
22 70 IVB Small  lymphocytic 4.2     42.2 
23 50 IIIB Follicular 7.9 2.5 
24 77 IIIA Small  lymphocytic 7.2   46.4 
25 46 IIIB Follicular 7.0  16.7 
26a 57 IE Small  lymphocytic 0.8    1.2 
Patient Gender Age (years) ECOG PS Stage Histology Time from diagnosis to study entry (years) Previous therapies prior to study entry
 
    Time from last therapy to study entry (months) 
       Radiotherapy Alkylating agent Anthracycline/ anthracenedione Purine analogue Rituximab  
65 IIIA Follicular 5.3    31.3 
67 IIIA Follicular 9.4    4.4 
46 IIIB Follicular 2.7    16.8 
4a 37 IVB Follicular 3.2   15.3 
75 IVB Follicular 10.7    3.8 
60 IIIE Follicular 14.6    1.4 
53 IIA Follicular 5.5    26.8 
44 IVA Follicular 1.8    13.6 
45 IVB Follicular 7.1     0.4 
10 46 IIA Follicular 3.3    23.3 
11 69 IIIA Small  lymphocytic 4.6     29.1 
12 46 IIIA Follicular 1.0     5.7 
13 46 IVB Follicular 0.8    6.6 
14 57 IVB Follicular 5.7   1.0 
15a 53 IIIA Follicular 4.9   16.9 
16 53 IVA Follicular 10.4    49.8 
17 51 IVA Follicular 3.2  31.4 
18 69 IIA Follicular 12.4  16.4 
19 57 IVA Follicular 2.6   6..3 
20 69 IIA Follicular 5.2   17.7 
21 45 IIIA Follicular 7.3    22.7 
22 70 IVB Small  lymphocytic 4.2     42.2 
23 50 IIIB Follicular 7.9 2.5 
24 77 IIIA Small  lymphocytic 7.2   46.4 
25 46 IIIB Follicular 7.0  16.7 
26a 57 IE Small  lymphocytic 0.8    1.2 
a

Indicates responding patients.

ECOG PS, Eastern Cooperative Oncology Group performance status; M, male; F, female.

Table 2.

Patient demographics

Demographic n (%) 
Total number entered 26 
Male/female 14/12 
Age, years [median (range)] 53 (37–77) 
Follicular NHL 22 
Lymphocytic NHL 
Staging  
    IE 1 (3.8) 
    IIA 4 (15.4) 
    IIIA/IIIB/IIIE 11 (42.3) 
    IVA/IVB 10 (38.5) 
ECOG performance status  
    0 16 (61.5) 
    1 8 (30.8) 
    2 2 (7.7) 
No. previous lines of chemotherapy  
    1 
    2 
    3 
    >4 
Demographic n (%) 
Total number entered 26 
Male/female 14/12 
Age, years [median (range)] 53 (37–77) 
Follicular NHL 22 
Lymphocytic NHL 
Staging  
    IE 1 (3.8) 
    IIA 4 (15.4) 
    IIIA/IIIB/IIIE 11 (42.3) 
    IVA/IVB 10 (38.5) 
ECOG performance status  
    0 16 (61.5) 
    1 8 (30.8) 
    2 2 (7.7) 
No. previous lines of chemotherapy  
    1 
    2 
    3 
    >4 

NHL, non-Hodgkin's lymphoma; ECOG, Eastern Cooperative Oncology Group.

Table 3.

Non-haematological and haematological toxicity

Toxicity Any grade [n (%)] Grade 3–4 [n (%)] 
Nausea 12 (46) 
Fever 6 (23) 1 (3.8) 
Somnolence 5 (19.2) 
Headache 5 (19.2) 
Diarrhoea 4 (15.4) 
Skin rash 3 (11.5) 
Pain 9 (35) 
Asthenia 7 (27) 1 (3.8) 
Anaemia 12 (46.2) 
Neutropenia 2 (7.7) 1 (3.8) 
Lymphopenia 15 (58) 12 (46) 
Thrombocytopenia 22 (84.6) 7 (26.9) 
Hyperbilirubinaemia 2 (7.7) 
Toxicity Any grade [n (%)] Grade 3–4 [n (%)] 
Nausea 12 (46) 
Fever 6 (23) 1 (3.8) 
Somnolence 5 (19.2) 
Headache 5 (19.2) 
Diarrhoea 4 (15.4) 
Skin rash 3 (11.5) 
Pain 9 (35) 
Asthenia 7 (27) 1 (3.8) 
Anaemia 12 (46.2) 
Neutropenia 2 (7.7) 1 (3.8) 
Lymphopenia 15 (58) 12 (46) 
Thrombocytopenia 22 (84.6) 7 (26.9) 
Hyperbilirubinaemia 2 (7.7) 
Table 4.

Response to ISIS 3521 oligonucleotides

Best response No. of patients Percent of assessable patients (n=23) Percent of all patients (n=26) 
Complete response 
Partial response 13 12 
Stable disease 10 43 39 
Progressive disease 10 43 39 
Not assessable  12 
Best response No. of patients Percent of assessable patients (n=23) Percent of all patients (n=26) 
Complete response 
Partial response 13 12 
Stable disease 10 43 39 
Progressive disease 10 43 39 
Not assessable  12 

This study was supported by ISIS Pharmaceuticals, Inc., Carlsbad, CA, USA.

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Author notes

1Royal Marsden Hospital, London and Surrey; 2St Mungo Institute, Glasgow; 3Southampton Hospitals NHS Trust, Southampton; 4St James University Hospital, Leeds; 5Weston Park Hospital NHS Trust, Sheffield; 6Birmingham Heartlands Hospital, London; 7Aberdeen Royal Infirmary, Aberdeen; 8Guy's Hospital, London; 9Belfast City Hospital, Belfast, UK; 10National Institute of Haematology and Transfusiology, Sofia, Bulgaria; 11ISIS Pharmaceuticals, Inc., Carlsbad, CA, USA