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© 2001 American Society for Clinical Oncology Results of a Pilot Study Involving the Use of an Antisense Oligodeoxynucleotide Directed Against the Insulin-Like Growth Factor Type I Receptor in Malignant AstrocytomasFrom the Kimmel Cancer Center, Departments of Neurosurgery, Radiology, Pathology, Internal Medicine, Radiation Oncology, and Neurology, Thomas Jefferson University Hospital, Philadelphia, PA. Address reprint requests to David W. Andrews, MD, Department of Neurosurgery, Thomas Jefferson University Hospital, 834 Walnut St, Suite 650, Philadelphia, PA 19107; email: david.w.andrews@ mail.tju.edu.
PURPOSE: Preclinical animal experiments support the use of an antisense oligodeoxynucleotide directed against the insulin-like growth factor type I receptor (IGF-IR/AS ODN) as an effective potential antitumor agent. We performed a human pilot safety and feasibility study using an IGF-IR/AS ODN strategy in patients with malignant astrocytoma. PATIENTS AND METHODS: Autologous glioma cells collected at surgery were treated ex vivo with an IGF-IR/AS ODN, encapsulated in diffusion chambers, reimplanted in the rectus sheath within 24 hours of craniotomy, and retrieved after a 24-hour in situ incubation. Serial posttreatment assessments included clinical examination, laboratory studies, and magnetic resonance imaging scans.
RESULTS: Other than deep venous thrombosis noted in some patients, no other treatment-related side effects were observed. IGF-IR/AS ODN–treated cells, when retrieved and assessed, were CONCLUSION: Ex vivo IGF-IR/AS ODN treatment of autologous glioma cells induces apoptosis and a host response in vivo without unusual side effects. Subsequent transient and sustained radiographic and clinical improvements warrant further clinical investigations.
MALIGNANT ASTROCYTOMAS are primary intracranial tumors that have resisted treatment with surgery, radiation, and chemotherapy. Median survival for World Health Organization (WHO) grade 4 tumors (glioblastomas) remains a dismal 47 weeks.1 Because of conventional treatment failure, we investigated a different approach that specifically targets the type I insulin-like growth factor receptor (IGF-IR) in glioma cells with an 18-mer IGF-IR antisense oligodeoxynucleotide (AS ODN). The IGF-IR is a tyrosine kinase cell surface receptor2 that shares 70% homology with the insulin receptor.2 When activated by its ligands (IGF-I, IGF-II, and insulin at supraphysiologic concentrations), it regulates broad cellular functions, including proliferation, transformation, and cell survival.3-5 Conversely, downregulation of the IGF-IR function provides a selective target for therapies aimed at the destruction of cancer cells. IGF-IR protects cancer cells from apoptosis induced by a variety of anticancer drugs6 and radiation,7,8 but when impaired by inhibitors such as antisense strategies, dominant negative mutants, or triple-helix formation, tumor cells undergo massive apoptosis even without adjuvant treatment, resulting in a dramatic inhibition of tumorigenesis9-14 and metastases6,15,16 in experimental animals. The IGF-IR is not an absolute requirement for normal growth, but it is essential for growth in anchorage-independent conditions3 that may occur in malignant tissues, thus rendering cancer cells uniquely susceptible to an IGF-IR manipulation that spares normal cells. This selective effect has been verified in animals bearing transplantable human tumors.17 IGF-IR/AS ODN targeting also unexpectedly seems to produce a host response that has many characteristics of an immune response.5,18-20 Animals harboring a syngeneic transplantable tumor, when subsequently challenged with IGF-IR/AS ODN–treated tumor cells, had no tumor growth at the test site and had complete tumor regression at the established control site.5,18-22 This was reproduced when the test cells were encapsulated in subcutaneously implanted diffusion chambers that excluded cell passage, suggesting a common soluble factor as mediator of this response.5,18-22 If IGF-IR was not downregulated, the cells failed to induce regression of the wild-type tumor, and thus irradiation of cells or injection of dead cells did not induce a similar host response. This host response has been confirmed in another laboratory, using a neuroblastoma cell line.20 To see whether similar host responses could be achieved in animals with brain tumors, BD-IX rats with established intracerebral C6 glioma were treated with a suspension of the C6 glioma cell line preincubated ex vivo with an IGF-1R/AS ODN. The suspension was loaded into diffusion chambers and implanted into the subcutaneous tissue of the rat at -7 days or 1 day after intracerebral inoculation of C6 glioma.23 Rats treated with the antisense 1 day after intracerebral inoculation of the C6 glioma survived three-fold longer than the control groups (random sequence ODNs, IGF-1R sense ODN, and no ODN), and 25% survived for more than 200 days. No tumor was present in this treatment group when the brains were examined microscopically at necropsy. These observations in experimental animals prompted us to explore whether a similarly indirect host response could also be observed in human tumors, specifically in human malignant astrocytomas, that had been treated unsuccessfully with previous therapy.
This clinical study was approved by the United States Food and Drug Administration (FDA; R.D.A. is the recipient of the physician-sponsored Investigational New Drug) and the Institutional Review Board at Thomas Jefferson University. Eligible patients had WHO grade 3 or 4 astrocytomas and failed all prior therapies with new clinical symptoms in eight patients and magnetic resonance imaging (MRI) progression in all 12 patients. To enroll, patients were also required to have a life expectancy of at least 8 weeks; Karnofsky performance status (KPS)  60 or Eastern Cooperative Oncology Group performance status of 0, 1, or 2; and age of at least 16 years. Patients who had received radiation therapy or chemotherapy less than 8 weeks before treatment were excluded. All tumors expressed the IGF-IR.
Clinical Intervention During resection, viable tumor tissue was confirmed by frozen section and sent to a BL-2 facility for disaggregation and treatment. Tumor cells were plated in complete culture medium (DMEM supplemented with 10% fetal bovine serum, penicillin, streptomycin, and glutamine). Once the cells attached, they were washed and shifted to serum-free medium (DMEM supplemented with 1 µmol/L of ferrous sulfate and 0.1% bovine serum albumin fraction V) and treated with an IGF-IR/AS ODN. We used an 18-mer phosphorothioate oligodeoxynucleotide (5'-GGACCCTCCTCCGGAGCC-3') starting six nucleotides downstream from the initiating methionine codon, synthesized by Lynx Therapeutics (Hayward, CA), lot no. LR4437-002A, the same lot as previously described.22 After a minimum 6-hour incubation with a dose of 2-mg IGF-IR/AS/107 cells, the cells were collected. The cells were detached from the plates, washed with calcium and magnesium-free phosphate-buffered saline, loaded into up to 10 Lucite (Belle, WV) diffusion chambers capped with 0.1-µ Millipore filters (Millipore Corporation, Bedford, MA), and implanted into a subcutaneous pocket in the patient’s abdominal rectus sheath that had been created by the neurosurgeon at the time of tumor resection surgery. Each diffusion chamber accommodated 106 cells/200 µL/chamber. The 2-mg IGF-IR/AS ODN treatment dose was derived from the preclinical experiments extrapolated on a weight-to-weight difference from rat to man. Immediately before implantation into human subjects, the suspension, which was already loaded into the sterilized chambers, was radiated with 5 Gy to comply with FDA requirements. On the first postcraniotomy day, the abdomen acceptor site was opened at bedside for diffusion chamber implantation. A time window was selected so that tumor cells treated ex vivo with the IGF-IR/AS ODN were detached, encapsulated in the chambers, and irradiated at the same time the acceptor site was opened. After local anesthesia and antibiotic prophylaxis, the rectus sheath pockets were exposed and up to 10 sterile diffusion chambers were implanted. On the second postcraniotomy day, the diffusion chambers were retrieved and transported to a BL-2 facility for analysis after recovery. The diffusion chambers were implanted in the patients for 24 hours based on our preclinical studies, which showed that by 3 hours of implantation, the maximal effects were observed in terms of induction of apoptosis and induction of host antitumor responses.18,22,23,25 We also wanted to abbreviate the patients’ exposure to a foreign body to minimize any treatment-related complications. Postimplantation surveillance included serial clinical and MRI examinations at 1-month intervals.
Compassionate Re-Treatment
Efficacy, Specificity, and Safety of the Treatment
Before implantation, autologous tumor cells were evaluated for bacteria (Fisher Diagnostics, Swedesboro, NJ) and Mycoplasma contamination (Sigma Chemicals, St Louis, MO). Integrity of the diffusion chambers was determined by measuring the volume before and after implantation and by microscopic inspection after retrieval. Recovered cell viability was assessed by trypan blue exclusion.18 When possible, acceptor site tissue was examined and the walls biopsied for the presence of glioma cells.26 Despite standard supportive medical measures, we observed that all of the first four patients developed deep venous thrombosis (DVT), a higher than expected rate. Thereafter, we amended the protocol to require daily 40-mg subcutaneous injections of enoxaparin beginning on the first postcraniotomy day and continuing for 3 months, including an additional 3 months for re-treated patients. Noninvasive vascular studies of the legs were performed immediately before and after the completion of enoxaparin treatment.
Posttreatment Evaluation Seven basic imaging characteristics were used to assess interval changes between imaging studies. These assessments were qualitative and based on common imaging parameters that are routinely evaluated in the clinical setting by the neuroradiologist. Because tumor burden is directly related to enhancement characteristics on MRI, the imaging characteristics were subdivided into primary and secondary features. Primary features included T1-weighted abnormalities: (1) size of the enhancing area using the broadest unambiguous diameter in any orthogonal plane, (2) characteristics of the enhancement (eg, progression from linearity to nodularity or regression from nodularity to linearity), and (3) intensity of enhancing area. Secondary features included T2-weighted abnormalities: (4) local mass effect, (5) size of T2-weighted abnormality (eg, edema, tumor, radiation change), (6) invasion of the deep white matter tracts, and (7) distal progression. Radiographic evaluation was performed by assessing changes in any of these characteristics when comparable images from serial studies were evaluated. Each imaging characteristic was rated as increased, decreased, or without change. Because distal progression including invasion of the contralateral hemisphere was either present or absent, this criterion was scored as either absent (stable) or present using fluid-attenuation inversion recovery (FLAIR) sequence ( Fig 2C). Comparisons were made between an MRI study performed within 48 hours of craniotomy documenting residual contrast-enhancing tumor28 and follow-up examinations performed weekly after initiation of therapy, which by 2 months were extended to 1-month intervals and by 6 months to 3-month intervals.
Serial clinical examinations were independently performed by a neurosurgeon (D.W.A.) and a neurologist (R.D.A.). Performance status was assessed according to KPS.29 Histologic examinations were performed by M.C. or L.K. We assessed response after treatment using the following criteria: Complete response. Improvement in all assessable imaging characteristics when compared at serial intervals to either complete resolution or to a stable image with features consistent with postoperative and/or radiation changes, associated with improvement or stability of neurologic and general physical examinations, off corticosteroid medication, for at least 3 months. Partial response. A net improvement in primary features (at least two of three features) with improvement in any or stability in all secondary features, or a net improvement in primary and secondary features (at least four of seven) with stability in the remaining three features, associated with improvement or stability of neurologic and physical examinations, and a stable or decreasing corticosteroid dose, for at least 1 month. Stable disease. No net improvement in primary features (one or 0 of three features) with no net improvement in primary and secondary features (three or fewer of seven) when compared at serial intervals with improvement or stability of neurologic and physical examinations, and a stable or decreasing corticosteroid dose, for at least 1 month. Progressive disease. A net increase in any primary and secondary features when compared at serial intervals with deterioration of neurologic and physical examinations, and with a stable or increasing corticosteroid dose.
Follow-Up Patient data at enrollment are summarized in Table 1. We treated 12 patients and the median survival from date of enrollment was 30 weeks. Patients no. 5 and 10 are currently alive at 168 weeks and 134 weeks, respectively ( Table 2 and Figs 3 and 4). With the exception of compassionate re-treatment in three cases, study patients received no other treatment except best supportive medical and/or surgical therapy. One exception was patient no. 5, who was treated with temozolomide after a distant intracerebral recurrence was surgically resected at 28 months. At treatment, tumor yields in six patients (patients no. 1, 2, 4, and 10 at first treatment, and patient no. 8, 10, and 12 at re-treatment) were too small to implant the maximum number of chambers intended (10 at initial treatment and 20 at re-treatment), and actual maximum implanted yields are as designated in Table 2.
Safety Assessments Complete blood cell count, liver function studies (ALT, AST, direct and total bilirubin), CD4+ and CD8+ counts, antinuclear antibody, anti-ssDNA and anti-dsDNA antibodies remained normal throughout the study period. Neither wound infections (n = 12) nor glioma tumor seeding (n = 6) were observed at the implantation sites after a total of 156 diffusion chambers were implanted (Table 2), and all chambers were recovered intact without membrane damage. Intact cell recovery was in all cases less than 2%, and only autologous tumor cells were recovered. In all cases, the recovered intact cells did not grow when replated in culture. The only possible treatment-related complication was the higher than expected incidence of DVT in patient no. 1 through 4. After enoxaparin prophylaxis, we observed no DVT in six of the last eight patients. Two patients compassionately re-treated developed DVT, one while receiving enoxaparin, the other after self-discontinuation of enoxaparin. We also treated two bone plate infections, which we did not ascribe to this treatment per se.
Posttreatment Observations The remaining four patients had disease progression, and one tumor in this response group had striking loss of central tumor contrast enhancement with volume expansion. At reoperation, pathology revealed massive tumor necrosis and lymphocytic infiltration not previously seen in tissues from prior resections (patient no. 8, Table 2). Among the patients who improved, three patients with intracerebral recurrence had unusual spontaneous or postsurgical regression at either the primary or distant site. In patient no. 5, we saw complete resolution of tumor at his primary left frontal resection site with corresponding complete recovery in function, but at 23 months discovered a small dural-based lesion with uniform enhancement in the left occipital pole initially thought to be a meningioma. At 27 months, however, this mass had increased in size and was resected at 28 months. This lesion was a malignant astrocytoma (WHO grade 3). He refused additional radiation and remains disease-free at 33 months follow-up after additional treatment with six cycles of temozolomide (200 mg/m2 every day, days 1 through 5 every 28 days). His radiographic course is featured in Fig 3. In patient no. 7, we observed a complete response at his primary left parietal tumor but at 8 months saw a small focus of new growth in the opposite right frontal lobe, which then grew rapidly. He underwent resection of the right frontal lobe tumor but died of disease progression at 9 months. At autopsy, brain sections revealed glioblastoma in the right frontal lobe but only scattered neoplastic cells at the original left parietal site with no evidence of WHO grade 4 malignancy. His radiographic course is featured in Figs 2 and 7, and the histopathology record is featured in Fig 6. In patient no. 10, we observed improvement at her primary left opercular tumor site but only after a compassionate re-treatment (Fig 4C). At 14 months a small focus of new growth was seen around the right lateral ventricle (Fig 4D), which resolved spontaneously along with improvement at the primary site at 24 months (Fig 4E). She remains neurologically stable with an expressive aphasia and right hemiparesis. Her radiographic course is featured in Fig 4. In nine cases we were able to analyze serial tumor tissues before and after treatment. These observations are summarized in Table 2. In all cases we observed abundant viable tumor at initial treatment when tumor was harvested for IGF-IR/AS ODN treatment, and in two cases of prior Gliadel implantation (polifeprosan 20 with carmustine implant; Rhône-Poulenc Rorer, Collegeville, PA), viable tumor cells were seen contiguous to wafer remnants. Residual viable tumor cells were identified in all postmortem examinations and in all posttherapy surgical biopsies. We saw individual cases in which the original tumor site either lost endothelial cell proliferation (patient no. 1), tumor cell number and pleomorphism (patient no. 7, Fig 6), or acquired massive central necrosis (patient no. 8 after re-treatment). Microthrombi limited only to tumor-associated blood vessels were identified in six of 10 cases for which posttherapy tissue was available. Also, varying degrees of tumor perivascular lymphocytic infiltration were identified in four cases in which no lymphocytic infiltrates were noted in pretreatment tumor. In brain sections without identifiable tumor cells, we did not identify inflammation, vasculitis, hemorrhage, necrosis, demyelination, or vessel thrombosis.
Preclinical experimental work has indicated that an antitumor host response occurs when the IGF-IR is downregulated by IGF-IR AS/ODN in syngeneic tumors. We performed the current trial to ascertain whether such a paradigm was feasible in humans. We reproduced the same preclinical experimental conditions using ex vivo IGF-IR AS/ODN tumor cell treatments and host reimplantation of treated tumor cells in small diffusion chambers. This paradigm proved to be safe and unexpectedly resulted in objective improvements in eight of 12 patients, in two cases only after compassionate re-treatment and in all cases with either a stable or tapering corticosteroid dose. Despite the phase I design of this study, we applied uniform response criteria for phase II studies of gliomas set forth by Macdonald et al.27 We acknowledge that uniform response criteria remain an important concept, but the range of responses we noted made judgments of responses according to these criteria difficult, particularly with current MRI imaging technology. The Macdonald criteria were initially developed as a method to standardize tumor response on cross-sectional imaging studies. Experience with gadolinium-enhanced MRI was limited at this time, and very little work had been done correlating the MRI characteristics of gliomas with histopathology. Imaging criteria were derived primarily from experience with contrast-enhanced CT, and freely acknowledging the shortcomings of CT images, broad response classes were defined. The stable disease response class included a broad range of tumor responses ranging from less than 50% size reduction to a 25% increase in size. This range overlaps substantially with responses that otherwise might be classified by MRI as partial response or progressive disease. As an example, a recent technique compared an automated MRI segmentation technique to these manually derived size criteria.30 Response classes differed considerably between techniques and, of interest, no consensus was reached in 30% of response class assignments derived from manual size measurements. Many variables contribute to the enhancement characteristics of a tumor bed, including type of modality (CT v MRI), contrast dose and delivery, scan timing, scan parameters, and field strength of the MRI scanner, as well as prior therapeutic interventions such as chemotherapy and/or radiotherapy. A broad experience has shown that CT enhancement characteristics do not correlate with those obtained with MRI images. MRI has many advantages over CT in depicting intracranial tumors. It has multiplanar capabilities, bone does not degrade image quality, the tissue contrast resolution of MRI is several orders of magnitude more sensitive than CT,31 and the sensitivity to contrast enhancement is inherently better than CT. Although the true size of enhancing and nonenhancing tumor is difficult to estimate with CT, it is more readily assessed with conventional MRI imaging. Today, it is well known that the volume of tumor burden does not predictably correspond to the volume of enhancing tissue.32,33 In fact, gliomas demonstrate variable enhancement characteristics and 10% of anaplastic gliomas exhibit no intrinsic enhancement. In cases of tumor enhancement, the size of the neoplasm has been demonstrated by stereotactic biopsy to be larger than the enhancing portion.32,33 Moreover, even the T2 envelope (the larger region of hyperintensity on the T2-weighted MR images), which has been shown to contain tumor cells, can underestimate the true tumor burden.34 Given these more recent imaging data, contrast-enhanced MRI provides a more comprehensive assessment of tumor size and morphology and should therefore be used to establish updated response criteria for gliomas. Additionally, the size and quality of the T2 envelope should be included with the enhancement parameters to give a complete evaluation of tumor burden through serial scan assessments after treatment. Because of the inherently ambiguous nature of tumor margins by any measure, we designed the use of seven commonly recognized MRI-based imaging characteristics to clarify response class assignments. We felt these qualitative evaluations would be more reproducible and less ambiguous in establishing response classes than any quantitative means of assessment. Using these criteria, any tumor size reduction must be accompanied by additional improvements in either T1- or T2-weighted images to count as a response. This eliminates the overweighting of size as a response indicator and mitigates the inherent ambiguity of quantitative serial size assessments. At the same time it permits a response score for minor or no volume reductions if there is a net improvement in primary features. By our criteria, we scored two patients with unchanged tumor volumes as partial responses that were scored as stable disease by the MacDonald criteria (Table 2, patients no. 9 and 11). In both cases, however, the patients had stable neurologic examinations and the MRI scans revealed unambiguous improvement in the appearance of the tumor, with no additional or ongoing treatments and stable or tapering corticosteroids. There are two obvious criticisms to the observations we have made. The first is that these responses may be purely accidental. The second criticism concerns the mechanism(s) involved if interpreted as a biologic response. The findings of this study cannot be construed as a possible therapy for treatment of malignant astrocytomas. Because this was a pilot study, we focused on the safety of this paradigm in a human trial, and no assessment of clinical efficacy was attempted or performed, including sham-operated or sense/random ODN controls, although these controls were performed in the preclinical animal studies.11,18,19,21,22,35 The findings have been so unexpected and so dramatic, however, that we think they should be brought to the attention of other investigators. We admit that we cannot presently offer an answer to the second question, but some possibilities will be discussed. We have previously addressed the importance of IGF-IR/AS ODN–induced apoptosis as a specific means of achieving a host antitumor response.25 Irradiated C6 cells, for example, were no longer tumorigenic in rats but they were not able to induce tumor regression or confer tumor resistance. Irradiated IGF-IR/AS ODN C6 cells were still able to do both, demonstrating that antitumor effects were specifically associated with IGF-IR/AS ODN–mediated downregulation of the IGF-IR, and this process was unaffected by irradiation.25 In the C6 brain tumor model, we were able to induce regression of intracranial C6 tumor utilizing C6 IGF-IR/AS ODN treated cells encapsulated in diffusion chambers and implanted for 24 hours.23 Tumor regression was not achieved with ODN sense controls, resulting in significantly shorter survival (24 V 221 days). At necropsy, brains from IGF-IR/AS ODN treated animals revealed no evidence of tumor at the inoculation site and instead revealed cystic cavities with associated gliosis, deposits from hemosiderin, and residual inflammatory infiltrates.23 In the animal model, we have assumed a soluble factor is released by the cells undergoing apoptosis, diffusing through the chambers and into systemic circulation. We can only assume a similar process is taking place in the current study, illustrated with histologic examination in the case of patient no. 7 (Figs 2, 6 and 7). It is possible that a peptide or peptides, generated as soluble by-products of a specific apoptotic pathway, serve as biologic effectors. Such peptides could have direct antitumor cytotixicity and/or they could induce cellular immunity. Synthetic peptides mimicking the sequences of antigenic tumor peptides are known to induce apoptosis directly, even at concentrations as low as 10-12 mol/L.36 In patients with metastatic melanoma, Marchand et al37 performed serial subcutaneous injections of the MAGE-3.A1 peptide, an antigen of melanoma cells that was anticipated to induce immunity. Surprisingly, the injection of this peptide caused regression of the melanomas before a cytolytic T-lymphocyte immune response could be detected. They were quite as puzzled by these antitumor responses as we are of ours, but perhaps the remarkable host responses that we have observed may be more common than we think. The induction of cellular immunity is also possible. A physical association between the IGF-IR and MHC-class 1 complexes has been previously described.38 Lafarge-Frayssinet et al39 found that suppression of IGF-1 production in a rat hepatoma cell line (by an antisense approach) caused a four-fold increase in the expression of MHC-class 1 antigens. Several papers have recently provided evidence that apoptosis of human tumor cells releases antigenic peptides that are subsequently taken up by dendritic cells.40 When compared with patients matched with the same tumors in the same location who had undergone previous treatments including radiation therapy,41,42 Gliadel wafer implants,43 stereotactic radiotherapy boost,44 surgery, or corticosteroid therapy, we could not identify comparable improvements (examples included in Figs 5 and 7). Corticosteroids in particular have been associated with CT-based radiographic improvements in recurrent malignant gliomas,45 but all observed improvements in the current study occurred either without a change or reduction in corticosteroid dose.
In three patients without initial improvement, compassionate re-treatments were performed using maximum yields of implantable chambers (range, 10 to 18), and only after re-treatment did we observe radiographic and clinical improvements in two of them (Fig 4 provides an example). These improvements suggest that repeated treatment and/or higher dosing might be required to achieve a response. Additionally, although tumor tissue is unavailable for analysis, the delayed and continuing responses noted in the absence of any subsequent treatments, particularly in the case of patient no. 10 (Fig 4), might suggest a host immune response. In preclinical studies, IGF-IR/AS ODN treatment seems to marshall a host response that has many characteristics of an immune response,10,12,35 and this may serve as an additional antitumor host-response mechanism. Using syngeneic rats, we demonstrated a cytotoxic CD8+ T-cell immune response related to IGF-IR AS/ODN treatment.25 C6 cells expressing either IGF-IR sense or IGF-IR antisense mRNA were injected and splenic lymphocyte function analyzed in vitro after 2 weeks. Cytotoxic, CD8+ lymphocytes from animals injected with IGF-IR antisense cells, but not from those treated with IGF-IR sense cells, proliferated in vitro in response to wild-type C6 cells. In our series, one patient had tumor lymphocytic infiltration before treatment. Previous studies have demonstrated naturally occurring cellular immune-mediated responses in glioma, as well as a positive correlation between lymphocytic infiltration and prognosis.46-50 After IGF-IR/AS ODN treatment an additional four patients acquired a marked lymphocytic infiltration (Table 2). This acquired lymphocytic infiltration noted only after treatment(s) represented a 36% incidence, which was a six-fold higher incidence than previously reported.51 Immunotyping of these samples revealed an infiltrating population of T cells (observations of M.C. and L.K.). If acquired lymphocytic tumor infiltration supports the contention that IGF-IR/AS ODN elicits an immune response, why was this not uniformly seen in all cases? Although the answer is unknown, a recent study by McCluskey and Lampson52 has revealed that the brain, usually described as immunologically privileged, actually displays vigorous immune activity. This activity, however, is variably and regionally controlled by mechanisms within its diverse microenvironments that could explain variability in cell-mediated immune responses in the brain, now confounded by the presence of tumor. As an additional histologic observation, we noted selective tumor microvessel thrombosis in six cases, which may provide an alternative clue regarding a possible mechanism of action and specifically provides an explanation for loss of tumor contrast enhancement seen on MRI. Antitumor effects have been recently documented in an animal model after selective occlusion of tumor vasculature.53 In summary, ex vivo IGF-IR/AS ODN treatment of autologous tumor cells induces massive tumor cell death in vivo and a host response. It is associated with radiographic and clinical improvements not typically seen after conventional treatments. At a molecular level, we are assessing possible mechanisms of antitumor activity in animals and hope such data will guide future translational IGF-IR/AS ODN strategies.
We thank David Abraham, PhD, Gerald Vandergrift, Debroski Herbert, and Ofra Leon for assistance; additionally we thank Carlo M. Croce, MD, Bruce A. Bach, MD, PhD, and Robert L. Capizzi, MD, for their helpful comments.
D.W.A. and M.R. contributed equally to this project.
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Copyright © 2001 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
2% intact by trypan blue exclusion, and none of the intact cells were viable in culture thereafter. Parallel Western blots disclosed IGF-IR downregulation to 
