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Adenovirus-Mediated p53 Gene Transfer in Sequence With Cisplatin to Tumors of Patients With Non–Small-Cell Lung Cancer

From US Oncology, Baylor University Medical Center, and Columbia Medical City, Dallas; and Section of Thoracic Molecular Oncology, Departments of Diagnostic Imaging, Pathology, Thoracic/Head and Neck Medical Oncology, and Biomathematics, The University of Texas M.D. Anderson Cancer Center, and Introgen Therapeutics, Inc, Houston, TX.

Address reprint requests to John Nemunaitis, MD, Physician Reliance Network Research, Inc, 3535 Worth St, Collins Building, 5th Floor, Dallas, TX 75246; email j.nemunaitis{at}usoncology.com

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: To determine the safety and tolerability of adenovirus-mediated p53 (Adp53) gene transfer in sequence with cisplatin when given by intratumor injection in patients with non–small-cell lung cancer (NSCLC).

PATIENTS AND METHODS: Patients with advanced NSCLC and abnormal p53 function were enrolled onto cohorts receiving escalating dose levels of Adp53 (1 x 10⁶ to 1 x 10¹¹ plaque-forming units [PFU]). Patients were administered intravenous cisplatin 80 mg/m² on day 1 and study vector on day 4 for a total of up to six courses (28 days per course). Apoptosis was determined by the terminal deoxynucleotidyl- transferase-dUTP nick–end labeling assay. Evidence of vector-specific sequences were determined using reverse-transcriptase polymerase chain reaction. Vector dissemination and biodistribution was monitored using a series of assays (cytopathic effects assay, Ad5 hexon enzyme-linked immunosorbent assay, vector-specific polymerase chain reaction assay, and antibody response assay).

RESULTS: Twenty-four patients (median age, 64 years) received a total of 83 intratumor injections with Adp53. The maximum dose administered was 1 x 10¹¹ PFU per dose. Transient fever related to Adp53 injection developed in eight of 24 patients. Seventeen patients achieved a best clinical response of stable disease, two patients achieved a partial response, four patients had progressive disease, and one patient was not assessable. A mean apoptotic index between baseline and follow-up measurements increased from 0.010 to 0.044 (P = .011). Intratumor transgene mRNA was identified in 43% of assessable patients.

CONCLUSION: Intratumoral injection with Adp53 in combination with cisplatin is well tolerated, and there is evidence of clinical activity.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
I N 1997, THERE WERE approximately 178,000 new diagnoses of lung cancer and 164,000 lung cancer–related deaths.¹ Therapeutic options that improve survival for non–small-cell lung cancer (NSCLC) are limited.^2-8 Surgical resection is the treatment of choice for patients in early stages of disease (I, II).⁶ Patients with a later stage of disease are treated palliatively with surgery, chemotherapy, and/or radiation therapy, all of which involve significant side effects that can be devastating to quality of life.^2-8 At 2 years, expected survival is 20% in patients with stage III disease and less than 5% in patients with stage IV disease.

The most active single agent frequently used in combination chemotherapy is cisplatin.⁸ Several new agents (navelbine, irinotecan, topotecan, gemcitabine, and docetaxel) have demonstrated activity in NSCLC.^9-14 However, median survival has not been improved, and toxicity to combination chemotherapy is significant.

The p53 gene is abnormal in 40% to 74% of NSCLC samples tested.^15-17 Therapeutic approaches involving gene therapy targeting the p53 gene have been explored in preclinical models.¹⁸ Genes transcriptionally activated by p53 include: BAX, a positive regulator of apoptosis¹⁹; MDM-2, a negative regulator of p53 function²⁰; thrombospondin I, an inhibitor of angiogenesis²¹; and GADD45, which plays a role in DNA repair.²⁰ A key function of p53 protein is to control progression of cells from G₁ to S phase. p53 protein transcriptionally activates a p21-Kd protein, which inhibits cyclin-dependent kinases.^22,23 Inhibition of cyclin-dependent kinase activity seems to block the release of transcription factor E2F, causing failure of activation of transcription genes required for S-phase entry.^23,24

Recent evidence suggests that normal p53 function prevents inherent mutability of the human genome in somatic cells. Interference with this function may render cells hypermutable.²⁵ Mutations involving the p53 gene typically occur with greater frequency in patients with late-stage cancer²⁶ and at sites of metastatic disease.²⁷ Additionally, undifferentiated tumors are more likely to harbor a mutant p53 gene.²⁸

Altered protein function (suggested by elevated expression) and/or mutation of the p53 gene is associated with poor prognosis in patients with NSCLC and a variety of other malignancies.^29-35 The presence of a p53 mutation may also identify patients more likely to be resistant to chemotherapy or radiotherapy.³⁶

Recent studies have reported the introduction of a wild-type p53 gene into human tumor cells with a mutant p53 genotype using a variety of delivery methods, including retroviral vectors, lipid complexes, and adenoviral vectors.^37-44 These results demonstrated expression of the transgene and have confirmed normal function of the expressed p53 protein, which caused tumor regression and survival improvement in animal models. Preclinical results also show that antitumor effects are enhanced by combined treatment with cisplatin.^44,45

Initial trials in humans using retroviral p53 gene transfer via intratumor injection showed no toxicity and demonstrated evidence of antitumor activity in three of nine patients with NSCLC.⁴⁶ However, low transduction efficiency associated with the retroviral vector was a major limiting factor.

The purpose of the current study was to explore the use of adenovirus-mediated (Ad) wild-type p53 gene transfer in combination with cisplatin in patients with NSCLC.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Objectives
The objectives of this trial were as follows: (1) to determine the degree of toxicity and its reversibility after monthly intralesional administration of Adp53; (2) to determine the maximum-tolerated dose of Adp53 administered after intravenous cisplatin; and (3) to document observed antitumor activity.

Study Design
This was an open-label, nonrandomized, phase I dose-escalation study. Patients were sequentially enrolled onto cohorts of three patients. Adp53 dose levels were as follows: 1 x 10⁶ plaque-forming units (PFU), 1 x 10⁹ PFU, 3 x 10⁹ PFU, 1 x 10¹⁰ PFU, 3 x 10¹⁰ PFU, and 1 x 10¹¹ PFU. Patients were treated by direct intratumor injection into a single indicator lesion. Each patient received an assigned dose level throughout the study; there was no dose escalation within the same patient. The first nine patients were planned to be treated as inpatients at M.D. Anderson Cancer Center (Houston, TX). If safety was acceptable, all subsequent patients would be treated in the phase I outpatient clinics at M.D. Anderson Cancer Center and Physician Reliance Network, Baylor University Medical Center, and Medical City Hospital (Dallas, TX). One course of study treatment consisted of a 28- ± 5-day period during which patients were administered intravenous cisplatin 80 mg/m² on day 1 and study vector on day 4, for a total of up to six courses. Patients were monitored for adverse events and clinical effects throughout the study. All patients were considered assessable for safety from the time of their first dose of study treatment. Treatment could be continued as long as there was no progression of the indicator lesion, no unacceptable adverse events, and no additional anticancer therapy administered to the indicator lesion. For the purpose of this trial, the term "study treatment" included Adp53, cisplatin, and the method of delivery of Adp53 (bronchoscopy or computed tomography [CT]-guided needle injection). Survival data were collected until death. This study was approved by the M.D. Anderson Surveillance and Biosafety Committees, the Physician Reliance Network Clinical Research Advisory Board, and by the M.D. Anderson and Physician Reliance Network institutional review boards. All participating patients gave written, informed consent.

Patient Clinical Evaluation
Baseline evaluation included medical history, physical examination, review of concomitant medication, hematology (complete blood count, differential, platelet count, prothrombin time, and partial prothrombin time), biochemistry (calcium, sodium, potassium, magnesium, phosphate, total protein, albumin and creatinine, AST, ALT, lactate dehydrogenase, urea, and total bilirubin), urine analysis, serum HCG, human immunodeficiency virus serology, and respiratory assessment (pulse oximetry and spirometry). Tumor assessment at baseline included chest radiograph, chest and abdominal CT scan, magnetic resonance imaging (MRI), and bronchoscopic photographs. Biopsies were performed at baseline to document p53 mutation, transgene expression, and histology. Samples of aerodigestive secretions, urine, and plasma were collected to determine baseline values for vector dissemination assays. Serum was also assessed at baseline for antiadenoviral type 5 antibodies and neutralizing activity.

On-study evaluations included monthly physical examination, medical history, concomitant medication review, assessment of adverse events, hematology, biochemistry, respiratory assessment, antiadenoviral type 5 antibodies, neutralizing activity, and tumor assessment. Biopsies were assayed by hematoxylin and eosin (H&E) staining, p53 immunohistochemistry (IHC) terminal deoxynucleotidyl-transferase-dUTP nick–end labeling (TUNEL), and reverse-transcriptase polymerase chain reaction (RT-PCR) of the Adp53 transcript. Aerodigestive secretions, urine, and plasma were assayed for vector dissemination by cytopathic effect assay (CPE), Ad5 hexon, enzyme-linked immunosorbent assay (ELISA), and PCR (see below).

At follow-up visits, each patient underwent a complete history assessment; a clinical examination, including vital signs (temperature, pulse blood pressure, respiration rate), height, weight, Zubrod performance status, hematology, and biochemistry assessment; and assessment of any residual clinical signs and symptoms.

Vector Administration
Adp53 vector was administered by fine-needle injection directly into the tumor. Administration was performed bronchoscopically or percutaneously via CT. Indicator lesions 4 cm in diameter were administered in a volume of 10 mL through a single injection site. For indicator lesions with a diameter less than 4 cm, the final volume administered was 3 mL. The Adp53 clinical formulation was diluted with Dulbecco’s phosphate-buffered saline to obtain the volume necessary for each dose.

Cisplatin Administration
Patients received cisplatin at a dose of 80 mg/m² administered intravenously over 2 hours on day 1 of every course. Cisplatin was diluted in 500 mL of normal saline plus 8 mEq of magnesium sulfate. Hydration with 500 mL of 5% dextrose in water and 0.45% normal saline plus 8 mEq of magnesium sulfate was given over 2 hours before cisplatin infusion and again for 2 hours after cisplatin infusion. All patients received antiemetics as per clinical indication.

Patient Population
Patients with histologically confirmed NSCLC who either had an endobronchial tumor accessible by bronchoscope or locally advanced malignancy in the lung or other organs accessible bronchoscopically or by CT-guided needle injection were enrolled onto the study. Numbers assigned to patients were not consecutive because NSCLC patients participating in research at the investigative sites were entered onto one of two trial arms: one investigating the effect of Adp53 alone and the other the combination of Adp53 and cisplatin. Patients must have had a life expectancy of at least 12 weeks and a Zubrod performance status 2 at the time of enrollment. All patients were required to have p53 dysfunction demonstrated by tumor DNA sequencing analysis or by elevated p53 expression by IHC before treatment. Patients were required to have adequate marrow (granulocyte count > 2,000/µL, platelet count 100,000/µL), liver (bilirubin level 1.5 mg/dL), and renal function (creatinine concentration < 1.5 mg/dL), and a forced expiratory volume in 1 second 40% of the predicted value. All patients of childbearing potential were required to have a negative serum pregnancy test result. Patients with prior platinum-based therapy were eligible for study. However, patients with symptomatic brain metastasis, prior gene therapy, use of any investigational agent within 4 weeks of study treatment, use of chemotherapy within 21 days before study entry, or use of immunosuppressive therapy (including corticosteroids 10 mg/d) were excluded from the trial.

Response Evaluation
Clinical signs and symptoms were assessed for each patient. The indicator lesion underwent biopsy for histology and was assessed for response before each course of treatment and/or at study day 28 (± 5 days). Comparability was ensured through the use of identical techniques when performing the baseline radiographs, CT scans, MRI, or photographic evaluation and subsequent follow-up evaluations. Patients were assigned a response category according to the following response criteria: (1) complete response was the complete disappearance of all clinical evidence of the indicator lesion by physical examination, roentgenography, MRI, and/or CT scans for a minimum of 4 weeks; (2) partial response was defined as decrease of 50% in the product of the perpendicular diameters of the indicator lesion for a minimum of 4 weeks by radiologic studies or bronchoscopic visual measurement; (3) progressive disease was defined as an increase of 25% in the product of the perpendicular diameters of a bidimensionally measurable indicator lesion (in patients with a unidimensionally measurable lesion, an increase of 25% in the single measurement qualified as progressive disease; in patients with assessable disease only, disease progression was determined clinically, eg, performance status decrease of two scale points, increase in tumor-related symptoms, and so on); and (4) stable disease was outlined as any variation of the indicator lesion assessment not meeting the criteria of a complete response, partial response, or progressive disease.

Time to progression was measured from the first dose of study treatment until there was evidence of progressive disease at the indicator lesion site. Survival was measured from first study treatment until death. Survival was evaluated quarterly after patient discontinuation from study.

Adenoviral Vector Construction and Testing
Adp53 is a replication-defective adenovirus serotype 5 vector. It carries a human p53 expression cassette containing the cytomegalovirus (CMV) promoter, human wild-type p53 cDNA, and the SV40 polyadenylation signal in place of the adenoviral E1 region. The wild-type sequence of the p53 cDNA in the vector was confirmed by dideoxy DNA sequencing.⁴⁷

The Adp53 stock used to produce the clinical vector lots for this study was qualified by testing for sterility, Mycoplasma, and replication-competent adenovirus. Lots of Adp53 used in this trial were manufactured under Good-Manufacturing Practices, formulated as sterile suspensions in phosphate-buffered saline containing 10% glycerol, and stored in 1.8-mL cryovials between -60°C and -80°C until use. Each lot was tested for contaminants of human immunodeficiency virus, CMV, human papillomavirus, Epstein-Barr virus, adeno-associated virus, human parvovirus, human T-cell lymphoma virus, Mycoplasma, bacteria, fungi, and endotoxin content. Two lots were used in this study. No contaminants or replication competent adenovirus were detected.

Tumor Biopsy Evaluations
H&E staining. Prestudy, day 3, day 7, and monthly biopsy specimens were evaluated for tumor content, necrosis, and the presence of inflammatory cell infiltrates after standard H&E staining. Specific inflammatory scores ranging from mild to severe were assigned at baseline and at follow-up posttreatment time points and indicated the degree of acute inflammation, chronic inflammation, and no inflammation. Results were determined on coded slides by a single, blinded observation.

p53 mutation analysis. Pretreatment biopsy specimens were used to determine tumor p53 mutation status by p53 DNA sequencing at the University of Texas M.D. Anderson Cancer Center in the laboratory of Jack Roth, MD; at the John T. Mallams Laboratory of John Nemunaitis, MD, supported by the Mary C. Crowley Foundation at Baylor University Medical Center (Dallas, TX); or at OncorMed, Inc (Gaithersburg, MD). DNA was extracted directly from sections of formalin-fixed paraffin-embedded needle biopsy specimens. If necessary, tumor cells were microdissected from sections of paraffin-embedded tissue. DNA was isolated from tumor cells using phenol/chloroform extraction and ethanol precipitation, and the p53 gene was amplified in several fragments by PCR. The amplicons were purified by gel electrophoresis and analyzed using either the DNA Sequencing Kit (Boehringer Mannheim, Indianapolis, IN) or the ABI model 310 automated sequencer (PE Applied Biosystems, Foster City, CA). Accurate reading of the sequencing gels and the sequencer printouts was confirmed by National Biosciences, Inc (Plymouth, MN).

Immunohistochemistry for p53. p53 expression was determined by an automated immunoperoxidase IHC technique (Ventana 320ES; Ventana Medical Systems, Tucson, AZ) as described previously,^48,49 using the p53 primary antibody clone Bp53-11 (Ventana). Subsequent p53 IHC testing was performed by Quest Diagnostics, Inc (San Juan Capistrano, CA), according to their standard operating procedure using the primary antibody DO-7 (Dako, Carpinteria, CA). A minimum of 200 tumor cells was evaluated whenever possible, and the number of cells with positive nuclear staining was reported. The degree of stromacell p53 staining was also evaluated, and the level of background staining in noncellular areas was noted to ensure low nonspecific staining. The results were reported as the percent of cells (tumor or stroma) with positive nuclear staining. A positive result required a minimum of 10% of tumor cells stained.

TUNEL assay. The degree of apoptosis was assessed by TUNEL^50,51 in biopsy samples collected before treatment and on day 7 of each course. The treatment status was blinded to the technicians who performed and read the assay results. The tests were conducted by Quest Diagnostics, Inc, using the ApopTag Plus Detection Kit (OncorMed). A minimum of 200 cells was evaluated whenever possible. The apoptotic index was recorded as the number of positive cells observed per number of cells evaluated. If a single cell was positive, this was interpreted as evidence of apoptosis. Results of posttreatment samples were compared with baseline values.

RT-PCR vector-specific PCR. Total RNA extraction, reverse transcription, PCR amplification, and blot hybridization were performed by a modification of a previously described technique.⁵¹ Tumors were analyzed for transgene expression by reverse RT-PCR using flash-frozen biopsy samples collected during each treatment course, at baseline, and 3 days after injection (day 7). Total RNA was extracted using the RNeasy Kit (Qiagen, Valencia, CA). The RNA was DNAse-treated and then reverse-transcribed using Ready.To.Go You-Prime First-Strand Beads (Amersham Pharmacia Biotech, Piscataway, NJ). A nested PCR procedure was used with vector-specific primers CMV3:5'-GGTGCATTGGAACGCGGATT-3' and Rev Ex3:5''-CAAATCATCCATTGCTTGGGA-3'' used for the first round, and CMV 3 + RN3:5'-GGGGACAGAACGTTGTTTTC-3'' used for the second round of amplification. Human glycoraldehyde-3-phosphate-dehydrogenase (GAPDH) primes (GAPDH-5:5''CAGCCGAGCCACATC-3'' + GAPDH-AS:5'' -TGAGGCTGTTGTCATACTTCT-3'') were used as a positive reaction control. All samples underwent PCR amplification without prior reverse transcriptase treatment to test for completeness of DNAase digestion. PCR products were identified by Southern blot hybridization.⁵²

Antibody response assay. Serum samples were tested for the presence antiadenoviral type 5 immunoglobulin G by Virolab, Inc (Berkeley, CA) using an indirect immunofluorescence assay to indicate the patient’s humoral immune response to the vector. The data are reported as the inverse of the dilution of patient serum required to give a positive result in the assay; larger numbers indicate higher levels of antiadenoviral type 5 antibody. Assays were conducted on serum samples collected at baseline and at day 28 of each course.

Neutralizing activity assays were performed at M.D. Anderson Cancer Center in the laboratory of Jack Roth, MD.⁵³ The assay measured the ability of patient serum to block adenoviral vector infection of cells in culture. The data are reported as the inverse of the dilution of patient serum required to give a positive result in the assay; larger numbers indicate higher levels of neutralizing activity. Assays were conducted on serum samples collected at baseline and at day 28 of each course.

Vector Dissemination and Biodistribution Analyses
CPE assay. The CPE assay is a bioassay designed to semiquantitatively detect the amount of vector contained in a biologic fluid.⁵³ The assay can also detect replication-competent adenovirus. CPE assays were performed on two cell lines, IT293 and A549. If a biologic fluid tests positive on IT293 cells and negative on A549 cells, that fluid contains Adp53. A positive CPE result with both IT293 and A549 cells indicates the following: (1) nonspecific toxicity; (2) the presence of replication-competent adenovirus; or (3) some other infectious agent that kills A549 cells.

Urine samples (first morning void) and aerodigestive secretion samples (sputum or saliva) were frozen and thawed once, sterile filtered, diluted, and added to monolayers of IT293 or A549 cells for the CPE assay. Plasma samples collected from cell preparation tubes (Becton Dickinson, Franklin Lakes, NJ) were added directly to the cell monolytes without filtering or dilution. The cell monolayers were examined daily for a cytopathic effect, indicating the presence of virus in the patient sample.

Ad5 hexon ELISA. Supernatants from positive CPE assays were tested for Ad5 hexon protein with a commercially available ELISA kit (Adenoclone EIA; Meridian Diagnostics, Cincinnati, OH), to confirm the presence of adenovirus in the bioassay.

Vector-specific PCR assay. DNA was isolated from plasma using the QIAamp Blood Kit (Qiagen) and analyzed for the presence of vector DNA by PCR. Oligonucleotide primers were obtained from Oligos, Etc. (Wilsonville, OR). The PCR product crosses a p53 open-reading-frame/adenoviral DNA junction and is therefore specific for the Adp53 vector.

Statistical Analysis
Exploratory and descriptive data analysis was applied as the primary statistical analysis tools. Summary statistics such as the mean, median, and SD were calculated to characterize the distribution of the data. Frequency tabulation was given whenever appropriate. Two-sided Wilcoxon signed-rank was applied to compare baseline and follow-up apoptotic indices. We took the pretreatment apoptotic index (AI) in course 1 as the baseline AI, and the average of all available AI after the p53 injection as the follow-up AI. The survival curves were computed using the Kaplan-Meier estimates.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Demographics
Twenty-four patients with advanced NSCLC whose prior radiation therapy or chemotherapy had failed were entered onto the trial (five women and 19 men; 23 white and one Hispanic). The mean age was 64 years (range, 47 to 77 years). All patients had a Zubrod performance status of 2. Eighteen patients (75%) had prior chemotherapy, and 22 patients (91%) had prior radiation therapy (see Table 1). Multiple sites of disease were observed in 16 patients (67%), and the mean area of tumors was 25 cm² (range, 3 to 80 cm²). The first nine patients were treated as inpatients, and all subsequent patients were treated in outpatient phase I clinic facilities.

The cisplatin dose was modified or discontinued as a result of toxicity in eight (33%) of the 24 patients. Six patients (patients no. 21, 40, 48, 50, 51, and 53) did not receive complete courses, and two patients (patients no. 5 and 21) had a dose modification because of cisplatin-related adverse events (Table 3). Two patients (patients no. 14 and 25) received an additional course of cisplatin, but Adp53 was discontinued because of bilateral brachicephalic vein thrombosis or disease progression, respectively. One patient (patient no. 19) was not assessable for cisplatin-related toxicity after course 1 because of exacerbation of previously existing cardiac difficulties.

The two patients (patients no. 40 and 49) who achieved a partial response were treated endobronchially. An additional patient (patient no. 15) achieved a partial response 28 days after his second CT-guided injection but was unable to undergo a 30-day follow-up CT scan to confirm response. Of the seven patients who received injections of Adp53 to endobronchial sites, five (patients no. 26, 35, 40, 48, and 49) achieved substantial reduction in the obstructing tumor mass to significantly relieve bronchial obstruction. Figures 1 through 4 show evidence of response in patients no. 26, 40, 48, and 49, respectively.

View larger version (79K): [in this window] [in a new window]   Fig 1. Evidence of response in patient no. 26 (A, before; B, 1 month after).

View larger version (73K): [in this window] [in a new window]   Fig 2. Evidence of response in patient no. 40 (A, before; B, 1 month after).

View larger version (47K): [in this window] [in a new window]   Fig 3. Evidence of response in patient no. 48 (A, before; B, 1 month after; C, 2 months after).

View larger version (67K): [in this window] [in a new window]   Fig 4. Evidence of response in patient no. 49 (A, before; B, 1 month after).

Tumor Biopsy Evaluations
p53 mutation analysis. Tumor biopsy specimens at baseline from 21 (91%) of the 23 assessable patients demonstrated p53 mutations by DNA sequencing (Table 4). No mutations were detected in exons 5 to 9 in patients no. 20 or 41. Insufficient tissue was available for p53 DNA sequencing in patient no. 40. All three of these patients showed evidence of abnormal p53 function based on elevated expression by IHC. Twenty-two tumor samples were assessable by IHC, and 18 (82%) had elevated expression. Fifteen (79%) of 19 assessable tumors had both a p53 mutation and elevated p53 protein expression. The most frequent amino acid change involved a tyrosine to a cysteine (patients no. 19, 27, 50, 52, and 53).

TUNEL assay. Fourteen (58%) of the 24 patients had TUNEL assay results available to assess apoptosis for both pretreatment and at least one posttreatment time point. Posttreatment time points varied from 2 to 24 days after treatment, and the number of malignant cells evaluated per sample ranged from 12 to 200. Ten patients were unable to provide baseline or follow-up samples because of limited availability of sufficient tumor tissue. TUNEL assays performed showed an increase in the number of apoptotic cells in 11 (79%) of the 14 patients, a decrease in one (7%), and no change in two (14%). Comparison of the mean apoptotic index between baseline and follow-up measurements showed a statistically significant increase from 0.010 to 0.044 (P = .011).

RT-PCR. RT-PCR analysis for transgene p53 mRNA was performed on at least one posttreatment biopsy specimen (with a corresponding pretreatment baseline sample) from 14 of the 24 patients. p53 mRNA was detected at follow-up evaluation in six (43%) of the assessable patients. One patient (patient no. 26) received 3 x 10⁹ PFU, two patients (patients no. 39 and 41) received 3 x 10¹⁰ PFU, and three patients (patients no. 48, 49, and 50) received 1 x 10¹¹ PFU. These results confirm vector-specific transgene expression and may suggest a dose-related effect because the majority of patients found to have intratumor transgene expression had received 3 x 10¹⁰ PFU. Ad5 neutralizing antibody (see following section) was also shown to be elevated at the time of transgene expression, confirming the potential for expression of the transgene product in the setting of elevated neutralizing antibody.

Immune Response to Adenoviral Vector
Titers of antiadenoviral type 5 immunoglobulin G antibodies and neutralizing activity were determined for 23 of the 24 patients, using serum samples collected before treatment and immediately before courses 2 and 3. An antibody response ( twofold increase) in 19 of 21 assessable patients before course 2 and in 15 of 15 patients before course 3 was observed (see Table 5). The two patients (patients no. 5 and 19) with no antibody response before course 1 both had elevated titers at baseline and showed a twofold and fourfold increase before course 2. A similar response was observed in neutralizing adenoviral antibody titers (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Use of intratumoral injection was performed to maximize the concentration of the viral vector within the tumor mass and to minimize potential toxicity related to systemic circulation of the vector. Administration of intratumoral injections of Adp53 after cisplatin was clinically well tolerated at dose levels of up to 1 x 10¹¹ PFU. Fever was observed as the most frequent treatment-related toxicity and may have been related to the transient systemic spread of the vector. Results of blood and body fluid analysis confirmed transient systemic spread of the vector.

The observation of systemic circulation of Adp53 in this trial after intratumoral injection may raise safety concerns. However, over the last few years, evidence has mounted that suggests that systemic administration of adenoviral serotype 5 vectors is relatively safe.⁵⁴ Eighty percent of adults have existing antibodies to adenovirus serotype 5,⁵⁵ but 15% of exposed patients are clinically symptomatic.⁵⁶ Oral adenoviral vaccines were given to thousands of military recruits in the 1960s without adverse effects.⁵⁷ Live adenovirus inoccula was also given intratumorally and intraarterially to patients with cervical carcinoma at the National Cancer Institute in the 1950s.⁵⁸ No significant toxicities (other than transient fever and malaise) were observed, even in subsets of patients in whom neutralizing adenovirus antibodies were not present and in patients concurrently treated with corticosteroids. Theoretical concerns regarding the oncogenicity of adenovirus are also unlikely. The life cycle of an adenovirus does not require integration into the host genome; thus, foreign genes delivered by adenoviral vectors are expressed episomally and have low genotoxicity in host cells.⁴² DNA from thousands of human tumors has also been analyzed for the presence of adenovirus DNA, no integrated viral DNA has been isolated from any human tumor,⁵⁹ and long- and short-term safety of adenoviral vector injections has also been shown in several animal models.^{18,39,42,43,60-73} Studies after preparation of the vector with repeat sequencing also show that the Adp53 vector does not undergo mutational changes, and it maintains wild-type p53 DNA throughout the manufacturing process.⁴⁷

Pathogenicity to high doses of bronchial airway administration of Adp53 in animals has been observed in the lung.^42,60,68,74 However, intratumoral or systemic administration with human group C adenovirus in animals showed no histopathologic changes or severe pulmonary or other organ site toxicity.^75-77 Inflammatory responses observed in animals after high-dose bronchial administration of Adp53 are characterized by interstitial infiltration of neutrophils and monocytes within 1 to 2 days of exposure.^74,75 This early inflammatory process is mediated by local elaboration of various cytokines such as tumor necrosis factor and interleukins 1 and 6.⁷⁸ Later, additional inflammatory response (within 3 to 7 days) involving peribronchial infiltration of lymphocytes occurs. This is believed to be mediated by cytotoxic T lymphocytes. However, direct exposure to the lung in animals with low concentration of adenovirus vector does not seem to be associated with pulmonary toxicity.^61,62 Treatment doses in this trial were below toxic levels observed in animals. However, inflammatory response not causing pulmonary symptomatology may, theoretically, contribute to local tumor regression, although biopsy of injected tumor tissue in patients who received Adp53 alone showed no inflammatory cell infiltration,⁷⁶ and in the present trial no inflammatory infiltration was observed after treatment in comparison to baseline.

Clinical activity was suggested in six of the 24 patients treated. This may be attributed to cisplatin alone, although all patients who showed evidence of activity previously experienced treatment failure with either cisplatin or carboplatin, and tumor regression occurred exclusively in the injected lesions. None of the patients who received platinum-based therapy after prior failure showed evidence of response in noninjected lesions. Preclinical data suggest that prior administration of cisplatin significantly increases tumor responsiveness to Adp53 injection.^43,44 Not all patients entered onto this trial received prior treatment with platinum-based agents; therefore, they may have different degrees of responsiveness related to tumor resistance to platinum-based agents. Other investigators have also shown the capacity of mitomycin and doxorubicin to increase sensitivity to Adp53, resulting in enhanced tumor regression when compared with either agent alone, but vincristine had no effect on Adp53 sensitivity.⁷⁷ Five of the patients who demonstrated antitumor activity of cisplatin/Adp53 were treated at endobronchial sites, which is consistent with activity observed with retroviral p53 injection in patients with NSCLC, where the only three patients who responded had received endobronchial injection.⁴⁶ The endobronchial lesions tended to be smaller in size and were visually accessible, thereby confirming injection access. Two of five patients in this trial met a formal definition of partial response ( 50% decrease in tumor measurement as compared with baseline), and three patients with endobronchial airway obstruction experienced significant opening of the airway but did not have a 50% reduction in overall tumor volume that extended beyond disease located in the endobronchial airway.

Several explanations are plausible for reasons as to why additional patients did not show evidence of antitumor activity and why activity may have been limited to these six patients. Variance of the intratumor dispersion of vector may limit gene transduction capacity and diffusion of the expressed product such that some differences in local tumor response may be related to intratumoral vector distribution.^41,78 Systemic variability of vector distribution from patient to patient was shown in the present trial, although an analysis of the tumor nodule for vector distribution was not performed. Intratumor and peritumor injection of Ad beta-galactosidase has been shown to be associated with nonuniform transgene expression after subcutaneous intratumor injection.^37,41 The degree of tumor regression and timing of tumor regrowth seem to be correlated with the efficiency of transduction. Additional factors such as the interaction of adenoviral coat proteins with cellular alpha_V integrins^43,79-81 and whether the malignant cells contain adenoviral surface receptors⁴¹ affect the efficiency of internalization of the adenoviral vector. Other factors also affect adenovirus binding and subsequent internalization. For instance, in vitro work with various breast cancer cell lines show efficient transduction of Adp53 in some but poor transduction efficiency in others despite transduction with the same viral load, equivalent levels of alpha_V integrin expression, and a similar mutation of p53 DNA.⁴¹ Data are also available that suggest that the particular mutation, which is contained within the malignant cells, may also influence clinical response even in the setting of adequate vector internalization and transgene expression.⁴¹ In addition, cells with increased levels of MDM-2 have also been shown to be resistant to Adp53 transduction.⁸² No consistent correlation between clinical activity and the specific p53 mutant or presence of elevated p53 expression within the baseline malignant cell samples from the injected patients were observed in this trial, and alpha_v integrin and MDM-2 status were not assessed.

Most patients showed an increase in apoptotic tumor cells after treatment with Adp53, and the mean apoptotic index was significantly higher at follow-up evaluation. Testing of tissue for evidence of apoptosis with histologic-based assays is difficult. Identification of as little as 2% of malignant cells undergoing apoptosis at any one time has been correlated with as much as a 25% regression of tumor tissue per day.⁷⁷ It has also been suggested that the optimal time to evaluate apoptosis after transduction with Adp53 is less than 3 days after injection.^80,83,84 The entire apoptotic process generally lasts 3 hours and is not synchronized from cell to cell.⁸³ Most samples were obtained 3 days after injection, which underestimates the degree of apoptosis induced by Adp53 in this trial. Cisplatin alone can induce a low degree of apoptosis, but apoptosis induced by cisplatin is far less than activity observed with Adp53^28,84-88 or the combination of Adp53 and cisplatin.⁸⁹ Patients who received injection retroviral p53 gene vector or Adp53 without cisplatin also showed significant evidence of apoptosis.^76,90 Thus, it is unlikely that cisplatin contributed significantly to apoptosis.

There are data that suggest that inhibition of angiogenesis may contribute to the antitumor effect of Adp53 because overexpression of p53 inhibits expression of vascular endothelial growth factor.⁹¹ p53 also increases expression of thrombospondin-1, which is an inhibitor of angiogenesis.²¹ BAX, BAD, and Bcl-2 expression also seem to affect p53-dependent apoptosis.⁹² These variables could have affected the clinical activity observed in this study.

The neutralizing antibody response observed in this trial may have limited transduction efficiency, to some degree, particularly with respect to the bystander effect.⁵⁰ However, transgene expression and evidence of clinical activity were evident after multiple sequential injections in the presence of elevated levels of neutralizing antibodies.

In conclusion, limitations observed with direct intratumor injection of Adp53 combined with the excellent safety observed during periods of systemic dissemination suggest that parenteral administration of Adp53 may be considered as an avenue of future investigation. Animal data exploring systemic administration of p53 when complexed with lyposomes have shown efficacy and good tolerability.^18,39

The antitumor activity observed in patients with endobronchial lesions and the associated morbidity with endobronchial obstruction may suggest further exploration of direct intratumor injection of Adp53 into endobronchial obstructed lesions containing a p53 mutation, and may be another potential area of clinical investigation. Because the activity of Adp53 has been fairly uniform within different solid tumors containing p53 mutations, it is likely that any nonhematologic malignancy (with a p53 abnormality) that obstructs a bronchial airway or possibly other orifices may be a reasonable direction for further investigation. Furthermore, in this trial only one lesion was injected, because the majority of disease, which progressed, occurred in noninjected sites. Injection of all visible lesions could also be considered for further investigation.

	ACKNOWLEDGMENTS

 
Supported by Introgen Therapeutics, Inc, Houston, TX; RPR Gencell, Hayward, CA; Rhône-Poulenc Rorer, Collegeville, PA; and the Mary C. Crowley Foundation, Dallas, TX.

We thank Ana Petrovich for excellent manuscript preparation; the following physicians for excellent patient treatment and care: Barry Brooks, Ronald Kerr, Steven Paulson, Claude Denham, Lisa Fichtel-Meyer, Svetislava Vukelja, Anuradha Gupta, Victor Hirsch, Lewis Duncan, Robert Ruxer, Victor Horadam, Robert Mennel, Allan Shulkin, Michael Savin, and Thomas Anderson; and Nadine Ognoskie for organization of trial performance.

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