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(Chest. 2000;118:966-970.)
© 2000 American College of Chest Physicians

Adenoviral-Mediated p53 Gene Transfer to Non-small Cell Lung Cancer Through Endobronchial Injection*

David Weill, MD; Michael Mack, MD; Jack Roth, MD; Stephen Swisher, MD; Scott Proksch; James Merritt, MD and John Nemunaitis, MD

* From the Medical City Dallas Hospital (Drs. Weill and Mack), Dallas; University of Texas (Drs. Roth and Swisher), MD Anderson Cancer Center, Houston; Introgen Therapeutics (Mr. Proksch and Dr. Merritt), Houston; and US Oncology (Dr. Nemunaitis), Dallas, TX.

Correspondence to: John Nemunaitis, MD, Associate Director, Clinical Research, 3535 Worth St, Collins Bldg, 5th Floor, Dallas, TX 75246; e-mail: John.Nemunaitis{at}USOncology.com


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Objective: The objective was to determine the degree of toxicity and antitumor activity following bronchoscopic injection of an adenoviral-mediated p53 gene (Adp53) into tumors causing airway obstruction.

Dosing: This was a subset analysis of a phase I dose escalation trial.

Setting: Patients were treated in the outpatient clinics at the University of Texas (MD Anderson Cancer Center, Houston, TX) and at Medical City Dallas Hospital (US Oncology, Dallas, TX).

Patients: Twelve patients (median age, 60 years) with advanced endobronchial non-small cell lung cancer (NSCLC) (squamous cell carcinoma, six patients; adenocarcinoma, six patients) were entered into trial. The median tumor area was 5 x 3.2 cm. All patient tumors contained a p53 gene mutation.

Interventions: Adp53 (dose range, 1 x 106 to 1 x 1011 plaque-forming units) was administered by bronchoscopic intratumoral injection once every 28 days.

Measurements and results: Toxicity attributed to the Adp53 vector was minimal. Six of the 12 patients had significant improvement in airway obstruction, and 3 patients met the criteria for partial response.

Conclusions: Direct bronchoscopic injection of Adp53 into endobronchial NSCLC is safe, with acceptable levels of toxicity. The initial clinical results demonstrating relief of airway obstruction warrant further clinical investigation.

Key Words: adenovirus • lung cancer • p53


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The incidence and mortality of lung cancer have increased over the past few decades. Resection is the treatment of choice, but it is not an option for the majority of lung cancer patients. Despite the variety of nonsurgical therapeutic options that are available to these patients, survival is still poor and declines with advanced stage of disease. Unfortunately, one third of patients who receive a diagnosis of non-small cell lung cancer (NSCLC) present with locally advanced, unresectable disease.

Many prospective and retrospective studies have shown that local disease control improves survival. Therapeutic approaches involving obstructed bronchial airways have limited efficacy. Palliative management includes focal radiation therapy, brachytherapy, laser therapy, or chemotherapy. Symptoms such as hemoptysis, dyspnea, and chest pain often can be effectively palliated by external beam radiation.1 In a large study by the Radiation Therapy Oncology Group, 24% of patients experienced complete relief of symptoms and an additional 47% had partial symptom relief.2 However, atelectasis, an indicator of airway obstruction, was relieved by external radiation in only 23 to 60% of patients.3 4 Endobronchial irradiation (or brachytherapy) involves either the surgical or bronchoscopic placement of a radioactive source near the tumor. Additionally, video-assisted thoracoscopic brachytherapy can be used to treat pleural and intrathoracic lesions. This therapy is particularly useful in patients whose disease has progressed despite aggressive external radiation treatment or in whom additional external radiation would not be well tolerated from a cardiopulmonary standpoint. In studies reviewing the efficacy of high-dose brachytherapy, the majority of the patients had a symptomatic response to the treatment. Hemoptysis, cough, and dyspnea were the most commonly relieved symptoms following brachytherapy. Complications were rare and included perforation, ulceration, and pulmonary hemorrhage.5

Light amplification by stimulated emission of radiation can be used to restore potency to an airway obstructed by a central lesion. The majority of patients (68 to 93%) achieved a subjective response, although the duration of benefit is unclear.6 7 8 Complications include bronchial wall perforation, hemorrhage, pneumothorax, and esophageal-bronchial fistulas. Additionally, one disadvantage of therapy with light amplification by stimulated emission of radiation is the need for general anesthesia, with its attendant risks.

The most active single agent against NSCLC frequently used in combination chemotherapy approaches is cisplatin. Several new agents (vinorelbine tartrate, topotecan, gemcitabine, irinotecan, and docetaxel) have demonstrated activity in patients with NSCLC; however, the median survival time has not been improved, and toxicity due to the combination of these agents is significant. Patients with obstructed airways also have a high incidence of postobstructive pneumonia; thus, neutropenia induced by chemotherapy makes this approach less attractive.

The p53 gene, a tumor suppressor gene, is often rendered nonfunctional in human NSCLC by mutation or deletion.9 By restoring the expression of p53, it is postulated that tumor growth could be suppressed. Preclinical data suggest significant antitumor activity in patients with NSCLC who have had direct intratumoral injections with adenoviral-mediated p53 gene (Adp53) vector.10 11 Preclinical studies have reported the introduction of wild-type p53 into human tumor cells via a variety of delivery methods, including retroviral vectors, lipid complexes, and adenoviral vectors.10 11 These results have demonstrated expression of the transgene product and confirmed normal function of the expressed p53 protein, which caused tumor regression and survival improvement in animal models without adverse effects on nonmalignant tissues with normal p53 function. More than 60% of NSCLC samples tested at baseline show evidence of mutation to the p53 gene. It is expected that a greater proportion of samples have abnormal p53 function.

Initial trials using retroviral p53 gene transfer via intratumoral injection have not been associated with significant toxicity, and evidence of antitumor activity was demonstrated in three of nine patients with NSCLC.12 The three patients who responded had endobronchial disease and received the intratumoral injections via a bronchoscopic approach. Unfortunately, the low transduction efficiency associated with the retroviral vector was a major limiting factor. Recently, the direct intratumoral transfer of the p53 gene using an adenoviral vector was explored in 52 patients with NSCLC.13 14 The results of these studies suggested the safety of the procedure, evidence of transgene expression and function, and clinical activity. Additional benefit was observed in a subset of patients with endobronchial airway obstruction. The results for this subset of patients are described later in this article.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
The objectives of this study were to determine the degree of and reversibility of toxicity following monthly intralesional administration of Adp53 via bronchoscopic injection and to document the observed antitumor activity.

Study Design
This is an open-label, nonrandomized phase I escalation study. Patients received Adp53 at dose levels of 1 x 106 to 1 x 1011 plaque-forming units (pfu). All patients treated were a subset of patients participating in a larger nonrandomized phase I trial involving Adp53 injection through CT-guided, ultrasound-guided, or direct visual-guided injections, or through endobronchial-guided injections.13 14 Patient clinical evaluation criteria have been previously described.13 14 The cohort of patients described herein are those who received endobronchial injections. Each patient received an assigned dose throughout the study. One course of treatment in the study consisted of a 28 (± 5)-day period (SE) during which patients were administered 80 mg/m2 of IV cisplatin, or no chemotherapy on day 1, and study vector on day 4 for a total of up to six courses of treatment. Treatment continues for as long as there is no progression of the indicated lesion, no unacceptable adverse events, and no additional anticancer therapy administered to the indicated lesion.

Vector Administration
Adp53 vector was administered by fine-needle injection directly into the tumor. Administration was performed bronchoscopically. Indicator lesions >= 4 cm in diameter were injected with a final volume of 10 mL Adp53 vector. Indicator lesions with a diameter of < 4 cm were administered a final volume of 3 mL Adp53 vector. Adenoviral vector construction and safety assessment have been previously described.13 14

Patient Population
Patients with histologically confirmed NSCLC who had endobronchial tumors accessible through bronchoscopic examination were enrolled. Patients must have a life expectancy of at least 12 weeks and a Zubrod performance status score <= 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 demonstrated by immunohistochemistry prior to treatment. Patients were required to have adequate marrow (granulocyte count, > 2.0/µL), liver (bilirubin level, <= 1.5 mg/dL), renal (creatinine level, < 1.5 mg/dL), and respiratory (FEV1, >= 40% of the predicted FEV1) function. Patients with symptomatic brain metastases, patients who had used any investigational chemotherapy agent within 4 weeks of study treatment, patients who had used chemotherapy within 21 days prior to study treatment, and patients who had used immunosuppressive therapy including corticosteroids were excluded from trial. This trial was approved by the Texas Oncology, P.A. Institutional Review Board, and participants were required to sign a board-approved consent form.

Response Evaluation
Clinical signs and symptoms were assessed for each patient. Biopsy specimens of indicator lesions were obtained for histologic assessment of the response prior to each course of treatment and/or at day 28. Patients were assigned a response category according to the response criteria previously described.13 14 Additional criteria to evaluate activity in endobronchially injected patients were applied as follows: bronchial airway obstruction was visualized and photographed, and an increase in airway opening > 25% was considered significant. An obstruction by lesions that inhibits air passage through the bronchial airway, as determined by the lack of bubbles when saline solution was placed at the site of the obstruction, was considered a complete obstruction. An opening sufficient to allow air passage, as witnessed on bronchoscopic video with two observations or more of brisk bubbles on saline solution flush, was considered an improvement.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Demographics
Twelve patients with endobronchial-accessible lesions were entered into the trial. Patient population characteristics are shown in Table 1 . Specific sequencing and histochemistry are shown in Table 2 .


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  		Table 1. Demographics*

 

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  		Table 2. Transgene Expression Results*

 
Safety
Four patients had grade 3 or 4 toxicity "possibly or probably" related to the treatment approach, although none of the grade 3 or 4 toxicity was attributed to the Adp53 vector. The events appeared to be related to tumor progression (anemia or obstruction), the bronchoscopic procedure (dyspnea, pneumonia, or abscess), and chemotherapy (hypocalcemia, anxiety, weakness, dehydration, or nausea). Specific grade I to IV adverse events are summarized in Table 3 . Transient grade I or II fevers appeared to be related to therapy with the Adp53 vector. No significant changes from baseline levels occurred in mean vital sign parameters, hematologic function, electrolyte levels, renal function, liver function, parameter values, or arterial blood gas levels with subsequent dose levels or through the course of treatments with Adp53 vector.


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  		Table 3. Treatment-Related Adverse Events (Grades 1 to 4)*

 
Tumor Response
Response data are summarized in Table 4 . Three patients fulfilled the definition of partial response, four patients were in stable condition for at least 2 months, two patients did not have follow-up bronchoscopic viewing because of rapid death related to disease progression (thus making it likely that they did not achieve response), and three patients showed evidence of progressive disease despite injection with Adp53. Six of the 12 patients who received endobronchial injection had < 10% obstruction after treatment. One of five patients who received Adp53 only achieved response compared to five of seven patients who received Adp53 plus cisplatin. The degree of tumor regression within the endobronchial airway involved minimal tumor volume, such that three patients failed to achieve a definition of partial response. Nevertheless, the relief of the obstructed airway was associated with median prolonged survival times of 213 days (Adp53-only group) vs 124 days (Adp53 plus cisplatin group) (Table 4) . The overall median survival time was 143 days (range, 16 to 619 days).


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  		Table 4. Antitumor Activity*

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Intratumoral injection was performed to maximize the concentration of the viral vector within the tumor mass and to minimize the potential toxicity related to the potential systemic circulation of the vector. Injections through a bronchoscopic approach were clinically well-tolerated at dose levels of up to 1 x 1011 pfu. No histologic evidence of adenoviral-induced infection or inflammatory response was observed despite direct exposure of the local bronchial airway site to Adp53. Grades 1 to 2 fever were observed as the most frequent toxic reaction and may have been related to the transient systemic dispersion of the vector. Toxicity related to endobronchial Adp53 delivery was limited to transient hypotension, which was associated with the usual sedative medications administered during bronchoscopy, and to mild temporary oxygen desaturation, which was due to the hypoventilation caused by the sedative medications and by temporary airway occlusion by the bronchoscope. Tumor hemorrhage caused by Adp53 injection was minimal and easily controlled.

The antitumor activity observed in this trial was consistent with the activity observed with retroviral p53 injection in NSCLC patients. Three of the six patients who had obstruction relief fulfilled the criteria for the formal definition of partial response. By comparison, only one partial response was observed in 40 patients who received Adp53 via a nonbronchoscopic injection route.13 14 One patient survived 619 days following the initial injection but eventually died of recurrent disease. Our previous results have shown that an increase in apoptosis was observed after treatment with Adp53 injection and that the mean apoptotic index was significantly higher at follow-up of multiple injections.13 14 It is unclear why endobronchially treated patients appear to show evidence of tumor regression at higher frequencies than patients treated by radiologic-guided injection. This may be related to a higher vector dose load that patients treated bronchoscopically generally receive (ie, 11 of 12 endobronchially treated patients received >= 3 x 109 pfu) or, possibly, to the larger bulk of tumors that were not endobronchially accessible. It is also unclear whether Adp53 actually enhances chemotherapy-induced apoptosis or inhibits it through induction of p21-mediated growth arrest. Further investigation will be required to address this issue. Additionally, only one of five patients who received Adp53 alone achieved endobronchial relief compared to five of seven patients who received Adp53 plus cisplatin. This difference could be related to the enhanced apoptotic activity of combination therapy with Adp53.15

The antitumor activity and potential survival advantage observed in patients with endobronchial lesions may suggest that further exploration of the direct intratumoral injection of Adp53 into the endobronchial obstructing lesions containing a p53 mutation, particularly in patients who are not candidates for surgery, brachytherapy, or laser resection, is warranted. More objective measures of response such as actual measurement of the occluded airway opening with known dimension probes and repeat pulmonary function testing will be required.


   Acknowledgements
 
The authors thank Ana Petrovich for manuscript preparation.


   Footnotes
 
Abbreviations: Adp53 = adenoviral-mediated p53 gene; NSCLC = non-small cell lung cancer; pfu = plaque-forming units

Received for publication January 21, 2000. Accepted for publication May 2, 2000.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Collins, TM, Ash, DV, Close, HJ, et al (1998) An evaluation of the palliative role of radiotherapy in inoperable carcinoma of the bronchus. Clin Radiol 39,284-286
  2. Simpson, JR, Francis, ME, Perez-Tamayo, R, et al (1985) Palliative radiotherapy for inoperable carcinoma of the lung: final report of a RTOG multi-institutional trial. Int J Radiat Oncol Biol Phys 11,751-758[ISI][Medline]
  3. Slawson, RG, Scott, RM (1979) Radiation therapy in bronchogenic carcinoma. Radiology 132,175-176[Abstract]
  4. Majid, OA, Lee, S, Khushalani, S, et al (1986) The response of atelectasis from lung cancer to radiation therapy. Int J Radiat Oncol Phys 12,231-232[ISI][Medline]
  5. Speiser, B, Spratling, L (1990) Intermediate-dose rate remote after loading brachytherapy for intraluminal control of bronchogenic carcinoma. Int J Radiat Oncol Phys 18,1443-1448[ISI][Medline]
  6. Hetzel, MR, Nixon, C, Edmonstone, WM, et al (1985) Laser therapy in 100 tracheobronchial tumors. Thorax 40,341-345[Abstract]
  7. Cavaliere, S, Foccoli, P, Farina, PL (1988) Nd-YAG laser bronchoscopy: a five-year experience with 1,396 applications in 1,000 patients. Chest 94,15-21[Abstract/Free Full Text]
  8. Gelb, AF, Epstein, JD (1984) Laser treatment of lung cancer. Chest 86,662-666[Abstract/Free Full Text]
  9. Xu M, Kumar D, Srinivas S, et al. Parenteral gene therapy with p53 inhibits human breast tumors in vivo through a bystander mechanism without evidence of toxicity. Hum Gene Ther 1997; 8:177–185
  10. Zhang, W, Alemany, R, Wang, J, et al (1995) Safety evaluation of AdCMV-p53 in vitro and in vivo. Hum Gene Ther 6,155-164[ISI][Medline]
  11. Fujiwara, T, Grimm, EA, Mukhopadhyay, T, et al (1994) Induction of chemosensitivity in human lung cancer in vivo by adenovirus-mediated transfer of the wildtype p53 gene. Cancer Res 54,2287-2291[Abstract/Free Full Text]
  12. Roth, JA, Nhuyen, D, Lawrence, DD, et al (1996) Retrovirus-mediated wildtype p53 gene transfer to tumors of patients with lung cancer. Nat Med 2,985-991[CrossRef][ISI][Medline]
  13. Swisher, SG, Roth, JA, Nemunaitis, J, et al (1999) Adenoviral-mediated p53 gene transfer in advanced non-small cell lung cancer. J Natl Cancer Inst 91,763-771[Abstract/Free Full Text]
  14. Nemunaitis, J, Swisher, SG, Timmons, T, et al (2000) Adenovirus-mediated p53 gene transfer in sequence with cisplatin to tumors of patients with non-small cell lung cancer. J Clin Oncol 18,609-622[Abstract/Free Full Text]
  15. Sauthoff, H, Heitner, S, William, R, et al (2000) Deletion of the adenoviral E1B–19kD gene enhances tumor cell killing of a replicating adenoviral vector. Hum Gene Ther 11,379-388[CrossRef][ISI][Medline]



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