Breathe in, breathe out: using the airway to combat cancer (Proceedings)

Primary lung cancer is uncommon in dogs, comprising around 1% of all cancer in this species. Yet dogs frequently succumb to metastatic carcinoma and sarcoma from distant solid tumors, just like people. Additionally, lung cancer in both dogs and people shows a propensity for metastasis to the central nervous system, and paraneoplastic syndromes are common in both species. Metastatic disease to the lungs is the life-limiting complication of most solid cancers in both dogs and people. Dogs have been used successfully to demonstrate proof-of-principle for inhalant therapy for primary and metastatic lung cancer (Khanna 1996, Hershey 1999, Khanna 2003, Selting 2008).

Breathe in

Inhaled chemotherapy is among several innovative therapies that are being explored for treatment of lung cancer. There are several aspects of inhalant therapy that must be considered in developing this as a novel treatment option. All nebulizers are not created equal and older jet and ultrasonic nebulizers are being replaced by newer vibrating mesh nebulizers. Vibrating mesh nebulizers are much more efficient at generating smaller droplets, so that less drug is needed to deliver the desired dose, which can help minimize the expense. Additionally, novel delivery catheters are in development and allow certain advantages over standard nebulizers, such as the ability to pass the catheter through the biopsy channel of a bronchoscope to deliver therapy to an isolated lung lobe. While metastatic disease requires whole lung treatment, solitary lung masses may be treated or downstaged using catheter-delivered chemotherapy via minimally invasive techniques. The author and collaborators have experience with various delivery instruments, and with two such catheters.

The main advantage of inhaled chemotherapy is that higher drug doses can be delivered to the lung and systemic effects minimized compared to intravenous administration. Clinical animal studies of inhaled chemotherapy exist and confirm efficacy and safety with some agents (doxorubicin, paclitaxel, and 5-fluorouracil). However prior to studies performed at the University of Missouri, cisplatin had not been investigated, nor had delivery directly to the lower airways with intratracheal catheters or probes been investigated in detail. Only inhalation via mask or endotracheal tube has been reported in dogs (Hershey 1999, Tatsumura 1993). In human medicine, mask delivery is known to greatly limit the amount of aerosol that reaches the lung tissue. This also increases the environmental hazard and potential for human (pet owner) exposure to cytotoxic drugs. Therefore delivery by endotracheal tube, either aerosol or catheter, would be superior to mask delivery.

Our group has recently completed two studies using targeted, intratracheal delivery of cisplatin with or without gemcitabine. There are several reasons to support our choice of chemotherapy agents. Cisplatin-based chemotherapy protocols are the treatment of choice for primary lung cancer in people, gemcitabine can work synergistically with cisplatin, gemcitabine added to a cisplatin protocol can increase the time to progression for people with primary lung cancer, and cisplatin is well suited to localized delivery, a favorite for intracavitary or intratumoral treatment. In normal dogs, we were able to show precise, localized delivery of drug, with no systemic adverse effects, and inflammation histologically in treated tissues only (Selting 2008).

The studies described in the previous paragraph were performed in normal dogs to determine the safe dose for inhalant delivery. We have also treated a handful of clinical patients. One miniature poodle with a primary lung tumor experienced stabilization of disease for almost a year. Another dog with metastatic thyroid cancer experienced regression of lung nodules when inhaled cisplatin was combined with radioiodine. We continue to gain experience with different instruments and have learned more about patient selection. An upcoming trial will evaluate a novel catheter-style delivery system in primary and metastatic lung cancer, with the advantage of shorter treatment times and the ability to perform targeted delivery.

In addition to cytotoxic chemotherapy, the airways have been used to delivery immunotherapy. In dogs with spontaneously-occurring primary and metastatic lung cancer, delivery of liposome-encapsulated interleukin-2 (IL-2) resulted in some unexpected responses. Two of 4 dogs with metastatic pulmonary osteosarcoma had complete regression of their disease, including regression of extrapulmonary metastasis in one dog. Both were durable responses lasting more than a year. Pulmonary metastatic osteosarcoma is highly resistant to any form of therapy so this response was remarkable.(Khanna 1996) Although this formulation of IL-2 is not commercially available, free IL-2 (Proleukin) can be purchased and administered by owners at home. This can be cost prohibitive but is very well tolerated.

Protocol for inhaled IL-2

1. Owners are asked to purchase a commercial nebulizer such as the Pulmo-Aide®.

2. Proleukin is purchase and comes in 22 million unit vials. (costs around $900/vial)

3. The dose is 1 million units twice daily.

4. The proleukin is reconstituted to a volume of 4.4 ml (5 million units per ml), and for each dose, 0.2 ml is withdrawn (1 million units) and qs to 1 ml with fluids used to reconstitute.

5. A total of 1 million units of IL-2 is instilled in the nebulization chamber and is further diluted by adding 4 more ml of fluid for a total of 5 ml.

6. The dog is fitted with a baggie over the muzzle with the end of the nebulizer tubing close to the nose (where a human would insert the adaptor in their mouth or over the nose and mouth)

7. The nebulizer is turned on and a fine aerosol is produced, which is then inhaled by the dog. This continues until the liquid is gone.

8. Dosing is continued daily for 3-4 weeks, then radiographs are repeated.

9. If masses are smaller or stable, the treatment is continued for a total of 60 days, then stopped regardless of response (to avoid neutralizing antibodies).

10. If the masses are larger, treatment is stopped.

Breathe out

Exhaled breath condensate (EBC) can be collected from breath humidity in a cooled chamber and used for non-invasive assessment of lung health. EBC contains proteins, nucleic acids, and metabolic byproducts and can be analyzed using nuclear magnetic resonance (NMR) spectroscopy to characterize chemical fingerprints of metabolic processes. Additionally, proteomic profiling using surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) may identify novel proteins present in the breath of diseased individuals.

Difference in metabolomics (profiling of metabolic pathway intermediates) in EBC have been seen in human patients with asthma and cystic fibrosis, as compared to normal. At the University of Missouri, we are collecting breath samples for analysis in healthy and tumor-bearing dogs. Our goal was to develop a method to collect and evaluate EBC from dogs with pulmonary neoplasia.

Dogs with spontaneously-occurring pulmonary neoplasia were recruited at the University of Missouri. Non-sedated patients were comfortably restrained with an anesthetic induction mask. A glass condensing column with ice water circulating in the outer chamber was attached to the mask. After 15-20 minutes of normal breathing, condensate was collected from the inner chamber of the column and aliquoted into two samples. These samples were stored at -80 degrees Celsius until assay by NMR and SELDI-TOF evaluation.

Two dogs with metastatic pulmonary neoplasia (thyroid and mammary carcinoma) and ten healthy control dogs were evaluated to develop collection and sample handling protocols. NMR data showed formate peaks unique to tumor samples. SELDI-TOF analysis showed an 8500 dalton protein that was also unique to tumor samples and not seen in the normal dogs.

Other methods to collect EBC include other tubing or piping surrounded by cooling systems. The RTube® is a unique tool used for EBC investigation and evaluation that may be able to be used for monitoring in remote locations or at the pet owner's home. A metal cylinder surrounded by a cooling sleeve is placed in the freezer. Once frozen, a plastic collection tube is inserted into the hollow center of the metal cylinder and this collection tube is affixed to a mouthpiece into which the patient breathes for 15-20 minutes. The tube is sealed and can then be shipped under cooled conditions to a laboratory for evaluation.

Conclusion

These evaluation and treatment methods show great promise because they are noninvasive and adaptable to clinical practice with minimal equipment. Further development is needed but preliminary results are promising. Collaborations at the University of Missouri have been formed to investigate inhalant therapies and exhaled breath condensate across species and across disease states including asthma and cystic fibrosis.

Selected references

Khanna C, Hasz DE, Klausner JS, Anderson PM. Aerosol delivery of interleukin 2 liposomes is nontoxic and biologically effective: canine studies. Clin Cancer Res 2 (1996) 721-734.

Hershey AE, Kurzman ID, Forrest LJ, et al. Inhalation chemotherapy for macroscopic primary or metastatic lung tumors: Proof of principle using dogs with spontaneously occurring tumors as a model. Clin Cancer Res 5 (1999) 2653-2659.

Khanna C and Vail DM. Targeting the lung: Preclinical and comparative evaluation of anticancer aerosols in dogs with naturally occurring cancers. Curr Cancer Drug Targets 3 (2003) 265-273.

Selting KA, Waldrep JC, Reinero C, Gustafson D, Mercier E, Branson KR, Kim DY, Henry CJ, Owen N, Hoopes PJ, Madsen R, Dhand R. Feasibility and safety of targeted cisplatin delivery to a select lung lobe in dogs via the AeroProbe® intracorporeal nebulization catheter. Journal of Aerosol Medicine and Pulmonary Drug Delivery 21(3):255-268, 2008 Sep.

Schiller et. al., NEJM 346:92-8, 2002