Angiogenesis is a process controlled by certain chemicals produced in

the body which stimulate cells to repair damaged blood vessels or form new ones... Angiogenesis plays an important role in the growth and spread of cancer. New blood vessels "feed" the cancer cells with oxygen and nutrients, allowing these cells to grow, invade nearby tissue, spread to other parts of the body, and form new colonies of cancer cells.

16.12 Size and Angiogenesis

The process of angiogenesis is necessary for tumors to survive, and conversely, shutting off or inhibiting the process may limit the spread of the cancer or even reduce the size of the tumor itself:

"One promising avenue of cancer research is the study of a group of compounds called angiogenesis inhibitors. These are drugs that block angiogenesis, the development of new blood vessels. Solid tumors cannot grow beyond the size of a pinhead (1 to 2 cubic millimeters) without inducing the formation of new blood vessels to supply the nutritional needs of the tumor. By blocking the development of new blood vessels, researchers are hoping to cut off the tumor's supply of oxygen and nutrients, and therefore its continued growth and spread to other parts of the body. Lycos.com, angiogenesis.

The process that we are talking about is angiogenesis. As we all know, in order for tumors to grow beyond the size of 2 cubic millimeters in volume, they must develop a vasculature so that, as the tumor cell population grows, the malignant cells secrete angiogenic factors which stimulate nearby vasculature endothelial cells to proliferate and to form tubes and capillaries to promote the continued growth and expansion of the tumor.

This new blood vessel formation also allows tumor cells to extravasate into the bloodstream,travel through the bloodstream, establish colonies again at distal sites, and grow metastatic disease and establish a vasculature once again. Teicher, Angiogenesis as a Target for CancerTherapy www.conference-cast.com/webtie/sots/lung/transcripts/teichertran.htm

16.13 Practical Difficulties with Anti-Angiogenesis Drugs

Teicher discusses some of the theoretical problems with these drugs and the importance of timing in the drug administration in animal studies:

We then looked at the antiangiogenic combination, starting therapy very early in the life of the tumor on day 4, when the tumor is just a seed and beginning to explode in its angiogenic activity. We are starting the angiogenic agent combination 3 days later on day 7, when the tumor is actually a fairly well-established nodule. The cytotoxic chemotherapy was administered on days 7-11. So we learned a lesson that cancer researchers learn again and again, and that is the tumor burden is very important. If we started the antiangiogenic therapy early on day 4 and treated at days 4-11 or 4-18, we obtained the greatest enhancement in tumor growth delay, but even if we had to limit the antiangiogenic therapy to the same 5-day period that we gave the cytotoxic therapy, we still had tumor growth delay of 29 days, which was better than cytotoxic chemotherapy alone. It was only when we administered the antiangiogenic therapy for the full 2-week period of days 4-18, which is really the full exponential growth phase of this tumor, that we obtained the greatest tumor response and with this therapeutic regimen Forty to fifty percent of the animals were cured of the Lewis lung carcinoma. Cancer Therapy. www.conference-cast.com/webtie/sots/lung/transcripts/teichertran.htm

 

16.21 Overview

Newer therapy attempts to frustrate cancer spread through molecular therapy. The task is to identify agent with tumor specificity and low toxicity. More specifically, we want to identify a molecular target

1) which drives tumor growth and or cancer progression,

2) which can be measured,

3) whose function can be reversed or altered significantly,

4) inhibition of that molecule will not alter normal body functions. Kelly (23).

Stating goals is either than meeting them. A May 21, 2002 article provides a summary of anti-angiogenic agents:

Like many experimental therapies, results from initial lab studies of antiangiogenic agents have appeared dramatic. But once they advance to human studies, the bloom tends to fall from the rose. Since 1988, more than 50 experimental antiangiogenesis drugs have been tested in 10,000 patients with few dramatic results.

Despite the challenges, antiangiogenesis remains a particularly appealing concept, because it may offer a less toxic alternative to chemotherapy and radiation, or, more likely, it will improve standard treatments when combined with them....

Since May 2001, 19 antiangiongenesis compounds have advanced to the phase III stage, which will determine whether a drug merits FDA approval. Seven of th 18 compounds were halted in development because interim results showed no effect or too little effect. Of the 11 remaining drugs, three distinguish themselves with extremely encouraging data, Li says.

The front runners are Avastin, by Genentech of San Francisco, Thalidomide, by Calgene Corp, of Warren, and Neovastat, by Aeterna Laboratories. (4)

16.22 FDA Approval Status

As of June, 2002, no anti-angiogenesis drug had been FDA approved for the treatment of lung cancer.

l

16.31 Neovastat

In laboratory studies, Neovastat was shown to be effective in inhibiting angiogenesis and improved the efficiency of standard chemotherapy drugs:

" A novel naturally occurring antiangiogenic agent isolated from cartilage, referred to as Neovastat (AE-941), was examined for its efficacy against tumor neovascularization and progression. Exposure to Neovastat results in ex ovo antiangiogenic properties in the chorioallantoid membrane of chicken embryo (71% decrease in the angiogenic index as compared to the basic fibroblast growth factor (bFGF) treated control embryos, P < 0.0001). Oral administration of Neovastat inhibits bFGF-induced angiogenesis in the Matrigel mouse model (87.5% decrease in hemoglobin as compared to the bFGF-treated control implants, P < 0.0001). Neovastat also induces a dose response decrease of lung metastases in the Lewis lung carcinoma model (oral administration; 69.1% of inhibition obtained at the maximal dose of 0.5 ml/day, P < 0.0001). Combined with a sub-optimal dose of cisplatinum (2 mg/kg, i.p.), Neovastat (0.5 ml/day) improved the therapeutic index by increasing the antimetastatic efficacy and by exerting a protective activity against cisplatinum-induced body weight loss and myelosuppression. In summary, our experimental data provide evidence of antiangiogenic and antimetastatic properties of Neovastat, following oral administration." Dupont, (5)

Neovastat is derived from cartilage of the dog-fish shark. Shark cartilage was a controversial experimental treatment advocated in the early 90's featured in a book, Sharks Don’t Get Cancer.

16.311 Human Studies

There are reports (as indicated above) of favorable human studies.

Neovastat is a complex of naturally occurring anti-angiogenic molecules exhibiting multi-functional mechanisms of action. It has been tested in Phase II clinical trials in non-small cell lung cancer and in renal cell carcinoma.... Several of its activities have been characterized and the antiangiogenic activity of Neovastat can be attributed to inhibition of several key pathways in angiogenesis making it an ideal candidate to test against metastasis formation in highly vascularized bone."

However, the citation is to an article in progress. An intelligent assessment of Neovastat must await published results. Based on the reports, it may be a plausible alternative for patients with advanced cancer.

16.32 Thalidomide

In the 60's, Thalidomide was source of birth defects, as it cut the supply of blood and the development of new vessels in the fetus. It is precisely this characteristic which indicates promise as a potent anti-angiogenic agent.

In addition to immunomodulatory and cytokine-modulatory properties, thalidomide has antiangiogenic activity. It has been investigated in a number of cancers including multiple myeloma, myelodysplastic syndromes, gliomas, Kaposi's sarcoma, renal cell carcinoma, advanced breast cancer, and colon cancer." Thalidomide (3).

 

16.33 Endostatin and Angiostatin

If nothing else, the story of Angiostatin and Endostatin is a lesson in the tendency of early clinical results to be given excessive weight, the tendency of news reporters to herald a new cancer cure from limited results in a single animal study, and the interrelationship between corporate goals and scientific research. After numerous early studies on the theory of angiogenesis, Dr. Folkman, a Noble-Prize winning Harvard researcher, reported that tumors in 50% of rats were markedly reduced by administration of Endostatin. The results became national, indeed, world-wide news as the new media profiled the new cancer cure.

Later scientists had difficulty reproducing the results and in any event, the promise was not translated to human studies. Today the future of Endostatin and Angiostatin remain uncertain, and are one of perhaps five hundred anti-cancer drugs which continue to be investigated.

Instead of trying to kill the cancer cells, one could try to prevent metastasis or spread to other organs. Recall that cancer cells metastasize because they do not have an enclosing shell and acquire the ability to penetrate the shells or basement membranes of normal cells. Matrix metalloproteinases (MMP) such as collagenase, help break down the extracellular matrix which protects other bodily structures. Some drugs are used to inhibit MMP, and thereby to prevent tissue penetration. We can review some of the recent information about these drugs. However, none have been shown to have proven, clear anti-cancer fighting properties in laboratory experiments and clinical studies. All have a theoretical basis for believing they could be used which are described below.

Experimental data indicate that angiogenesis is crucial for growth and persistence of solid tumor and of their metastasises. Another agent to display anti-angiogenesis properties is suramin . Although suramin has shown, at least in vitro (in cells), to inhibit lung cancer growth (7), clinical studies in NSCLC have yielded negative results. Angiostatin, a component of plasminogen and endostatin, inhibits metastasis by inducing a balance between metastatic and primary tumor cells defined as "dormancy". Both agents are capable of inhibiting tumor growth in vivo in animal models. Although tumors re-grow when anti-angiogenic treatment is discontinued, experimental tumors, remain sensitive to a second cycle of treatment with the same agent. Different from conventional chemotherapeutic agents, no drug-resistance was observed even after multiple repeated cycles of antiangiogenic therapy (9). These agents hold considerable promise for the treatment of a number of tumor types including NSCLC. Other drugs which, in preclinical models, have been found to be angiogenesis inhibitors and that are currently entering clinical trials include TNP-470 (a synthetic fumagillin derivative), thalidomide, vitamin and squalamine.

16.41 Cox 1 and 2

16.42 The role of Cox-2 in Lung Cancer.

Cox-2 is involved with the development and spread of lung cancer.

Developing a new drug poses two important challenges. First, how does one deliver the drug to the relevant area. This is where many anti-angiogenic drugs have failed; in cells tests, the drug suppresses cancer cells in the laboratory or in large quantities upon an animal, but in the complexity of the human body, little impact is seen. Secondly, how do we avoid disruption of normal cells and bodily functions, the obstacle facing many chemotherapy drugs. These problems have been solved to some extent with Celebrex and other drugs.

16.44 Celebrex

Maspherer (17) showed that Celebrex suppressed growth of lung and colon tumors in mice, "Celecoxib supplied in the diet continuously from date of implant at doses between 160-3200 pip significantly retarded the growth of these primary tumors. The inhibitory effect of celecoxixib was dose dependent and ranged from 48% to 85% when compared with untreated tumors."

Aspirin and other NSAID’s suppress both cox- and cox 2. Scientists continue to aim at developing cox-2 treatments.

16.46 Cox 2 and Radiation

Cox-2 inhibitors may make radiation more effective. "Inhibitors of cyclooxygenase (COX)-2 can be effective in combination with conventional chemotherapy and radiation therapy," Ferrario (19).

Some suggest Cox-2 should be used early in the treatment process to prevent spread of the tumor.

16.47 Summary

In short, there are many powerful arguments in favor of Cox-2, scientific evidence showing its significance in carcinogenic processes, the demonstrated ability of drugs to suppress Cox-2, and the comparatively few side effects, particularly when contrasted with conventional chemotherapy. While one could not recommend it blindly or suggest its use for particular patients, patients will want to discuss possible use with their oncologists.

Vascular endothelial growth factor (VEGF) is associated with angiogenesis and metastasis and is thus a target for new drugs. "VEGF stimulates new blood vessel formation, or angiogenesis, by binding to specific receptors on nearby blood vessels to stimulate extensions to existing blood vessels." Gene.com (12)

16.51 The Rationale for VEGF Receptor Drugs

A growth factor comes into contact with a receptor, like a key and lock, and begins a process of cell reproduction and changes. Some newer drugs attempt to prevent the two from connecting. The process is complex since related growth factors and receptors are activated also. As with other forms of gene therapy, targeting the offending protein rather than all cells can reduce harmful side effects. "The high selectivity achieved with neutralizing antibodies, soluble receptors and ribozymes reduces the risk of adverse reactions not related to VEGF inhibition itself." Investigational Drugs (13)

16.52 Flovopirodol

16.521 Origin

Results in a phase 2 trial were modest. No complete or partial responses were observed. Thus, the drug is unlikely to be effective alone. However, a limited impact was seen and toxicity was also limited. Flovopirodol holds some promise as a treatment in conjunction with chemotherapy. See ElSayed (12). Clinical trials comparing chemotherapy with and without the drug can be performed.

16.53 Avastin

Avastin also known as rhuMAb VEGF or bevacizumab, is ia promising an anti- VEGF drug:

Blood and heart-related side effects have been reported including thrombosis and hypertension. (14)

16.81 P-53's Functions

P-53 is perhaps the most important tumor suppressor gene.

Consider its importance in cancer research:

"Between January 1993 and July 1996, more than 4300 papers have been published in which the term "p53" appears in the title! This massive interest in a single protein is almost unprecedented and reflects the central place of p53 in the regulation of cell number and the frequency with which abnormalities of p53 occur in human tumours. p53 has been named Molecule of the Year by the editor's of the journal Science (262, 1958-59)...,

This protein plays a major role in the transcription ("reading") of DNA, in cell growth and proliferation, and in a number of metabolic processes. Because p53 suppresses abnormal cell proliferation (it acts like an 'emergency brake' in the cell cycle), it may represent an important mechanism for protection against cancer. It also appears to be involved in programmed cell death, or apoptosis. When a mutation in the p53 gene results in the substitution of one amino acid for another, p53 loses its ability to block abnormal cell growth. Indeed, some mutations produce a p53 molecule that actually stimulates cell division and promotes cancer. Almost 50% of human cancers contain a p53 mutation -- including cancers of the breast, cervix, colon, lung, liver, prostate, bladder, and skin -- and these cancers are more aggressive,..." P53 Weitzman (2)

16.811 Description of P-53

P-53 is a protein of 53 kilodaltons (hence the name). It is located on chromosome 17 (p13). There are two types of P-53. First, we have normal P-53 also called wild-type P-53. This is P-53 in its normal condition, serving various tumor suppression functions outlined above. Mutant P-53 means the gene has been damaged. Not only will the gene not perform its tumor suppressor function, it may even contribute to carcinogenic processes:

"Mutant p53 loses its original function but may acquire a new potentially oncogenic activity. Conversion of p53 protein from a normal to a mutant phenotype alters its histochemical features; the half-life of the protein is prolonged from 6-20 minutes to several hours. This increases the amount of p53 protein in affected cells,... Detection of excessive amounts of p53 protein is useful as a marker of mutation because the amount of wild-type p53 protein is too low to be detected in nonmutant cells." Gemba, (3) at 23-31.

P53 protein is located in the nucleus of cells and is unstable. In a normal person, the body activates P-53, it performs its function of either correcting cell damage or inducing the death of damaged cells, and disintegrates. Malfunctioning or mutant P-53 can maintain itself in cells with the finding of P-53 indicates an abnormality . P-53 damage is seen in approximately 50% of breast colon, stomach, bladder, and non-small cell lung cancers.

16.812 p53 and Non-Small Cell Lung Cancer

In non-small cell lung cancer (NSCLC), most series report that 50% to 60% of tumors have identifiable mutations." A 1999 study found that over 50% of small cell patients had traces of P53 in bronchial specimens.

16.813 When Do P-53 Mutations Occur in the Cancer Process.

Scientists have not been able to determine precisely when P-53 mutations occur. Curie suggests that P-53 alteration is an early event:

" p53 alteration is an early event in lung cancer, several years before the clinical diagnosis of the tumor. Recently, p53-Ab were detected in sera of two patients who were heavy smokers without diagnosed lung malignancy. Both of these patients developed invasive squamous lung cancer 5 and 15 months, respectively, after detection of serum p53-Ab.

In one patient, the level of serum antibodies directed against different epitopes of p53 protein was shown to progressively increase during the 15 months of follow-up before the diagnosis. In this patient, p53 overexpression was detected in tumoral cells from bronchial biopsy specimens. Since p53 alterations represent an early genetic changes in lung carcinogenesis, it is suggested that p53-Ab detection represents a new and sensitive tool for detection of preneoplastic and microinvasive bronchial lesions in patients with a high risk of lung cancer, i.e., heavy smokers. This finding was confirm by Trivers et al. using three types of assays to detect p53-Ab. They were able to find p 53-Abs before diagnosis in several patients with Chronic Obstructive Pulmonary disease."

Szak suggests that P53 mutations are about midpoint in tumor development, just as damaged tissue becomes cancerous, "Most studies indicate that in the development of squamous cell lung cancer, loss of heterozygosity at 17p (suggesting loss of wild-type p53 function) occurs at the transition of preneoplasia to carcinoma in situ." (7)

16.82 P-53 and Patient Prognosis

Determining P-53 impact upon lung cancer prognosis has been unclear:

16.821 Early stage Lung Tumors

One hypothesis is that even after a tumor has developed, P-53 can play a role in limiting cell proliferation. We know that there is a group of tumors where apoptosis or cell death, equals proliferation, and those patients have a good prognosis. Under this theory, P-53 mutations would affect survival for early stage patients, but only minimally impact patients with advanced tumors, where P-53 would be limited in its function:

"Horio et al. analyzed resected Japanese lung cancer cases and found adverse prognostic significance, especially in early-stage tumors...Damic published on 408 consecutive resected stage 1 NSCLC patients and showed a modest prognostic impact of P53 overexpression." Szak (7), at 126.

Even, here, though results are equivocal. Szak states,

"An even larger study of 515 resected stage NSCLC showed no difference in survival for p53 positive immunostaning... the isolated impact of having a mutant p53 or p53 overexpression on time to recurrence or coverall survival in lung cancer is unclear. Szak (7) at 126.

If damage to P-53 could be repaired, then tumor spread could be limited and perhaps P-53 could again perform its function of preventing cell duplication. This type of treatment has worked in a laboratory setting: \

Reintroducing a wild-type p53 gene into lung cancer cells, including Bronchioalveolar lung cancer (BAC), dramatically inhibits tumor cell growth and promotes tumor cell death despite the presence of mutations in multiple other genes. Lee, (1) at 324.

In NSCLC it was initially shown that introduction of a vector containing the wt-53 (wild type) into cell lines, which {had} either a deletion or a missense mutation in p53, markedly reduced cell proliferation and tumorigenicity.

16.84 Studies and Trials Using P-53

Some limited, promising results were seen in phase 1 P-53 clinical trials. In one trial, tumor regression was seen, though it did not affect survival since the patients had advanced metastatic cancer. A recent trial found no impact of P-53 treatment, "There was no difference between the response rate of lesions treated with p53 gene therapy in addition to chemotherapy (52% objective responses) and lesions treated with chemotherapy alone." (1) The treatment cannot be disposed of based upon one clinical trial. In the study, P-53 patients had slightly more favorable results. More importantly, it may be possible to develop more effective methods of delivery.

One strong argument for gene therapy is that the therapy will have limited side effects. Chemotherapy targets or affects different groups of dividing cells, cancerous and normal. On the other hand, gene therapy is designed to create new sources of P-53, and the presence of additional P-53 in the body, or the performance of ordinary functions by P-53, should not involve significant side effects.

16.85 Hurdles to Successful Treatment

It is difficult to repair the gene, the solution being studied is transferring another P-53 gene to the cancer area. Lee writes:

The complexities of the three-dimensional structure of the p53 tumor-suppressor gene product and the radical changes in this structure induced by a single point mutation makes it extremely difficult to restore its function with pharmaceuticals. Thus, the basic concept of tumor suppressor gene therapy utilizing p53 is to reintroduce a functionally active copy of the deficient genes in the cancer cell by direct gene transfer to directly induce cell death by apoptosis. Lee, (1) at 324.

Getting enough P-53 to the tumor to have significant results is difficult:

"In all of these studies, it has been difficult to deliver recombinant virus into solid tumors so as to transduce a significant fraction of the tumor cells, and the effect is limited to the injected nodule, which may be of limited real clinical benefit." Lee, (1) at 324.

16.86 Use in Conjunction with Chemotherapy

The optimal use of P-53 treatment may be to compliment chemotherapy:

"The function of p53 is critical to the way that many cancer treatments kill cells since radiotherapy and chemotherapy act in part by triggering cell suicide in response to DNA damage. This successful response to therapy is greatly reduced in tumors where p53 is mutant so these tumors are often particularly difficult to treat." Weizman (2)

Theoretically, P-53 compliments chemotherapy, and examination of laboratory cells showed a complimentary role in attacking cancer cells:

That type of role must be confirmed in human clinical trials which is one reason why at the time of publication, P-53 was not an FDA approved drug. In one well-known study, that complimentary impact was not demonstrated.

The suppression of normal P-53 and expression of its mutated form, expression of Cox-2, the production of epidermal growth factor, expression of VEGF, are steps in the development and spread of lung cancer, with each step a target for research and treatment. Lung cancer has been difficult to cure, probably because so many genes are disrupted, and repair of a small part does not restore normal functioning. Thus future treatment may focus on the use of a variety of drugs, to the extent possible without substantial side effects.

16.91 Impact Upon Treatment at Various Stages

We review treatment by stage in later chapters, but a brief summary is appropriate here. One difficulty with today’s treatment is that newer drugs are tested upon the patients with the most advanced illnesses, and thus the toughest to cure. Lung cancer patients tend to enter clinical trials when other forms of treatment have been exhausted.

Unfortunately, the only reliable cure has been surgical removal of tumors in stage 1 patients, and even then here is approximately 30-40% recurrence. Patients may want to consider promising newer forms of treatment at earlier stages. The downside is that the drugs are unproven. The upside is that if the side effects are limited, the patient can benefit at a time when the disease is not advanced.

Drugs like Celebrex which are FDA approved and widely used in the population at large with few side effects, should be considered. More intrusive experimental drugs present tougher choices.

2. Galligioni, Angiogenesis and Antiangiogenic Agents in Non-small Cell Lung Cancer, Lung Cancer 34 (2001) S3-S7.

3. Thalidomide in Cancer, Potential Uses and Limitations, BioDrugs 2001;15(3):163-72

4. Friend, Starving a Tumor, May 21, 2002, Daily Record (New Jersey)

5. Dupont, Antiangiogenic and antimetastatic properties of Neovastat (AE-941), an orally active extract derived from cartilage tissue Clin Exp Metastasis 2002;19(2):145-53

6. Weber, The Effect of Neovastat (AE-941) on an Experimental Metastatic Bone Tumor Model, Intl Journal of Oncology, 20: 299-303, 2002.

7. Frequent co-localization of Cox-2 and laminin-5 gamma2 chain at the invasive front of early-stage lung adenocarcinomas, Am. J. Pathol,2002 Mar;160(3):1129-41

10. Shapiro, A Phase II Trial of the Cyclin-dependent Kinase Inhibitor Flovopirodol in Patients with Previously Untreated Stage IV Non-Small Cell Lung Cancer,Clinical Cancer Research Vol. 7, 1590-1599, June 2001.

13. www.gene.com/gene/pipeline/status/oncology/avastin/index.jsp

14. Manley, Therapies directed at Vascular Endothelial Growth Factor, Expert Opinion on Investigational Drugs 2002, vol. 11, no. 12, pp. 1715 - 1736

15. Phase III Trial With Avastin in Relapsed Metastatic Breast Cancer Does Not Meet Primary Endpoint; Results from Lead Phase III Study in Colorectal Cancer Due in Mid-2003 www.salesandmarketingnetwork.com

20. Hida, Cyclooxygenase-2 Inhibitor Induces Apoptosis and Enhances Cytotoxicity of Various Anticancer Agents in Non-Small Cell Lung Cancer Cell Lines, Clinical Cancer Research Vol. 6, 2006-2011, May 2000

1. Lee, Gene Therapy, in Pass, Lung Cancer: Principles and Practice (Lippincott 2000).

2. P53 http://bioinformatics.weizmann.ac.il/hotmolecbase/entries/p53.htm

3. Gemba, Immunohitochemical Detection of Mutant P53 protein in Small Cell Lung Cancer: Relationship to Treatment Outcome, Lung Cancer, vol 29 (1) (2000) pp. 23-31.

5. Schuler, Adenovirus-mediated wild-type p53 gene transfer in patients receiving chemotherapy for advanced non-small-cell lung cancer: results of a multicenter phase II study, J Clin Oncol 2001 Mar 15;19(6):1750-8

6. Curie, P-53 Mutation in Lung Cancer http://perso.curie.fr/Thierry.Soussi/p53_mutation_in_%20lung.html#Bronchopulmonary%20cancers.

7. Szak, Gene Therapy, in Pass, Lung Cancer: Principles and Practice (Lippincott 2000).