Lung Cancer and Cystic Fibrosis: Diagnosis and Care

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Population Statistics and General Overview

Lung Cancer is the most prominent form of morbidity due to cancer, and 90% of all cases are caused by cigarette smoking. In Germany From 2000 to 2005, the incidence of lung cancer in men and women are decreasing and increasing respectively (Alberg, Ford et al. 2007). In the United States, it accounts for 15% of all new cancer diagnosis and 28% of all deaths due to cancer.(Mallick, Patnaik et al., 2010).

The main types of lung cancer are squamous cell carcinoma, adenocarcinoma, non-small cell lung carcinoma (NSCLC), and small cell lung carcinoma (SLC). SLC and NSCLC are differentiated histologically in order to define prognosis and treatment. (Hammerschmidt and Wirtz, 2009). The genetic causes of lung cancer are very diverse and can arise from defects across multiple genes that protect the body from the formation of cancerous cells. If you add up the deaths from breast cancer, prostate cancer, and colon cancer combined, the total will still be less than that for lung cancer(Reyes 2010).

The first reported case of Cystic Fibrosis (CF) was in 1938, and it is one of the most common life threatening genetic diseases in the Caucasian population (Voter and Ren 2008). The life expectancy of CF individuals is about 37 years, and 1 in 20 Caucasians carry the CF mutation on the Cystic Fibrosis Transmembrane Receptor (CFTR) Gene (Guggino and Stanton 2006). In the U.S., 30,000 adults are affected by it, and 1,000 new adults are found to have it each year (Wiehe and Arndt 2010).

In European descent populations, there are 1 in 25 carriers of the gene and 1 in 2000-3000 live births worldwide. The gene itself was discovered in 1989, and through much resistance from obstetricians has become a common screening in prenatal care (Grody 2009). CF is an autosomal recessive disorder due to mutations in a trans-membrane chloride ion transporter called CF Transmembrane Conductance Regulator (CTFR).

Unlike lung cancer, CF arises mainly from a single gene. The interruption of this chloride ion exporter reduces the osmotic potential across the cell membrane and causes water to remain in the cell. This leads to a higher viscosity in the mucous lining exocrine passageways. This thick mucous cannot be efficiently cleared from the lungs and results in increased infections from Pseudomonas aeruginosa and an auto-immune cytokine inflammatory response (Torpy, Lynm et al. 2009).

Through extensive research, it has become apparent that both of these diseases are highly dependent on familial genetics. Individuals who have a family history with either CF or Lung Cancer have an increased risk of inheriting or expressing the disease, respectively. Thus Genetic profiling has become an important tool in differentiating the types of treatments that individuals should receive. The use of molecular markers such as microRNA, mutations and genomic signatures allow the tailoring of treatments to specific causes of each disease. This is especially important for lung cancer as many types of genetic changes: mutation, deletion, amplification, translocation, and methylation on many different genes can give rise to uncontrolled cell proliferation.

Cell Biology Aspects

The CFTR protein consists of two transmembrane binding domains TMD1 and TMD2 that each has six-membrane spanning alpha-helices. Two nucleotide-binding domains NBD1 and NBD2 are attached to each TMD, and they are regulated by ATP. When activated, they form a heterodimer which forces the pore to open and Cl^– to flow down the electrochemical gradient. The CFTR protein also has secondary phosphorylation regulation in an R domain. This is controlled by a cAMP cascade mechanism and directly by protein kinase A (PKA) and PKC. PKA activation is necessary before ATP phosphorylation can occur (Mueller and Flotte 2008).

Mucous viscosity overall is also regulated by two other channels, the epithelial sodium channel (ENaC) and the outward rectifying chloride channel (ORCC). The CFTR channel releases Cl-, HCO3- and ATP, which in turn down-regulates ENaC activity and up-regulates ORCC activity.

Without the CFTR channel, ENaC then increases its sodium absorption, and ORCC decreases its chlorine output. Due to a defect in the CFTR gene, these two additional ion channels create a cascade of osmotic events that result in a decrease of water in the airways; and thus lead to a higher viscosity mucous. To date, due to the high number of hydrophobic regions, the CFTR structure has not been crystallized (Uppall 2010).

Lung cancer has been focused around the Epidermal Growth Factor Receptor Gene located at 7p12; which controls many aspects of cell growth and death (Sharma, Bell et al. 2007). Mutation and amplification of this gene are associated with many NSCLC patients and is indicative of uncontrolled cell growth. The receptor is a transmembrane protein that has extracellular binding sites for Epidermal Growth Factor (EGF) and transforming growth factor-alpha.

Once EGF binds to the EGFR it activates a small guanosine triphosphatase (GTPase)-mediated protein-tyrosine kinase signal transduction cascade that leads to increases in calcium molarity, protein synthesis and glycolysis as well as an auto feedback loop that increases expression for EGFR. Eventually, its ultimate goal is to replicate DNA and cause cell division. The complex architecture of the signal map for EGFR can be seen in reference Oda, Matsuoka et al. 2005. The structure of the signal pathway has been called a bow-tie or hourglass structure.

Basically, a numerous amount of ligands become available to bind to erythroblasts leukaemia viral oncogenes homolog (ErbB) receptors that activate molecules like EGFR in a large network of receptor molecules. The path then narrows down to a smaller subset of molecules such as nonreceptor tyrosine kinase (non-RTK), small GTPase, and PIPs which then cause the pathway to enlarge again as they activate several cascades. These cascades then lead to transcriptional regulation. These many cross-talking pathways and feedback control loops set up a highly robust and redundant system (Oda, Matsuoka et al. 2005). Evolutionarily this is important as cell division is crucial to an organism’s survival and uncontrolled cell division will ultimately lead to death.

Genetic Background

In Lung Cancer family genetic history can provide the first evidence of susceptibility to lung cancer as well as germ-line mutations in the tumour suppressor TP53 gene, retinoblastoma, and the epidermal growth factor receptor gene. A recent study found more than 80% of lung cancer lines carry alterations at TP53 in both NSCLCs and SCLCs (Sanchez-Cespedes 2009). More recently, the effect of nicotine exposure has been confirmed by single-nucleotide polymorphism of 15q24 – 15q25.1, which includes two genes that are responsible for the nicotinic acetylcholine receptor alpha.

This receptor has been found to be regulated by exposure to nicotine. (Herbst, Heymach et al. 2008). Changes to 15q24 – 15q25.1 have been associated with higher susceptibility to cancer. LC risk also increases with a decrease in DNA repair, and the excision repair gene ERCC1 has been shown to be damaged by tobacco smoke. Many more mutations across several chromosomes can lead to lung cancer. These can be seen in the following diagram from Cespedes et al.

Diagram of affected genes in Lung Cancer (Sanchez-Cespedes 2009)

Such a wide variety of genes will require a broad spectrum of individual drugs to target each protein or gene. However, the use of microRNAs which will be discussed later may be able to target several genes at once and provide an effective measure of treatment.

CF is an autosomal recessive disease where, in 70% of all cases, is due to a deletion in the CFTR gene located on chromosome 7 at q31.2. It consists of three base pairs between the 507^th and 508^thtriplet base pair position. At the 507^th position, we have the three base pairs T-A-G, and at the 508^th position, we have A-A-A. The mutation results in the G from the 507^th position and 2 A’s from the 508^th position being deleted, leaving the three base pairs T-A-A.

In the non-mutated CTFR gene, the 508^th position T-A-G is translated into A-U-C, which results in Isoleucine. In the mutated from the deletion results in a T-A-A triplet base pair that is translated into A-U-U which also is translated into Isoleucine. However, the phenylalanine that would have been expressed by the A-A-A at the 508^th position is never created. This results in a mutated CFTR protein that becomes unstable and is usually degraded faster than the normal CFTR protein. In general, there are four main classes of CF disease mutations and these are:

mutations that cause CF phenotype;
CFTR Related Disorders (CFTR-RD) mutations;
Non-Clinical mutations;
Unknown consequence mutations.

Mutations that lead to Classical CF Phenotype Expression are ΔF508del Mainly nonsense, frameshift, splicing (invariant dinucleotide): G542X, R553X, W1282X, 2183AA4G, 3659delC, 1717-1G4A, 3120+1G4A Missense that severely affects CFTR synthesis or function: G551D, N1303K, R347P, 2789+5G4A, 3849+10kbC4T, 3272-26A4G, L206Wa, D1152Ha, (TG)13(T)5a (Dequeker, Stuhrmann et al. 2009). From this, we can that any change in the amino-acid sequence that severely affects CFTR synthesis or function will result in CF disease. An early termination signal, GT/AG intron and exon splicing sites and deletion of one or more exons will also result in CF disease.

Symptoms of Lung Cancer

Unfortunately, in the early stages, lung cancer has no symptoms, and thus it is usually diagnosed at advanced stages. Lung cancer can occur throughout different parts of the lung and can metastasize to other parts of the body. Endobronchial growth: on the lining of the bronchi, intrathoracic extension: a mass protruding into the thoracic cavity, or distant metastases: Cancer that has spread from the original tumour site are all possible forms of lung cancer(Hammerschmidt and Wirtz 2009).

If Endobronchial growth is present, the patient will most likely experience coughing, hemoptysis: coughing up blood, pain, wheezing, post stenotic pneumonia, dyspnea, and stridor: high pitched wheezing. For intrathoracic extension of cancer cells, you will encounter chest pain, hoarseness, upper airway inflow obstructions, Horner’s triad, pleural effusion, pericardial effusion, dysphasia, raised diaphragm. Horner’s triad occurs when a lung tumour presses on the nerves leading to the eye and face from the upper intercostals.

This may cause the eye to not open completely and prevent sweating on the affected side of the face. Outward or systemic signs of cancer include weight loss, night sweats, fatigue and fever. If cancer has metastasized: spread to other parts of the body, you will see bone pain, headache, neurological or psychiatric abnormalities, paraplegia, hepatomegaly (enlarged liver), and pathological fractures.

Symptoms of Cystic Fibrosis

Problems in CF develop from interruption of the major exocrine glands for sweat, tears, saliva, digestive juices and mucus. This causes symptoms from different parts of the body, including the intestines, the lungs like LC, and the pancreas. A newborn child will typically present with intestinal pain which is the result of meconium ileus (bowel obstruction). A child will present with stomach pains, greasy stools, a persistent cough with thick sputum and salty skin. From European folk stories, the curse that “a child that taste salty when kissed will soon die” comes from encounters of CF when the cause of the symptoms was not fully understood (Quinton 2007).

Additionally, the nasal sinuses secrete thicker mucus and can result in the formation of polyps. The main adult clinical features are endobronchial infection, pancreatic insufficiency, and infertility resulting from the seminal vesicles. Additional features that may be present are distal clubbing (a result of bronchiectasis) and nutritional abnormalities that result in failure to thrive or steatorrhea: the presence of excess fat in faeces.

Diagnosis of Lung Cancer

Histology

Lung cancer histology, how far the tumour has spread and a determination of the patient’s functional status and ability to endure treatment must be taken into consideration. For example after a confirmation of metastasis to other areas of the body, it would not be logical to conduct an invasive lung procedure (Hammerschmidt and Wirtz 2009). The most important histology aspect is differentiating between small cell and non-small cell lung cancer.

Once this is done a selection of appropriate chemotherapies, radiation therapies, and targeted therapies can be made. International staging classification ranges T, N, and M are given to thoracic, lymph node and metastatic lung cancer respectively (Hammerschmidt and Wirtz 2009). Bronchoscopy can reveal T-Staging classification in which the position within the thoracic cavity and size are specified. Bronchoscopy can also provide cytology samples for N-Staging classification.

Non-Invasive Detection Methods

T-Descriptors can be revealed through contrast-enhanced CT. MRI as well is helpful in showing more detail in the affected area. For N-Descriptors CT is inadequate for this type of classification which is why positron emission tomography (PET) is often used. PET becomes 100% with a specificity of 78% if a large tumor (>1%) is present. Once confirmed the lymph node can be biopsied using video-assisted mediastinal lymphadenectomy. For metastatic M-Descriptors it is best to image the brain, liver, skeleton, lungs and adrenals.

This can be done using contrast-enhanced cranial CT or MRI, bone scintigraphy, ultrasonography, CT or MRI of the liver and adrenals, and PET or PET-CT. This last imaging technique is especially important in the diagnosis of non-small cell lung cancer (Hammerschmidt and Wirtz 2009).

Diagnosis of Cystic Fibrosis

There are three main levels of diagnostic intervention: the “sweat test”, genetic testing, and newborn screening. Not all cases are caught during the prenatal stage, and many CF parents will describe their baby as tasting “salty.” Genetic counseling should be included as well.

Sweat Test

The sweat test is currently the “gold standard” for cystic fibrosis and was established in 1959. Commonly the test is performed three times to ensure an accurate diagnosis. A child with CF will have sodium or chloride sweat concentrations greater than 60 mmol/L and adolescents and adults will be greater than 70 mmol/L (Wallis 1997). During the test Pilocarpine is used to stimulate sweating from the skin and is delivered using iontophoresis. The sweat test should not be the only measure of confirmation if apparent CF symptoms exist.

Genetic Testing

If an abnormal CF gene is found in both parents then the decision can be made to go ahead and test the fetus. There have been found 1500 sequence variations in the CF gene (Dequeker, Stuhrmann et al. 2009). When searching for the mutation the geographic and/or ethnic background is taken into consideration. The same mutations can also occur in CFTR-related disorders (CFTR-RD). The two main methods of molecular testing are direct gene analysis which tests DNA for known specific mutations and “scanning methods” which statistically look for a change in the standard nucleotide sequence. 1-5% of CF alleles remain unknown and therefore genetic testing can sometimes miss these individuals.

These mutations may lie within introns or possible functional and regulatory chromosome regions. Additionally CF symptoms may appear however these may be due to abnormal sequences on non-CF genes. SCNN1 genes which express the sodium channel (ENaC) subunits are possible candidates for non-classical CFTR phenotype genetic mutations (Dequeker, Stuhrmann et al. 2009). Methods for the known mutations include: heteroduplex analysis, restrictions enzyme analysis, reverse dot blot hybridization, amplification refractory mutation system and oligonucleotide ligation assay.

For unknown mutations it is possible to use: denaturing gradient gel electrophoresis, denaturing high performance liquid chromatography, single strand conformation polymorphism and quantitative fluorescent multiplex PCR. Most importantly the lab running the diagnostics must have experienced technicians to analyze and choose the correct tests to run.

Immunoreactive Trypsin

Newborn screening is typically done by testing for an immunoreactive trypsin that is measured to be 2 to 5 times higher than normal. Due to decreasing levels this test becomes unreliable after 1 to 2 months. This test is best done in combination with CFTR mutation analysis.

Current Treatments

The common treatment for lung cancer is surgery combined with chemotherapy and radiation therapy which can be administered simultaneously (Hammerschmidt and Wirtz 2009). If maximum oxygen consumption is less than 40% of the normal rate then surgery to remove part of the lung will not provide improvement of quality of life. Due to the many cell death regulating proteins affected by lung cancer the chemotherapy is polychemotherapy.

If NSCLC and SCLC is encountered then the treatment should follow the path of SCLC. For NSCLC after resection of affected lung platinum-based adjuvant chemotherapy is recommended for stages II and III. Stage IV NSCLC can only be treated palliatively. For SCLC stages I to III platinum-based chemotherapy and radio therapy should be done simultaneously. For SCLC stage IV only palliative chemotherapy is performed with a recommended combination of etoposide and irinotecan.

In CF, current treatments involve a myriad of drugs, respiratory exercises and situational considerations of accompanying diseases that often impair other bodily systems. Nebulized and intravenous drugs, anti-mucus coagulation agents such as rhDNase, and inhaled corticosteroids and bronchodilators are used to treat the primary respiratory complications.

New Genetic Treatments

There are many types of genetic therapies that are being sought after to treat CF and Lung Cancer. They present cheap methods of detection and hopefully effective means of curing CF and Lung cancer. They also potentially present a less toxic form of treatment to the patient. The current literature describes many avenues including the use of MicroRNAs, gene therapy and protein folding correction.

Lung Cancer and microRNAs

Recent advances in Micro-RNAs (miRNAs) have aided in the prognosis, diagnosis and treatment of lung cancer. MicroRNAs are about 18 to 25 nucleotides long and inhibit translation from mRNA or cause their breakdown. MicroRNAs play a role in tumorgenesis when they are expressed incorrectly and they are stable in serum which makes them excellent clinical biomarkers (Wang, Xu et al. 2009). Additionally there are other RNAs including small interfering RNA (siRNAs) and Piwi-interacting RNAs (piRNAs). microRNAs can influence the expression of hundreds of genes and thus have a great effect on the phenotype.

The microRNA known as let-7 and one of its family member’s mir-84 has many genes that are deleted in lung cancer. The high expression of let-7 and mir-84 has is known to indicate a good chance of survival for LC individuals.(Eder and Scherr 2005). Together they form a silencing complex for the RAS protein family which, if unregulated, can lead to overproduction of cell growth. Additional microRNAs wrongly over expressed in lung cancer include: miR-21, -191, -210, -155, -205, -17-3p, -214, -212, -106a, -192, -197, -146, -203, and -150.

In body fluids, such as serum, plasma, saliva, urine and semen secreted by individuals with cancer microRNAs can be detected by extracting them from exosomes secreted by cancer cells. They are also stable when cooled down to 4°C and stored for 4 days. (Mallick, Patnaik et al. 2010). Analysis and diagnosis of microRNAs provide a much faster, less costly and efficient method of detection for especially small tumors that can’t be detected by imaging techniques.

In Vitro studies have shown that microRNA mir-7 targets the EGFR protein and reduces activation of cellular growth (Mallick, Patnaik et al. 2010). Mutations in EGFR also cause the over expression of miR-21, that when inhibited cause cell death of the EGFR-mutated cell lines. Also miR-145 will cause the growth of LC cells to stop. We can see that the use of microRNA will prove useful however certain experiments have shown that mutated genes may not be affected by microRNAs.

This can be due to post-transcriptional alteration leading to the inability to regulate mRNA. Therapeutically microRNAs can be repressed using antisense oligonucleotides or enhanced by virus-based gene therapy as well as by administering man-made microRNA. Human testing has yet to occur however mouse models show that injection of let-7 has shown to reduce lung cancer tumors. The reluctance to conduct human trials is partly because microRNA can affect such a wide range of mRNA and have undesirable effects. Nevertheless microRNA represent novel biomarkers and will eventually allow the creation of highly specialized diagnostic and personalized approaches to treating LC.

Cystic Fibrosis and Gene Therapy

New CF treatments are required because many types of treatment come at a cost to living a normal life. The treatments are highly time consuming and costly. CFTR gene therapy is a possible an viable route to permanently improving the lives of CFTR patients. These therapies fall into treating five categories:

defective protein synthesis;
impaired processing;
defective regulation
impaired function; and
reduced synthesis of normal functioning CFTR (Jones and Helm 2009).

There are many types of gene therapy delivery including: recombinant adenovirus, recombinant adeno-associated virus (AAV), cationic liposimes, and cationic polymer vectors (Mueller and Flotte 2008). The methodology of gene therapy is to use a gene transfer agent to deliver the genetic information to the cell of interest. This type of therapy seems to be a good fit for CF because the CFTR is a single gene protein.

However the cells of interest are the pseudostratified columnar superficial epithelium do not differentiate and instead are naturally terminated. This presents a problem for application since the cells do not divide and thus treatment would have to be almost continuously administered. Therefore the focus has shifted to the earlier stem cells called pulmonary stem cells. The thick mucus generated by the pathology of the disease also presents a barrier between the airway passage and the underlying cells.

Finally the immune system has been shown to attack the transduced cells with Cytotoxic T lymphocytes and Neutralizing antibodies against viral vectors (Mueller and Flotte 2008). Recently it was shown in primates that AAV serotype 2 (AAV2) when delivered as an aerosol could improve pulmonary function and inflammatory mediators (Warrington and Herzog 2006). The following diagram shows the delivery of the cDNA by AAV2.

cDNA Delivery by AAV2

However there is a limit to the packing size of AAV and it cannot accommodate the full-length CFTR cDNA in combination with an active promoter and relevant 3’ end signals. A work around was achieved by deliver of half of the cDNA in two different AAV vectors.

When the two vectors infect the same cells, the complementary sequences at the 30 and 50 noncoding regions located upstream and downstream respectively cause the two pre-mRNAs to join together. With this trans-splicing technique chloride conductance was observed in vitro with an efficiency of 12.1 %. This type of viral deliver seems promising and additional studies must be carried out in animal models.

Animal Homologues

In order to carry out experiments it is important to recognize that the expression of diseases are located on different chromosomes and thus different nucleotide sequences within non-human animals. For lung cancer the most common animal homologue is the mouse Murine model (Colunga 2010). The EGFR gene is located on chromosome 11 A1-A4; and is 11 9.0 cM long. In Cystic Fibrosis the mouse model is also used. The CFTR gene is on chromosome 6 and is located at NC_000072.5 (18,268,986..18,269,760) which is approximately 775 base pairs. This gene has been knocked out and used to study the effects of viral delivery drugs. Both of these homologues can be found on the NCBI.org Entrez Gene website.

Summary

In summary the treatment for lung cancer must be multi-factorial and the diagnosis or screening must be able to detect the formation of cancer cells at a very early stage. microRNAs seem to promise a solution to both of these requirements. The treatment for cystic fibrosis must be able to overcome the physical disease barriers and the multi-defense strategies imposed by the immune system. The use of adeno-associated viruses possibly hold the key to solving these problems. The use of mouse models that effectively represent the conditions of the human expression of CF will bring about quicker and more realistic results in respect to drug development and delivery.

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