Saturday, December 17, 2022

Notes from "Cancer: Emperor of All Maladies" Part - II

Hormones typically work by binding to receptor in a target cell. ER was the estrogen receptor in cells for binding estrogen. Some breast cancer cells had high levels of ER while some didn't (ER+ve and ER-ve tumors). Thats why only some breast cancer cases responded with ovary removal (i.e hormonal treatment) while others didn't. There was little enthusiasm to treat cancer with hormonal treatment, in favor of chemotherapy.

Adjuvant Chemo: Treating with chemo even after all traces of cancer cells are gone. 50% of women without therapy relapsed but only 33% relapsed with adjuvant chemo.

Both hormonal and adjuvant treatment were not cures. Eventually patients relapsed, even after long remission period.

The death rates for other diseases like cholera, TB, malaria, typhus, scurvy, pellagra etc had dwindled because humans have learned about how to prevent these. The same couldn't be said for Cancer.

Between 1962-85, cancer deaths had increased by 8.7%, primarily attributed to increase in smoking rates in 1950s resulting in increase in lung cancer.

For vast majority of cancer, more intensive regimens of standard chemo treatment did not necessarily mean more survival.

NCI (national cancer inst.) had neglected prevention of cancer over treatment of cancer.

If nearly all men smoked and only some of them developed cancer, then how can one connect the link? Such cause effect relationship was only reserved for diseases where there is a known pathogen and a known carrier, like malaria. To establish linkage, studies were conducted by grouping people into smokers and non-smokers and watching them. After 10-15 years, we count the number of people having cancer in both groups. Doll and Hill created master list of doctor's cohort, dividing it into smokers and nonsmokers. Each time a death was reported, the cause of death was noted and this tabulation was maintained for extended period of time.

Watching evolution in action: Collecting moths across marshy areas of oxford. Mark them and release, then recapture them next year the marked moths and their descendent unmarked moths. There were changes in moth color, wing size, shape etc. Dark colored moths - better camouflaged on pollution-darkened trees - tended to be spared by predatory birds, thus demonstrating "natural selection" in action.

By 1960s, gross annual sale of cigarettes in America peaked at $5billion. Tobacco company began to tout the benefits of filters added to the tips of their cigarettes, after they were concerned of the tightening link between tar, tobacco and cancer. Tobacco company released "A frank statement", a full page ad saying "Experiments with mice showed link between smoking and lung cancer and not humans" which was incorrect because Doll and Hill had performed the studies on humans, not mice. The double standard was evident a decade later when more and more 'human' studies showed the link, the tobacco company countered that smoking had never been effectively shown to cause lung cancer in , of all things, mice.

Strategy of tobacco companies: Obfuscation of facts and reflection of self-doubt.

Tobacco companies were pouring money into research of faction of scientists who believed all diseases, including cancer, were hereditary. Smoking was just exposing the inherent aberration. 

In lung cancer patients, the lung contained layer upon layer of precancerous lesions in various states of evolution. As smoke travelled through the lung, the outermost layers, exposed to highest concentrations of tar, began to swell and thicken. Within this thickened layers, next stage of malignancy was found - abnormal cells with ruffled or dark nuclei in irregular patches. 

Nearly 1mil men and women were involved in the trials to establish connection of smoking with lunch cancer. Relationship was found to be the strongest. Ever since the failure to regulate alcohol during prohibition era, congress had conspicuously disabled the capacity of any federal agency to regulate an industry. Few agency wielded direct control over industry, with the FDA being an exception, but cigarettes have escaped being categorized as a drug. 

Tobacco industry were claiming freely that new filter tips were safe from cancer. Federal agency could not directly control tobacco but FTC, federal trade commission, could regulate tobacco advertisements. And this debate forced advertisements to also accompany the health advisory warning. Tobacco industry wanted to instead be regulated by congress rather than FTC, believing they could pay their money to congressmen for buying their loyalty easily. Tobacco was the economic lifeblood of southern states and the industry had financed multiple political campaigns before. Congress blunted FTC strong message in label to a mere "It 'may' cause health hazard". And so it was proved. The politicians were far more protective of narrow interests of tobacco than of broad public health interest. 

1949, congress had passed Fairness Doctrine, i.e since airwaves are public resources, any public broadcast media had to allow fair airtime to opposing viewpoints on controversial issues. FCC started regulating airtime given to tobacco ads and forcing broadcasting networks to give equal airtime to anti-tobacco ads. Internal report leaked from tobacco industry showed the threat from FCC and it proposed "Doubt is our product since it is the best means of competing with the body of fact".

Frustrated, in 1970, tobacco industry voluntarily pulled tobacco ads from networks. Last cigarette commercial was broadcast on TV on Jan 1 1971 at 1159pm.

Between 1954-84, 300 cases were filed against tobacco company by smokers asking for compensation for cancer but not a single case went against them. Then Rose Cipollone filed charges saying tobacco company knew about the risks but they kept this info hidden from public. This case allowed attorneys to scourge internal documents of the tobacco companies. They found that many cigarette makers not only knew about the cancer risks, but they actively tried to quash internal research that proved it. Fred Panzer, the PR manager at Tobacco Research Instt had laid out their strategy to combat the growing health concern. (1) Create doubt about the health charge without actually denying it (2) advocate the public's right to smoke (3) encouraging objective scientific research to resolve the question of health hazard.

In 1994, in a first, the state of Mississippi filed suit against several tobacco companies seeking to recover over a billion dollars of health care costs incurred by the state from smoking related illness. Tobacco industry is now targeting developing countries due to loose legislations and overall poverty.  

When mesothelioma cases were compared to controls, this cancer appeared to be clustered in certain professions: insulation installers, firefighters, shipyard workers, heating equipment handlers and chrysolite miners. The causal agent: exposure to asbestos.

In 1971, another carcinogen was found. Diethylstilbestrol (DES). DES was prescribed to pregnant women in 1950s to prevent premature deliveries (although it wasn't proved conclusively to be of any effect). A generation later, women diagnosed with vaginal and uterine cancer were questioned, it was found that their mothers had been given DES. DES had skipped a generation. It caused cancer in DES-treated women, but in their daughters, who were exposed to the drug in utero.

Is there an intrinsic property of all carcinogens, which could help us detect them apriori?

Sometimes a bacteria could acquire a gene mutation. Exposure to some substance might cause a higher rate of mutation. Ames found that chemicals that scored as mutagens were also carcinogenic: Dye derivatives, X-rays, benzene, nitrosoguanidine derivatives etc. The test wasn't perfect however because known carcinogens like asbestos or DES didn't cause mutation in bacteria cells. Common property of carcinogens: They altered genes.

Not only chemicals, but in 1960s it was found that chronic, smoldering inflammation caused by a human hepatitis virus could also cause cancer.

One blood antigen was present in several australian aboriginals and also found frequently in asians and africans but markedly absent in europeans and americans. It was called Australian antigen or au. Individuals carrying au often suffered from chronic hepatitis (inflammation of liver). These livers showed signs of injury and repair. Au was found to be neither a human protein or blood antigen. Au was a piece of viral protein floating in the blood, the sign of infection, therefore a person infected with this virus could turn from au-ve to au+ve. This virus turned out to be HBV. It was spreading through blood transfusion. HBV infection increased likelihood of liver cancer. It was therefore a live carcinogen. However, later it was found that the virus doesn't actually cause cancer, but the inflammation induced by the virus in the liver cells, and a cycle of death and repair was responsible for cancer.

Stomach inflammation, called gastritis was precursor to peptic ulcers and stomach cancer. In 1970s, it was common knowledge that bacteria do not grow in stomach. Upon examining biopsies of gastritis patients, the cause was found to be a new bacteria species called helicobacter pylori. But inoculated pigs didn't show signs of ulcers. So Marshall drank a solution of this bacteria himself and within few days fell violently sick. Biopsies proved he got gastritis. And thus the bacteria was associated with the disease and soon after it was linked with stomach cancer as well. An antibiotic was developed which reduced occurrence of gastric cancer in young men and women but not much useful for older patients in whom the inflammation had progressed to the point that eradication of bacteria made little effect.

Question is how could DES, asbestos, radiation, HBV, H.pylori all converge on cancer in different populations and in different organs?

As hormones rise and fall cyclically, the cells shed by cervix changed their shapes and sizes cyclically as well. Observing this you can foretell precise stage of menstrual cycle. In nearly every case of cervical cancer, when Papanicolaou brushed cells of the cervix, he found aberrant and bizarre forms with abnormal, bloated nuclei, ruffled membranes and shrunken cytoplasm that looked nothing like normal cells. Thus a new test for malignant cells was born: Pap smear. Cervical cancer typically arises in outer layer of cervix, then grows in a flaky, superficial whirl before burrowing inward into surrounding tissues. Pap smears were used to detect early and therefore cure cancer by simple surgical procedure. 

Primary prevention: disease prevented by attacking its cause i.e stop smoking or vaccine against HBV. Secondary prevention: disease prevented by screening early, like pap smear, mammography. However with screening tests like these, there was always a debate of overdiagnosis and underdiagnosis. 

Women above 55 had benefited from mammogram screening with a reduction in breast cancer deaths by 20%. Not much benefit in younger women. 

"If a man dies, it is because death has first possessed his imagination".

The toxicity of chemotherapy drugs was limited by the sensitivity of bone marrow. Sometimes patients would be left with no normal blood forming cells because the chemotherapy drugs were too toxic to kill cancer as well as all remnants of normal bone marrow cells. This ceiling was breached when doctors could transplant bone marrow from donors. Sometimes even the new bone marrow cells would attack any residual leukemia left in the patient, thinking it as foreign pathogen. This trifecta of assaults, obliterative chemo, marrow transplant and attack on tumor by foreign cells was fashioned into a treatment procedure of its own, called STAMP (Solid Tumor Autologous Marrow Program) . Soon this treatment was applied for host of cancers, not just leukemia, and remissions were more durable. However tests over the next 2 decades proves that this treatment was also not the cure, as patients often relapsed. 

March 1981, 8 cases of highly unusual form of cancer called Kaposi's sarcoma was reported. However all these cases were violent variants that had exploded into bleeding, metastatic blue-black macules spread all over the bodies. All 8 were homosexuals. The 8th person even had a very rare pneumonia called PCP. PCP only occurs when immune system becomes severely compromised. And then more such cases arose from other parts of the country. This was AIDS. In the early days, the first doctors responsible for treating AIDS patients were oncologists. AIDS patient were treated with chemo, just like any cancer patient.

Jan 1983, in the biopsy of a lymph node of an AIDS patient, signs of a virus were found. It was a retrovirus. An RNA virus that could convert its genes into DNA and lodge into human genome. HIV. 

Genes can move from one generation to next, being carried on chromosomes. In certain bacteria species, genes could also be transmitted laterally between two organisms, even from dead inert bacteria, which means some inert chemical was responsible for carrying genes. This chemical was DNA. 

Central dogma of molecular biology: The unidirectional flow of genetic information. DNA->RNA->Protein. This is universal rule, found in bacteria to humans. 

Some forms of cancer, like breast and ovarian, tended to run in families. 

X-rays was found to vastly increase rate of mutation in genes. X-rays also caused cancer. So a link came to surface. Could cancer be a disease of mutations?

Most of time, when a virus leaves the body, it doesn't leave any footprint behind, our DNA is left untouched. However, RSV (Rous Sarcoma Virus) was different. After infecting cells, it physically attaches itself to cell's DNA thereby altering the genome. The virus was nothing but a single strand of RNA. Thus it was found that RNA could generate DNA. The genes of retroviruses exist as RNA outside cells. When they infect a cell, they make DNA copy of their genes and attach this copy to cell's genes. This DNA copy, called a provirus, makes RNA copies and virus is regenerated to form new virus. Finally the single gene in RSV that was causing the cancer was identified. This gene was called src. The first oncogene. Src worked by encoding a protein which works by modifying other proteins by attaching a phosphate group, to these proteins. This phosphate group attachment acts like a ON switch for the protein. Often one turned on protein would then turn on another protein and so on. A confluence of many such activated switches produced powerful signal inside a cell to change its state from non-dividing to a dividing state. 

Later a nearly identical version of viral Src was found to be lodged firmly in normal cell's genome. RSV had likely picked up an activated Src gene from a cancer cell and carried it in the viral genome, creating more cancers. Virus then acted as a courier. 

Proto-oncogene: precursor of a cancer causing gene. They are normal cellular genes until a mutation induced by chemical or x-rays caused cancer not by inserting foreign genes into cells, but by activating such endogenous proto-oncogenes.

  • Humans have 46 chromosomes - 23 matched pairs, one inherited from each parent. In CML, the 22nd chromosome has its head lopped off. The missing head had attached itself to tip of chromosome 9, and a piece of 9 had conversely attached itself to 22. This flip-flop transposition of two pieces is called Translocation. Abl gene was on 9th and bcr gene was on 22nd chromosome. Bcr-abl was the oncogene causing CML.
  • To find out correlation of genes and cancer a particular type of hereditary related cancer was chosen I.e retinoblastoma. Its a variant of eye cancer with the tendency to erupt in same family across generations.RB has 2 variants: an inherited familial form and a sporadic form. The familial one is the inherited form, with family history I.e fathers, mothers, cousins, siblings etc also showing the cancer. This form of RB affects both eyes. But then RB was also seen in some patients who had no family history (sporadic form) and this one typically affects only one eye. Familial form typically happens at 2-6months after birth and develops rapidly. Sporadic one developed at 2-4yrs old children and grows slowly. Why? Only 1 genetic change required to develop ‘inherited’ RB but 2 genetic changes required to trigger sporadic RB. Every human has 2 pairs of chromosomes therefore 2 copy of every gene, including ‘rb’ gene. In sporadic RB, both copies of rb gene needs to be inactivated through mutation in each copy of the gene. Therefore it develops later in life. Children with inherited RB, they are already born with one defective rb gene therefore require only one mutation to trigger, therefore develops much earlier. (2-hit theory of cancer)
  • Why single mutation in src causes cancer but 2 mutations for rb? Src activates a function in cell division and rb performs suppression of cell division. So 2 classes of genes. (1) Oncogenes which acts by virtue of abnormal cell division (2) anti-oncogenes (or tumor suppressor) which acts by suppression of division. Accelerator and brakes in car. 1 mutation needed for oncogene and 2 needed for antioncogene to activate(or inhibit) their functionalities.Jammed accelerator and failed brakes.
  • ras gene was derived out of a human cancer cell (an oncogene), from a patient who died of bladder cancer. rb gene (for retinoblastoma) was also isolated from human cancer cells which is anti-oncogene
  • rb gene was also found to be mutated in other cancers like lung, bone, breast, bladder and esophageal etc. between 1983-93, many other cancer genes were isolated-myc, neu,fos,ret,akt (all oncogenes) and p53, vhl, apc (all anti-oncogene).
  • Experiment to  induce cancer in mouse by mutating gene: Myc gene was activated in mouse but mice developed only small unilateral breast cancers and that too, late in life. Mice typically developed cancer only after pregnancy, suggesting that environmental influences such as hormones are also strictly required to achieve full transformation. Only active myc gene isn’t sufficient.
  • How cancer grows: Transitions in the stages of cancer was paralleled by same transitions in genetic changes. Transition from Premalignant to invasive state was correlated with same sequence of activation and deactivation of genes.cancer is slow march of one gene mutation to next. Cancer cells not only divide uncontrollably but also migrate and invade other organs. Genes encode proteins. Proteins work like small molecular switches. Protein A may turn off protein B which will then turn on C which will turn off D and so on. Such pathways exists in normal cells for normal growth. Similarly in cancer cells, a permanent activated gene will turn on one protein which will then permanently activate another protein which will then permanently deactivate another protein and so on. Certain activated signaling pathways in cancer cells could also induce neighboring blood vessels to grow and then the tumor grows in grape like clusters around those vessels. This is called angiogenesis.The “motility genes” activated by cancer cells are the very genes normal cells use when they require movement through the body such as when immuno-logical cells move to sites of infection. Tumor angiogenesis exploits the same pathways when blood vessels are created to heal wounds. 
  • 6 rules of malignant growth
    • Self sufficiency in growth signals.
    • Insensitivity to growth inhibitory signals
    • Evasion of programmed cell death(apoptosis)
    • Limitless replicative potential
    • Sustained angiogenesis
    • Tissue invasion and metastasis.
  • Treating cancer: achilles’s heels of cancer
    • Accumulation of mutations are driving factor of cancer.We’ve to target the hyperactive genes.
    • Cancer cells depend on activated pathways for propagation. We’ve to find a way to inhibit the pathway.
    • Cancer cells have developed resistance to drugs over the course of cell division. We’ve to mix and match the drugs
  • APL cancer cells are left immature. Then a maturation agent, trans-retinoic acid was found. APL patients were given this drug. The cancer cells matured in 4 days, and then having fully matured, the cells began to die out. Remission lasted weeks and months.A combination of chemotherapy and trans-retinoid acid produced no relapse at all in 75% of patients. APL oncogene encodes a protein that is tightly bound by trans-retinoid acid. This binding immediately extinguishes the oncogene’s signal in APL cells.
  • Genentech could engineer a human gene into a bacterium, say, and use bacterial cell as a bioreactor to produce vast quantities of that protein. Thus in lab proteins could be made instead of extracting from animals.
  • Cancer cells that become dependent on the activity of a gene for their growth can amplify that gene by making multiple copies of the gene in the chromosome. This phenomenon is called oncogene amplification. Her-2 was highly amplified in breast cancer samples. We could divide breast cancer into 2 categories. Her-2 amplified (her-2+ve) and her-2 unamplified(her-2-ve). Then came a anti-Her-2 drug.When her-2 antibody was injected into a her-2+ve cancer patient, the tumors disappeared.
  • Women treated with Herceptin along with standard chemotherapy drugs showed remarkable benefit.
  • Every kinase attaches a phosphate tag to unique set of proteins in the cell, thus they act like master switches in cells, turning “on” some pathways and turning off some other.Staurosporine inhibited dozens of kinases by binding to a pocket present in most kinases.could such a drug be developed to inhibit cancer specific kinases? A drug was found that could cure bcr-abl positive leukemias.This drug is Gleevec
  • In some cases, patients had developed resistance to Gleevec and therefore were relapsing. How can cancer cell become resistant to a drug that directly inhibits its driving oncogene? These cells acquire mutations that specifically alter the structure of bcr-abl, creating a protein still able to drive the growth of the leukemia but no longer capable of binding to the drug. The a new kinase inhibitor was developed to target air-all, dasantib, and this one helped cure leukemia in Gleevec resistant patients.
  • In individual specimens of breast and colon cancer between 50-80 genes are mutated, pancreatic cancers had 50-60 and brain cancers had 40-50. Only few cancers like AML, 5-10 genetic mutations found.Set of mutated genes can vary from one breast cancer sample to another. But not all mutations actually cause cancer. Most of them are bystander/passenger mutations which has no impact on cancer. “Driver” mutations are the ones which actually contribute to cancer. 
  • Cancer’s immortality is borrowed from normal physiology. Human embryos possess a tiny population of stem cells that are capable of immortal regeneration. In few weeks, a single hematopoietic stem cell can replenish the entire human organism with new blood and then through yet unknown mechanism, lull itself back to sleep.

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