Appunti Medicina Interna (Internal Medicine, Biomolecular Basis of Organ Pathologies)

Internal Medicine 1 – 06/03/2023

Colorectal cancer – CRC

CRC risk in relatives of patients with adenoma or CRC → x age (independent value) → y RR (relative risk) in general population

• RR > 1 = increased risk
• RR < 1 = protected

Population risk → general population RR = 1 → increased risk RR > 1 → decreased risk RR < 1

Molecular pathogens for CRC

• ↑ Oncogenes = increased expression, point mutation
• ↓ Oncosuppressors = inactivation of both alleles by deletion and/or point mutation
• DNA methylation = inactivation (silenced expression) of genes by methylation (i.e. mismatch repair genes)

Internal Medicine 2 – 07/03/2023

Molecular changes – Cell proliferation

6 m of alimentary canal apart from the esophagus (which must be ready to defend itself), that has a thick epithelium, the rest of the GI tract is made up by a single layer of cylindric cells → leaky gut: beginning of huge number of gastric diseases.

One normal cell become hyperproliferative [giving rise to an] adenoma (benign tumor) that has its own life, moving from a small to a medium adenoma becoming bigger with increasing of mutations to the point where we have a malignant tumor (carcinoma)

APC and hMSH involved more frequently in the early phases → APC oncosuppressor inactivation (Cr 5) present in hyperproliferative mucosa (early stage) APC + methylation are at the beginning of the process → metaplasia: normal cells located in the wrong region (gastric cells in the esophagus) → dysplasia: cells are abnormal, different grades of dysplasia (severe dysplasia equals to in situ cancer) → neoplasia.

APC (adenomatous polyposis coli – tumor suppressor gene) long arm Cr 5 → gatekeeper gene cloned in 1991 through

• Identification of interstitial deletion on Cr5q in FAP pts (loss of heterozygosity – LOH)
• Linkage analysis

Later studies demonstrated that APC plays a pivotal role also in sporadic CRC (>70% – peculiar feature of CRC) → > 90% APC gene mutation premature STOP signal trunked protein. This abnormal protein typical of APC mutations can be used for genetic screening.

APC Gene and β-Catenin

What is the role of the “non-mutated” APC gene product (i.e. APC protein) in physiological conditions?

What has β-catenin to do with?

β-catenin: protein necessary to keep a monolayer cylindric type of epithelium together. It belongs to a protein complex that contains GSK, axin, APC (destruction complex). APC is the transporter protein, the other components are “skeleton” components, necessary for cell survival because it is the transporter, it does NOT directly cause cancer. APC is responsible for β-catenin proteolysis. By 2 hit mutation or deletion, APC is downregulated. If β-catenin in the cytoplasm goes over the threshold level, it translocates into the nucleus going over the nuclear membrane (protein overflow).

Gut Role and Absorption

Gut role: absorption

• Transcellular
• Intracellular (sort of phagocytosis) → cells next to each other open and close necessity to have “muscles”

In physiological condition, APC is linked to GSK/axin/β-catenin leading them to destruction → mutated APC β-catenin accumulation with consequent translocation into the nucleus, linked to the TCF (= tumor cell factor), this complex leads to oncogenes activation (transcription) in the nucleus.

Are APC mutations the only cause of β-catenin accumulation? What happens in patients with normal APC and high levels of β-catenin?

Wnt Pathway

Wnt = wingless/intern gene whose recessive mutation is characterized by wingless Drosophila Melanogaster. Human genes coding for Wnt proteins: in the absence of Wnt ligands, β-catenin is destructed → Wnt frizzled dishevelled (Dsh) activation high affinity to the destruction complex. Wnt is the ligand for frizzled receptors: when activated, this receptor activates an internal cytoplasmic protein called dishevelled that, once activated, gets to the destruction complex (high affinity).

• Wnt overexpressed leads to tumor onset due to β-catenin accumulation
• Absence of Wnt ligands, β-catenin is destructed

β-catenin linked to TCF induces oncogenes activation in the nucleus (COX-2, PPAR-δ, C-Myc, cyclin D1, etc.) except for C-Myc, the use of NSAIDs downregulates these genes.

Arachidonic Acid and COX

Arachidonic acid: metabolized by COX-1 (constitutive) and/or COX-2 (inducible by trauma/inflammation) → arachidonic acid is metabolized by COX-1 leading to the production of protective Pg such as PgI2 involved in gastric mucosal protection and platelet aggregation → arachidonic acid metabolized by COX-2 (induced by trauma) leads to pro-inflammatory Pg responsible for inflammation: they create overflow of the blood.

NSAIDs and CRC Prevention

NSAIDs rectal adenoma regression in FAP (familial adenomatous polyposis) → prevent metabolism of arachidonic acid into prostaglandin → inhibit both COX-1 and COX-2 no gastric mucosa protection → protection against CRC.

COX-2 selective inhibitors → created on animal models → induced overexpression of COX-1 causing myocardial infarction → now are more effective and with fewer side effects.

Clinical trials → parallel treatment one group always treated with placebo and the other treated with the active drug → cross-over treatment switch in treatment within the treatment period; one group starts treatment with the placebo and then switch with the active drug whilst the other group starts with the active drug and then switch with the placebo.

Internal Medicine 3 – 14/03/2023

Cigarette Smoke and CRC Risk

Cigarette smoke/inhaling smoke can lead to CRC, e.g. death risk (RR) in firemen

• >30 years on duty → 1.51 (95% CI = 1.05-2.11) CRC
• 2.53 (95% CI = 1.27-7.07) CNS
• 2.87 (95% CI = 1.43-5.14) leukemia
• 6.25 (95% CI = 1.70-16.00) kidney
• >40 years on duty → 4.71 (95% CI = 2.03-9.27) CRC
• 36.12 (95% CI = 4.03-120.42) kidney

RR = relative risk → probability of being different from those that have a particular characteristic → comparative number (compare this to that). CI = confidence intervals (variability of the risk) do not need to get across the 1 line (1 means that risk is identical to control group) in order to be statistically significant, higher/lower compared to the control group, you need the CI to be completely above or below the 1 line significance depends on the position of the values compared to the 1 line (must be all on the same side of the line, all above or all below) → importance of the 95% value we have to NOT consider the outliers so the smallest and the highest number (each corresponding to 2.5% 100 – (2.5 x 2) = 95%) outliers, values not in the “average”.

Prolonged cigarette smoking increases cancer risk → x3 adenomas → x2 deaths from CRC → regular physical activity decreases disease, why? May be linked to decrease of inflammation.

CRC Risk in Obese People

Increased CRC risk in obese people → epidemiological studies confirm a strong association between obesity and CRC in industrialized countries → possible mechanisms: hyperinsulinism, chronic inflammatory condition (PgE2) ...?

Metabolic syndrome: combination of obesity (particular abdominal obesity) + hypertension + diabetes + hyperuricemia + hepatic steatosis cardiovascular diseases and increased deaths → metabolic diseases and tumors are the two main cause of deaths.

CRC Chemoprevention Diet

CRC chemoprevention diet → animal fat increases the risk of CRC (RR > 1) → vegetable fibers decrease the risk of CRC (RR < 1) → calcium has a protective role: a major intake of calcium reduces CRC risk.

DNA Methylation

DNA methylation → link of a methyl group to the DNA chain biological process → methylation can change the activity of a DNA segment without changing the sequence (epigenetic) → when located in a gene promoter, DNA methylation acts to repress gene transcription → in mammals, DNA methylation is essential for normal development and is associated with a number of key processes including genomic imprinting, X-chromosome inactivation, repression of transposable elements, aging, and carcinogenesis.

K-Ras Oncogene

K-Ras oncogene – Cr18 part of the RAS/MAPK pathway → it relays signals from outside the cell to the nucleus growth/proliferation/differentiation to specialized functions → modulate response to growth factors → K-Ras protein is a GTPase: GTP GDP (active inactive), acting like a switch that is turned on and off by GTP and GDP molecules → K-Ras-GDP does not relay signals to the cell's nucleus → mutated K-Ras is constantly bound to GTP → K-Ras mutation poorer prognosis, no benefit from anti-EGFR therapies.

DCC Deletion

DCC deletion – Cr18 → initiates apoptosis in a caspase 9-dependent pathway → tumor suppressor gene (or ligand-dependent suppressor frequently epigenetically silenced), a transmembrane receptor that can promote an existing tumor growth → DCC is a receptor for netrin-1 (axon guidance/cell migration) reduced cell growth in the absence of netrin-1.

Oncosuppressor p53

Oncosuppressor (activator) p53 – Cr17 guardian of the genome → activated whenever the cell is suffering → probability that mutations occur when the cell is dividing are very high: cell might be in danger and the probability of erroneous replication is higher thus leading to p53 activation → regulation/progression through cell cycle, apoptosis, genomic stability by activating > 20 oncosuppressors → reduce growth by holding cell cycle at the G1/S point on DNA damage recognition DNA repair proteins can fix the damage and cell will be allowed to continue cell cycle → increases DNA repair proteins in damaged DNA (aging, hypoxia, etc.) → initiate apoptosis if irreparable DNA damage → exerts a tissue-level anticancer effect by reducing angiogenesis.

Cancer Screening

What is “cancer screening”? Population screened in order to prevent cancer, e.g. prostate/uterus/ovaries/breast screenings → e.g. search for blood in feces/stools: if blood present free colonoscopy → cancer screening detecting cancer before symptoms (blood tests, urine tests, DNA tests, imaging) → screening can lead to false positive results invasive procedure and false negative results cancer missed.

Benefits (cancer prevention, early detection and treatment) must be weighed against any harms → universal screening usually with a specific age group → selective screening higher risk of developing cancer (i.e. family history).

CRC Screening

CRC screening → > 80% of CRCs arise from adenomatous polyps → diagnosis of CRC through screening occurs 2-3 years before diagnosis of cases with symptoms → detected polyps can be removed by colonoscopy → screening has the potential to reduce CRC deaths by 60% → the main screening tests are fecal occult blood testing (FOBT) and colonoscopy.

Internal Medicine 4 – 15/03/2023

Familiar Forms of CRC

Many familiar forms of CRC (not all forms will be discussed) → e.g. one form is characterized by adenomas preceding the cancer (sporadic) → familiar form in which cancer starts in the absence of adenomas everything said in prevention cannot be taken into consideration in this particular form.

Take into consideration two forms of CRC:

1. FAP (familial adenomatous polyposis)
2. Lynch syndrome/HNPCC (hereditary nonpolyposis colorectal cancer) different names based on the type of associate cancer (characterized not only by CRC but also cancer in other parts of the body)

FAP and Lynch Syndrome

FAP, tumor initiation = between 30-50 years of age (it takes 20 years to change from adenoma to carcinoma) → colonoscopy no later than the age of 50 → starts earlier in familiar forms of polyposis → even 5-year-old kids have adenoma.

Lynch syndrome, tumor initiation = nothing happens → mucosa remains normal → progression takes approx. 20 years, in sporadic forms in FAP the onset of adenoma is accelerated, but progression is the same → quick passage from normal mucosa to cancer (takes short time).

Difference between familiar forms and sporadic forms → cancer appears early not because the mechanisms transforming an adenoma into a carcinoma are different, they are the same, the difference is that the benign tumors appear when the individual is very young → it still takes approx. 20 years to become a cancer.

Risk of developing a carcinoma is 6% overall (in general population) → risk of developing a carcinoma in members of FAP is 100% they will invariably develop carcinoma → they will develop CRC before dying of anything else → age around 40 years when they develop CRC (in both FAP and HNCC) → earlier onset in aggressive types.

FAP – Familial Adenomatous Polyposis

FAP familial adenomatous polyposis → 1:8,300-14,025 live births → prevalence 2.3-3.2/100,000 → M : F = 1 : 1 → >100 colon adenomas and/or multiple adenomas + I° FAP relatives

• Diagnosis made when you see multiple adenomas in the colon or >20 in a member of FAP family
• If a member of the FAP family develops more than 20 adenomas then it means that we have APC mutations (long arm 5q)
• All colonocytes with a mutated allele on Cr5
• Adenomas onset by deletion/mutation of the opposite originally normal allele

75%-80% FAP patients with an affected parent → 100% malignancy risk → 0.5%-1% of all CRCs (decreasing due to early diagnosis).

What are the characteristics of all members of a FAP family? Genetic problem in long arm of chromosome 5 (5q) in all members deletion of 5q, the place where APC gene is located → all members of FAP family have a deletion of the long arm 5q, no matter whether they have or not an adenoma all present the deletion → individuals will remain healthy until they develop a second mutation on the opposite allele (two-hit mutations model).

Difference in Cancer on Adenoma

Main difference between cancer on adenoma in the sporadic form → sporadic forms start later while in the familiar forms the tumor starts very early and pretty soon the cancer will also appear.

95% of patients will have the presence of polyps by the age of 35 mean age of the appearance of cancer is 40 years (ranges between 34-43).

FAP – Gastrointestinal and Extraintestinal Features

FAP – gastrointestinal lesions → polyps particularly frequent in the distal part of the colon, especially rectum and sigmoid colon → they can show up everywhere in the colon → rare in small valve and duodenum → frequent in ampulla of water.

FAP – extraintestinal features → congenital hypertrophy of retinal pigment epithelium (CHRPE) → brain tumors → abnormal dentition → thyroid tumors → epidermal cysts → osteomas → desmoid tumors.

Genetic Tests for FAP

When should genetic tests be performed? No test has a 100% sensitivity/specificity, which means that it invariably detects the abnormalities it is supposed to detect and it never detects it when the abnormality is not there.

1. People with uncertain features of FAP (≥ 20 polyps, no relative involved) in the case of a large number of polyps (20-30) they are identified but there are no relatives known to be affected by FAP
2. People with overt FAP (≥ 100 polyps) but no other cases in the family (uncertain paternity?) in this case the diagnosis is made up because the number of polyps is such that it can identify a familiar form of adenomatous polyposis but again the family doesn’t show any other member, therefore it is important for the family/other people to be informed that in their family they have this problem.
3. Relatives of FAP patients FAP without mutation we identify a patient with a familiar form of cancer and we must test all the family members in order to screen the family and to understand where this abnormality in chromosome 5 comes from.

All the tests have the possibility of having false positive/false negative results → notably lack of evidence of genetic mutation does not exclude FAP (10% probability of having a false negative result).

Available Genetic Tests

• FAP
• HNPCC
• Peutz-Jeghers syndrome (PJS)
• Familial juvenile polyposis (JPS)

DNA-MMR – DNA mismatch repair genes

HNPCC – Lynch Syndrome

HNPCC hereditary nonpolyposis colorectal cancer (Lynch syndrome) → 0.8%-1% of CRC → rare (if any) colon adenomas → 45% develops other cancers in other parts of the colon and/or in other organs within 10 years from first CRC in particular females (e.g. endometrial cancer) → microsatellite instability (MSI) mismatch repair genes and MI work together to increase the probability of developing cancer → MMR mutations (MLH1, MSH2, etc.) in > 95% of cases.

Lynch syndrome has an early onset + can have multiple locations since every colonocyte can start the cancer and, unlike FAP, it is predominant in right colon more difficult to control.