This document provides an outline for a presentation on colorectal cancer (CRC). It discusses the etiology, types, molecular pathogenesis and diagnosis of CRC. Key points include: CRC accounts for 9% of cancer deaths worldwide and arises through genetic and epigenetic changes. The main types are sporadic, inherited syndromes like Lynch syndrome, and familial CRC. Molecularly, CRC arises through chromosomal instability, mismatch repair defects, and epigenetic silencing. Important genes mutated include APC, KRAS, BRAF, p53 and DCC. Familial adenomatous polyposis and Lynch syndrome are inherited CRC syndromes associated with APC and mismatch repair gene mutations respectively.
Target Audience: Oncology fellows and Oncologists
Carcinoma of unknown primary is a challenging scenario often encountered in Oncology practice. This slide presentation discusses favorable and unfavorable presentations of CUP and it's management
Target Audience: Oncology fellows and Oncologists
Carcinoma of unknown primary is a challenging scenario often encountered in Oncology practice. This slide presentation discusses favorable and unfavorable presentations of CUP and it's management
Potentiality of a triple microRNA classifier: miR- 193a-3p, miR-23a and miR-3...Enrique Moreno Gonzalez
MicroRNAs (miRNAs) are short, non-coding RNA molecules that act as regulators of gene expression. Circulating blood miRNAs offer great potential as cancer biomarkers. The objective of this study was to correlate the differential expression of miRNAs in tissue and blood in the identification of biomarkers for early detection of colorectal cancer (CRC).
Clinical molecular diagnostics for drug guidanceNikesh Shah
1. Be familiar with next generation molecular diagnostic techniques that can provide guidance in clinical decision making
2. Identify the utility of these diagnostic approaches with some examples
3. Be aware of the challenges that exist in implementing these tools as part of the routine clinical decision making process, especially in resource limited settings
Each January, the best and brightest minds in colorectal cancer research meet at the Gastrointestinal Cancers Symposium. Fight Colorectal Cancer and the Colon Cancer Alliance are partnering to bring you the big news in colorectal cancer from the 2013 symposium.
Join us to learn more about these topics:
- Can aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) keep cancer from returning?
- The relationship of body mass index (BMI) and exercise in colorectal cancer
- What scientists are learning about how your immune system can fight cancer
- The latest on what biomarkers can tell us about your cancer
- Rectal cancer treatment that is based on your biological make-up
The webinar will be led by Dr. Richard Goldberg, an internationally renowned gastrointestinal oncologist who specializes in colorectal cancer. He is a tenured professor in the Department of Internal Medicine at The Ohio State University and serves as physician-in-chief at Ohio State’s Comprehensive Cancer Center – Arthur G. James Cancer Hospital and Richard J. Solove Research Institute (OSUCCC – James).
In this webinar our Medical Advisory Board member Dr. Dennis Ahnen will cover the basics of colorectal cancer – the hows, whats, and whys.
This August 2015 webinar is brought to you by Fight CRC’s Research Advocacy Training and Support (RATS) program. http://fightcolorectalcancer.org/do-something/support-research/research-advocacy-training-and-support-rats/
ISMB Translational Medicine SIG 2016: Reproducible Genomic Interpretation Too...Katie Fisch, Ph.D.
TransMed 2016 SIG presentation for ISMB 2016 in Orlando, Florida (https://www.iscb.org/ismb2016program/ismb2016-sigs#transmed) profiling a precision medicine case study and open source notebooks developed for translational medicine (Jupyter-Genomics https://github.com/ucsd-ccbb/jupyter-genomics).
Please share this webinar with anyone who may be interested!
Watch all our webinars: https://www.youtube.com/playlist?list=PL4dDQscmFYu_ezxuxnAE61hx4JlqAKXpR
Cancer care is increasingly tailored to individual patients, who can undergo genetic or biomarker testing soon after diagnosis, to determine which treatments have the best chance of shrinking or eliminating tumours.
In this webinar, a pathologist and clinical oncologist discuss:
● how they are using these new tests,
● how they communicate results and treatment options to patients and caregivers, and
● how patients can be better informed on the kinds of tests that are in development or in use across Canada
View the video: https://youtu.be/_Wai_uMQKEQ
Follow our social media accounts:
Twitter - https://twitter.com/survivornetca
Facebook - https://www.facebook.com/CanadianSurvivorNet
Pinterest - https://www.pinterest.com/survivornetwork
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ONCOGENE AND PROTOONCOGENE
P53 GENE AND ITS APPLICATION IN CANCER ETIOLOGY
TUMOUR SUPPRESSOR GENE AND BCA AND BAC GENE AND ITS APPLICATION ON THE APOPTOSIS AND DEATH RECEPTORS
Answer:
Cellular oncogene formation: Carcinogenesis, tumorogenesis or oncogenesis are developed from
normal cells into a lethal cancer due to the following mechanisms inside the cell.
1. Point mutation: This mutation induces alteration in cellular DNA sequence through
synonymous nucleotide substitution result in normal gene becomes protooncogene finally
become oncogene due to chromosomal DNA replication. For example normal Ras protein
synthesizing gene become mutant Ras oncogene due to point mutation.
2. Gene amplification: This is the process of abnormal amplification of DNA in chromosomes
and generates protooncogenes into oncogenes due to numerous replications of chromosomal
DNA in succession. Example is presence of MYC gene family in lung and breast cancer cells
due to amplied DNA homogeneously.
3. Chromosomal translocation: BCR-ABL leukemia is the due to chromosomal translocation and
meticulous translocation of chromosmomal regions leading to coding of a fusion protein from an
oncogene. Ex. Philadelphia chromosome
4. DNA rearrangement: Insertions, deletions, transposition & inversion of nitrogen bases of
nucleotide sequences in DNA rearrangement generates cellular oncogenes. These rearrangements
are due to exposure of cells to carcinogenic agent
5. Insertional mutagenesis: This type of oncogene development is mainly due to retrovirus as
explained
below
For example:
The genetic changes are expected to alter the activity of the gene product --> loss of
heterozygosity ---> abnormal phenotype
Mutations result in genetic drift, which in turn results in the loss of heterozygosity. This loss of
one parental copy of nucleotide gene bases may lead to the lethal and dangerous consequences of
the living being in the following of life. This loss or drift may result in the occurrence of cancer
(breast cancer) hereditary retinoblastoma, because there may be nonexistence of the functional
tumor suppressor gene in the lost region. One parental copy only can be noticed at that lost
region there by hemizygosity.
But however there is one more functional gene copy inside the genome copy thereby offspring
can get resistance proteins synthesis against the mutation induced cancer.
If both copies are bad or one copy is become badly due to mutation the remaining gene copy
responsible for the tumor growth (the tumor suppressor gene example p53) can be denaturated by
another point mutation, result in complete suppression of gene to protect the body.
A lot of catastrophic events and consequences may occur with loss of heterozygosity (LOH).
Genetic drift results in loss of heterozygosity which may reduce and limit longevity of asexual
organisms in a population there by low population size.
During intermittent cancer development, usually inactivation of the TSG induced due to the two
somatic mutations acting on respective 2 alleles. In contrast, during the familial cancer
development merely one somatic mutation is considerably enough to generate inactivation of
TSG (p.
Prix Galien International 2024 Forum ProgramLevi Shapiro
June 20, 2024, Prix Galien International and Jerusalem Ethics Forum in ROME. Detailed agenda including panels:
- ADVANCES IN CARDIOLOGY: A NEW PARADIGM IS COMING
- WOMEN’S HEALTH: FERTILITY PRESERVATION
- WHAT’S NEW IN THE TREATMENT OF INFECTIOUS,
ONCOLOGICAL AND INFLAMMATORY SKIN DISEASES?
- ARTIFICIAL INTELLIGENCE AND ETHICS
- GENE THERAPY
- BEYOND BORDERS: GLOBAL INITIATIVES FOR DEMOCRATIZING LIFE SCIENCE TECHNOLOGIES AND PROMOTING ACCESS TO HEALTHCARE
- ETHICAL CHALLENGES IN LIFE SCIENCES
- Prix Galien International Awards Ceremony
Lung Cancer: Artificial Intelligence, Synergetics, Complex System Analysis, S...Oleg Kshivets
RESULTS: Overall life span (LS) was 2252.1±1742.5 days and cumulative 5-year survival (5YS) reached 73.2%, 10 years – 64.8%, 20 years – 42.5%. 513 LCP lived more than 5 years (LS=3124.6±1525.6 days), 148 LCP – more than 10 years (LS=5054.4±1504.1 days).199 LCP died because of LC (LS=562.7±374.5 days). 5YS of LCP after bi/lobectomies was significantly superior in comparison with LCP after pneumonectomies (78.1% vs.63.7%, P=0.00001 by log-rank test). AT significantly improved 5YS (66.3% vs. 34.8%) (P=0.00000 by log-rank test) only for LCP with N1-2. Cox modeling displayed that 5YS of LCP significantly depended on: phase transition (PT) early-invasive LC in terms of synergetics, PT N0—N12, cell ratio factors (ratio between cancer cells- CC and blood cells subpopulations), G1-3, histology, glucose, AT, blood cell circuit, prothrombin index, heparin tolerance, recalcification time (P=0.000-0.038). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and PT early-invasive LC (rank=1), PT N0—N12 (rank=2), thrombocytes/CC (3), erythrocytes/CC (4), eosinophils/CC (5), healthy cells/CC (6), lymphocytes/CC (7), segmented neutrophils/CC (8), stick neutrophils/CC (9), monocytes/CC (10); leucocytes/CC (11). Correct prediction of 5YS was 100% by neural networks computing (area under ROC curve=1.0; error=0.0).
CONCLUSIONS: 5YS of LCP after radical procedures significantly depended on: 1) PT early-invasive cancer; 2) PT N0--N12; 3) cell ratio factors; 4) blood cell circuit; 5) biochemical factors; 6) hemostasis system; 7) AT; 8) LC characteristics; 9) LC cell dynamics; 10) surgery type: lobectomy/pneumonectomy; 11) anthropometric data. Optimal diagnosis and treatment strategies for LC are: 1) screening and early detection of LC; 2) availability of experienced thoracic surgeons because of complexity of radical procedures; 3) aggressive en block surgery and adequate lymph node dissection for completeness; 4) precise prediction; 5) adjuvant chemoimmunoradiotherapy for LCP with unfavorable prognosis.
Explore natural remedies for syphilis treatment in Singapore. Discover alternative therapies, herbal remedies, and lifestyle changes that may complement conventional treatments. Learn about holistic approaches to managing syphilis symptoms and supporting overall health.
Flu Vaccine Alert in Bangalore Karnatakaaddon Scans
As flu season approaches, health officials in Bangalore, Karnataka, are urging residents to get their flu vaccinations. The seasonal flu, while common, can lead to severe health complications, particularly for vulnerable populations such as young children, the elderly, and those with underlying health conditions.
Dr. Vidisha Kumari, a leading epidemiologist in Bangalore, emphasizes the importance of getting vaccinated. "The flu vaccine is our best defense against the influenza virus. It not only protects individuals but also helps prevent the spread of the virus in our communities," he says.
This year, the flu season is expected to coincide with a potential increase in other respiratory illnesses. The Karnataka Health Department has launched an awareness campaign highlighting the significance of flu vaccinations. They have set up multiple vaccination centers across Bangalore, making it convenient for residents to receive their shots.
To encourage widespread vaccination, the government is also collaborating with local schools, workplaces, and community centers to facilitate vaccination drives. Special attention is being given to ensuring that the vaccine is accessible to all, including marginalized communities who may have limited access to healthcare.
Residents are reminded that the flu vaccine is safe and effective. Common side effects are mild and may include soreness at the injection site, mild fever, or muscle aches. These side effects are generally short-lived and far less severe than the flu itself.
Healthcare providers are also stressing the importance of continuing COVID-19 precautions. Wearing masks, practicing good hand hygiene, and maintaining social distancing are still crucial, especially in crowded places.
Protect yourself and your loved ones by getting vaccinated. Together, we can help keep Bangalore healthy and safe this flu season. For more information on vaccination centers and schedules, residents can visit the Karnataka Health Department’s official website or follow their social media pages.
Stay informed, stay safe, and get your flu shot today!
Pulmonary Thromboembolism - etilogy, types, medical- Surgical and nursing man...VarunMahajani
Disruption of blood supply to lung alveoli due to blockage of one or more pulmonary blood vessels is called as Pulmonary thromboembolism. In this presentation we will discuss its causes, types and its management in depth.
These lecture slides, by Dr Sidra Arshad, offer a quick overview of physiological basis of a normal electrocardiogram.
Learning objectives:
1. Define an electrocardiogram (ECG) and electrocardiography
2. Describe how dipoles generated by the heart produce the waveforms of the ECG
3. Describe the components of a normal electrocardiogram of a typical bipolar leads (limb II)
4. Differentiate between intervals and segments
5. Enlist some common indications for obtaining an ECG
Study Resources:
1. Chapter 11, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 9, Human Physiology - From Cells to Systems, Lauralee Sherwood, 9th edition
3. Chapter 29, Ganong’s Review of Medical Physiology, 26th edition
4. Electrocardiogram, StatPearls - https://www.ncbi.nlm.nih.gov/books/NBK549803/
5. ECG in Medical Practice by ABM Abdullah, 4th edition
6. ECG Basics, http://www.nataliescasebook.com/tag/e-c-g-basics
Ozempic: Preoperative Management of Patients on GLP-1 Receptor Agonists Saeid Safari
Preoperative Management of Patients on GLP-1 Receptor Agonists like Ozempic and Semiglutide
ASA GUIDELINE
NYSORA Guideline
2 Case Reports of Gastric Ultrasound
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Ve...kevinkariuki227
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Ve...
Molecular diagnostics of colorectal cancer
1.
2. Outline of Presentation
Introduction
Etiology
Types of CRC
Molecular pathogenesis of CRC
Molecular diagnosis for colorectal cancer
3. CRC accounts for about 9 percent of
all cancer deaths; ranks third in both
incidence and cause of cancer death
in both men and women.
Approximately 782,000 new cases are
diagnosed worldwide each year, of
which 70% originate in the colon and
the rest in the rectum
4. Etiology
The only certain way to avoid cancer is not to be born, as to
live is to incur risk.
Genetic mutations
inherited
can be transmitted from parent to offspring
occurs at or before fertilization
Acquired
occurs spontaneously in the sperm, ovum, or zygote -
future progeny may inherit such mutation
somatic mutation - during the growth and/or
development
Common in CRC (65%)
5. Types of CRC
Sporadic = 70% of cases
there is no family history, common above age of 50
Inherited = 1 – 6% of cases
Hereditary NonPolyposis CRC (HNPCC, Lynch syndrome)
Multiple polyps CRC
familial adenomatous polyposis (FAP)
hamartomatous polyposis syndromes (e.g. Peutz-Jeghers, juvenile polyposis)
MUTYH-associated polyposis (MAP)
Familial CRC = 25% of cases
have a family history of CRC, but the pattern is not consistent and the risk
is not as high as with the inherited syndromes.
single affected first-degree relative = 1.7 ↑ed risk
two affected first-degree relatives or if Dx before age 55 = Further ↑ risk
6. The adenoma-carcinoma sequence
Most CRCs arise from adenomas
(adenomatous polyps) which are formed
when normal mechanisms regulating
epithelial renewal are disrupted.
the accumulation of multiple germ-line
or somatic mutations determines the
behavior of a tumor
clonal nature of tumors
cancers result from the stepwise
accumulation of multiple somatic
mutations. Therefore, many CRCs
remain asymptomatic for years before
diagnosis.
7. Comprehensive exome sequencing
has revealed that individual CRCs
harbor an average of 76 gene
mutations and the mutated genes in
the 2 tumors overlap to only a small
extent; a few genes such as APC are
mutated at high frequency, whereas a
much larger number of genes are
mutated at relatively low frequency.
9. 1. The chromosomal instability (CIN/APC)
pathway
encompasses 80% to 85% of all CRC and adenoma
Result in abnormal karyotypes, gross chromosomal
abnormalities, such as aneuploidy, chromosome
rearrangement, oncogene activation and loss of
heterozygosity of tumor suppressor genes.
Results "gain of function" mutations which may result in
loss or mutation of tumor suppressor genes such as APC, TP53
activation of oncogenes such as KRAS
apoptotic pathways
CRC caused by CIN usually have poor prognosis
10. In 90% of CRCs inactivation of the Wnt signaling pathway
/APC or β-catenin gene/; usually by mutation of one copy
of the APC gene (70%).
a tumor suppressor adenomatous polyposis coli gene
mutation + (allelic deletion or an additional mutation →
inactivation of the other allele) → development of dysplasia
in aberrant crypt foci and early adenomas → accumulation
of additional genetic mutations (KRAS, DCC, p53 and
others) → tumor progression
11. kRAS oncogene
encode proteins that regulate cellular signal transduction of
extracellular growth signals(e.g., epidermal growth factors) to the
nucleus
point mutations resulting in a constitutively active GTP-bound protein
and a continuous growth stimulus.
The activation of RAS genes can promote cell survival and suppress
apoptosis
RAS mutations are found in up to 50% of sporadic CRCs and 50% of
colonic adenomas larger than 1 cm; they are rarely seen in smaller
adenomas → acquired during later adenoma progression
The presence of a RAS mutation in CRC is significantly associated
with the absence of response to agents targeting the epidermal growth
factor receptor (EGFR) such as cetuximab and panitumumab.
12. Causes mutation of serine/threonine protein
kinase which is involved with that acts as a
downstream effector of the KRAS gene.
substitution of valine forglutamate
Not detected in any LS/HNPCC tumors
occurs in approximately 12% of all CRC,
mutually exclusive of KRAS mutation
a prognostic and predictive marker to predict
resistance to anti-EGFR therapy
BRAF gene mutation
13. Role and Expression Patternof Epidermal GrowthFactor
Receptor in Colon Cancer
• EGFR is expressed in normal
colon epithelium and in 80-
100%of colorectal cancers
(by IHC)
• Up to 40%response rate to
anti-EGFR in wild type tumors
• BRAF, NRAS, and PIK3CA exon
20 mutations are significantly
associated with a low
response rate
14. P53
DNA damage → ↑ p53 protein levels intracellular → up
regulation of cyclin dependent cell cycle inhibitor
p21Waf1/Cip1→ cell cycle arrest → cells undergo DNA
repair
if the cell is unable to repair the DNA injury, p53 leads
to an increase in the levels of the proapoptotic protein
BAX and the cell undergoes apoptosis.
p53 may also promote apoptosis by baseline
suppression of the inhibitor of apoptosis (IAP)
15. Somatic mutations in both alleles are present in 80 percent
of sporadic CRCs, and a single germ-line mutation in this
gene is responsible for FAP
Loss of heterozygosity (LOH) can be defined as the
manifestation that results from the loss of large genomic
regions.
Lack of amplification of a particular segment of DNA can be
identified by PCR.
Amplification of two to ten segments of interest is performed.
Loss of heterozygosity (LOH)
16. DCC (deleted in colorectal cancer)
Tumor suppressor genes, affected as a result of 18q
mutations
DCC is an adhesion molecule of the immunoglobulin
family, responsible for homotypic binding between cells.
DCC protein loss can be examined by immunohistochemistry.
17. 2. mismatch repair pathway
/The mutator phenotype /
accounts for 15% of CRC
Mismatch repair corrects errors made when DNA is
copied. For example, a C could be inserted opposite an
A, or the polymerase could slip or stutter and insert two
to five extra unpaired bases.
If a mismatch or small loop is found, endonuclease (MSH2
protein recognizes, MSH6 or MSH3) cuts the strand bearing
the mutation and exonuclease (MLH1 and PMS2) then
digests this strand.
Finally it will be filled in by normal cellular enzymes.
18. microsatellites are short,
sequences of 1 to 6 nucleotide base
pairs which are repeated dozens to
hundred times throughout the
genome.
aka simple sequences or short tandem
repeats
are a ubiquitous component of the
genome of higher organisms.
The most common microsatellite in
humans is a dinucleotide repeat of
the nucleotides C and A, which
occurs tens of thousands of times
across the genome.
microsatellite loci with many
repeats are rarely detected in the
genome
Microsatellites
19. Microsatellites…
due to their repetitive nature
they are liable for backward
slippage during DNA
replication so that the same
short sequence is copied
twice
a higher number of repeats
causes a higher mutation rates
single-stranded loop or
insertion/deletion loops
20.
21. Microsatellite instability (MSI)
due to the presence of the mismatch repair system, in vivo
microsatellite mutation rates range from 10-6 to 10-2 per
generation.
Mutation of both alleles of mismatch repair (MMR) system
→ DNA strand (microsatellites) might be displaced, and
realigns out of register creating small loop of unpaired DNA
→ unable to remove the loops → microsatellite increases
or decrease in size due to either insertion or deletion of
repeating units when compared to the normal cell’s =
Microsatellite instability (MSI)
MSI is implicated in 50–60% of inherited condition
HNPCC(Lynch syndrome) → germline mutations
22. How does faulty mismatch repair result in
colon cancer?
tri- (and hexa-) nucleotide repeats are overrepresented
in coding sequences.
If the mutation do NOT encompassed a codon triplet →
frameshift mutations → affecting critical areas of cell
growth regulation genes → promotion of tumorigenesis.
These kinds of tumors are diploid
23. 3. epigenetic gene silencing
(Hypermethylation phenotype (CIMP+)) pathway
DNA methylation is involved in normal cellular control of gene
expression
(CpG island) are usually found in the regions close to promoters
characterized by methylation of a number of genes rich in
CpG islands → silencing of multiple genes or promoter region
→ loss of gene function or transcriptional inactivation (e.g.
tumor-suppressor genes, APC)
If MMR system is silenced→ MSI
CRCs with high frequency of methylation of some CpG
islands are referred to as CpG island methylation phenotype
(CIMP) positive tumors
24. 3. epigenetic gene silencing…
encompasses 35%-40% of sporadic CRC
begin with serrated polyps, especially sessile serrated
adenomas (SSAs) bearing an activating mutation in the
BRAF gene.
Epigenetic changes are potentially reversible by drugs.
CIMP+ CRCs
microsatellite stable (MSS) cancer (60% of CIMP+ ) or
MSI-H (40% of CIMP+)
Methylation occur in hMLH1
methylation may occur in tumor suppressor genes
25.
26. FAP
FAP is the most common polyposis syndrome.
the risk of CRC by the age of 40 years is almost 100%.
It is autosomal dominant and is caused by de novo germline
mutations.
The presence of FAP can be diagnosed by direct sequencing
of the germ-line mutations in APC gene on chromosome
5q21.
Somatic APC mutations are also present in most sporadic
colorectal adenomas and cancers.
27. HNPCC (Lynch syndrome)
The presence of HNPCC is defined as the presence of germ-line
mutation found in one of the four MMR genes, namely
MLH1, MSH2, MSH6 and PMS2.
HNPCC is the most common hereditary colon cancer syndrome.
It is autosomal dominant.
2-hit hypothesis = germline mutation in 1 copy of 1 MMR gene
represents the “first hit,” and somatic inactivation of the wild
type allele the “second hit.”
The BRAF gene is almost never mutated in Lynch syndrome–
associated CRCs; however, KRAS and p53 mutations can be
present.
29. Bethesda guidelines
Bethesda guidelines can be used as a screening tool for
HNPCC.
(1) CRC in a patient younger than 50 years of age;
(2) synchronous or metachronous colorectal or other HNPCC-
related tumors,
(3) CRC with histologic features associated with MSI-H status in a
patient younger than 60 years of age;
(4) CRC in 1 or more first-degree relatives with an HNPCC-
associated tumor, with one of the patients being diagnosed
before age 50 years; and
(5) CRC in 2 or more first- or second-degree relatives with
HNPCC-related tumors, regardless of age.
30. Molecular testing of CRC
Screening
Diagnosis
Prognosis
Targeted drug therapy (personalized cancer care)
Follow up
Molecular Diagnosis for CRC
31. Screening of CRC
The risk of recurrence and subsequent death due to CRC is closely
related to the stage of the disease at the time of the first diagnosis.
A good marker helps the detection of disease at earlier stage so
that diseases can be cured effectively.
Screening of CRC
1. Fecal occult blood testing (FOBT)
is simple, non-invasive and inexpensive
false positive results might be yield by diet and medication.
2. Immunological FOBT
detects human haemoglobin
Specific but low sensitivity at detecting adenomas and CRC
3. Colonoscopy and sigmoidoscopy
more effective and sensitive in screening
invasive, high cost and inconvenience (privacy, extensive bowel preparation…)
32. Screening of CRC…
4. molecular markers testing
Following the better understanding in the molecular basis
of CRC, Molecular markers that detect gene mutation in
the early stages of CRC can be used as non-invasive
screening tests for early detection of CRC, followed by
invasive confirmatory tests such as colonoscopy for
individuals with positive results.
e.g. Carcinoembryonic antigen (CEA) and carbohydrate
antigen (CA)
33. Screening of CRC…
4.1 Carcinoembryonic antigen (CEA)
glycoproteins initially found in embryonic tissue and colon
malignancies.
may not be detected until the cancer is in advanced stages
found in patients with IBD, malignancies of breast, pancreas,
stomach and lung.
So, better used as prognostic markers and for follow up (Half
life of CEA is approximately 2 weeks) rather than screening
34. Screening of CRC…
4.2 Blood samples
apoptotic release of DNA from tumor cells into the
bloodstream → elevated level of circulating methylated
SEPT9 DNA (oncogen) sequences was found.
35. Screening of CRC…
4.3 Fecal DNA Tests (Cologuard™)
during apoptosis of normal colonic cell some DNA fragments of 180-
200 bps long is released into stool.
However, CRC cells show a decreased rate of apoptosis and DNA
sequences with 1800-2400 bp are detected in stool samples
CRC cells gene assay for
APC (mutated in up to 70% of specimens),
Kras (mutated in up to 60% of specimens) and
p53 (mutated in up to 60% of specimens).
for screening
specificity 95%; sensitivity 60 - 90%
not currently used – expensive, low sensitivity
36. Detection of gene mutations with known positions and
base changes
o Sanger Sequencing
o Allele Specific PCR
37. Name/Metho
d Target
Intended Use Detected
Property
Source
Material
Molecular Method Use
/Availability
KRAS therapeutic decision EGFR
targeted therapy
KRAS mutations FFPE or snap
frozen tissue
Sequencing clinical routine
BRAF chemotherapeutic
susceptibility
BRAF mutations >> >> sequencing, Real-time PCR clinical routine
BRAF chemotherapeutic
susceptibility
BRAF mutations >> >> Digital PCR, COLD-PCR studies
MSI status PCR chemotherapeutic
susceptibility
MSI status >> >> PCR clinical routine
MSI status IHC chemotherapeutic
susceptibility
MSI status >> >> IHC clinical routine
TP53 mutation screening p53 mutation
analysis
>> >> sequencing clinical routine
TP53 mutation screening >> >> >> >> oligonucleotide microarray studies
CIMP probable screening
/staging
methylation >> >> methylation microarray studies
miRNA assay for
blood/stool
screening miRNA expression
level
plasma, stool micro array studies
Multitarget stool
DNA test
screening, increasing
sensitivity
for colonoscopy
KRAS mutation,
NDRG4, BMP3
methylation,
hemoglobin
immunoassay
stool mutation and methylation
analysis, immunoassay
under approval
for clinical use
Epi proColon
early detection
assay
screening, increasing
sensitivity for colonoscopy
Septin 9 DNA
methylation assay
blood plasma Real-time PCR available for
clinical use
CIMP = CpG island methylator phenotype, FFPE = formalin fixed paraffin embedded, IHC = immunohistochemistry, miRNA = micro
ribonucleic acid,MSI = microsatellite instability, PCR = polymerase chain reaction. ARMS= amplification refractory mutation
system
38. Sanger sequencing
uses dideoxy nucleotides to terminate
DNA synthesis, yielding a series of DNA
fragments whose sizes can be measured
by electrophoresis. The last base in each
of these fragments is known, because we
know which dideoxy nucleotide was used
to terminate each reaction.
directly detects nucleotide sequences of
regions of interest.
Base substitution, insertion and deletion
mutations, could be detected
long turnover time and high running cost
39. Allele Specific PCR
allele-specific PCR, also known as amplification refractory
mutation system (ARMS) that detects specific known mutated
form of a gene i.e. primers bind to mutant DNA but not WT DNA
so that you only get amplification if the mutant allele is present..
could be adopted to a real-time PCR platform in order to
increase the speed and accuracy of the detection
Employs probe consists of a fluorophore and a quencher attached
covalently on both ends; that is specific to the targeted DNA sequence.
As this probe is broken down by the 5’ to 3’ exonuclease activity of DNA
polymerase, a fluorescent tag is separated from a quenching tag, so
fluorescence increases, and this increase can be measured in real time in
a fluorimeter.
40. the amount of target DNA
molecules can be measured.
turnover time is shorter than that
of Sanger sequencing
Compared to conventional PCR,
real-time PCR is quantitative, fast
and sensitive.
As no post-PCR manipulation,
such as gel or capillary
electrophoresis, is required, the
risk of laboratory cross-
contamination can be minimized.
41. Identification of MSI-H CRC
microsatellites are marker of choice
are highly liable for mutation
high variability in size and number of repeats from one individual to another,
co-dominance and distribution over the euchromatic genome
MSI status determination could be done by
immunohistochemical staining for MMR protein expression
PCR (A fluorescent multiplex PCR-based method, RT-PCR)
42. TESTING MSI IN TUMORS PROTOCOL
1. Unstained sections from formalin fixed paraffin
embedded tissue (5-7 micron); overlap with H&E
stained section
• From same patient select one or two tissue blocks
containing
o Viable Tumor
o Noneoplastic tissue
• Scrape selected areas for DNA
extraction at least 1cm2
o Tumor Area (T); Nonneoplastic tissue (N)
43. PCR Identification of MSI-H …
2. A tumor tissue specimen and normal tissue specimen are
amplified using PCR for 5 to 7 microsatellite markers
3. Fluorescently labeled products are sized by capillary
electrophoresis
mononucleotide loci
dinucleotide loci
44. PCR Identification of MSI-H …
4. Analyze 5 loci (2 mononucleotide repeats and 3
dinucleotide repeats) for change of any length
1. microsatellite-high(MSI-H) = instability ≥2 loci
5-fluorouracil is not effective in treating
betterprognostic factor
sporadic (~12%) or Lynch syndrome–associated (~3%)
2. microsatellite-low (MSI-L) = instability at 1 locus
If that locus is dinucleotide → additional analysis of one
mononucleotide repeat with BAT-40 ) is recommended
3. microsatellite-stable (MSS) = no microsatellite
mutations
45. DNA hypermethylation detection
DNA hypermethylation can be detected in primary colorectal
carcinomas using bisulfite conversion of DNA samples
followed by methylation-specific PCR.
Methylation-Specific PCR (MSP), which is based on a
chemical reaction of sodium bisulfite with DNA that converts
unmethylated cytosines of CpG dinucleotides to uracil or
UpG, followed by traditional PCR. However, methylated
cytosines will not be converted in this process, and primers
are designed to overlap the CpG site of interest, which allows
one to determine methylation status as methylated or
unmethylated.
46. Microarray Analysis
is a technique that permits the study of up to thousands of genes
concurrently in a single experiment. Multiplex lab on chip
Genes are represented by 15–20 different 25mer oligonucleotides on
GeneChip® that serve as unique, sequence–sequence detectors
Evolved from southern blotting
47. Is collection of microscopic DNA
spots attached to solid surface
(glass or silcon chip).
The identity of the the feature is
known by its position
Each DNA spot contain 10-12
moles of specific DNA probs
which is used to hybridize c DNA
cRNA
Prob target hybridization is
detected & quantified by
fluorophore labled targets
48.
49. microRNAs in CRC
are a class of small non-coding single-stranded RNAs with
important posttranscriptional regulatory functions by binding
to their target mRNAs.
E.g. involvement of miRNA-133a in regulating the EGFR
pathway