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CAPP-Seq Research

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on 30 November 2014

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Transcript of CAPP-Seq Research

CAPP-Seq Research

What is CAPP-Seq?
Cancer Personalized Profiling by Deep Sequencing
an economical and ultrasensitive method for quantifying ctDNA
analyzing levels of ctDNA is a patient-friendly method for detecting, monitoring, and treating cancer, as compared to a biopsy
CAPP-Seq can detect one molecule of tumor DNA among 10,000 molecules of healthy DNA in the blood
Step 5: Prepare Samples for DNA Sequencing
Step 4: Extract cfDNA from Plasma
Through a series of elutions by adding and vacuuming buffer solutions, cfDNA is purified from plasma.
Step 2: Patient-Level Analysis
A blood sample is periodically drawn from each patient, before and/or after chemotherapy treatment.
"Cancer is a genetic disease...
... for most cancers it's very difficult to identify any one particular genetic aberration or mutation that is found in every patient. Instead, each cancer tends to be genetically different from patient to patient." - Ash Alizadeh, MD, PhD, Assistant Professor of Medicine
Lymphoma
Stage I: The disease involves only one group of lymph nodes or one section of organ tissue
Stage II: The disease has spread to two lymph groups on the same side of the diaphragm, or to a lymph group and nearby organ
Stage III: The disease includes lymph nodes on both sides of the diaphragm
Stage IV: The disease involves multiple internal organs, including the liver, bone marrow, and blood
Step 1: Population-Level Analysis
Specific, recurrent mutations are identified and selected from the CAPP-Seq selector library.
Step 7: Quantification
After sequencing specific regions of DNA, we measure the yield of DNA (how DNA there is in a sample).
Biopsy vs. Blood Draw
What is ctDNA?
circulating tumor DNA is a noninvasive biological molecule in blood that indicates the presence of a disease
dying tumor cells release small pieces of DNA (cell-free circulating tumor DNA) into the bloodstream, which are then sequenced
What is lymphoma?
Lymphoma, the most common type of blood cancer, occurs when lymphocytes (white blood cells that help protect the body from infection and disease) behave abnormally.
Lymphoma may develop in the lymph nodes, spleen, bone marrow, blood, or other organs, and can spread to almost any other part of the body.
There are two main types of lymphocytes that can develop into lymphoma: B-cells and T-cells.
B-Cells
T-Cells
1) T helper cells assist the maturation of B cells into memory cells and the activation of cytotoxic T cells and macrophages.

2) Cytotoxic T cells destroy virally infected cells and tumor cells.

3) Memory T cells are antigen-specific T cells that can reproduce a faster and stronger immune response because they become "experienced" after encountering an antigen from a prior infection.
Follicular Lymphoma
Follicular lymphoma (FL) is an indolent (slow-growing) cancer that affects B-Cell lymphocytes.
30-40% of patients with FL eventually develop a transformed, more aggressive lymphoma.
Symptoms of FL:
enlargement of the lymph nodes in the neck, underarm, stomach, or groin
fatigue
shortness of breath
fever
night sweats
weight loss
More common in adults over the age of 60
FL is NOT an inherited disease
Small Lymphocytic Lymphoma
Step 3: Spin the Sample
Samples from patients are spun in a centrifuge, separating the blood from plasma.
Since blood has a greater density than that of plasma, it is found at the bottom of the tube after spinning in the centrifuge.
White blood cells and platelets are less dense than blood but more dense than plasma, so they are found in the middle of the tube.
cfDNA
Cell-free DNA (cfDNA) is released from tumors primarily due to apoptosis or necrosis (death of living cells or tissues).
During malignant conditions, the level of cfDNA increases due to the increased release from the tumor cells (and normal cells in the surrounding tissue).
cfDNA is extracted from plasma samples and quantified.
To distinguish tumor cfDNA from non-tumor cfDNA, specific somatic DNA mutations (which were previously selected by CAPP-Seq) are identified.
Qubit Fluorometer
A fluorescent dye is intercalated with the DNA.
The Qubit Fluorometer detects the intensity of the fluorescence, therefore indicating the concentration of DNA in a sample.
A qubit (quantum bit) is a unit of quantum information.
Bioanalyzer
Samples are placed into tiny wells on a chip containing micro-channels which create interconnected networks among the wells.
The channels are filled with a fluorescence dye.
An electric current charges the DNA (similar to gel electrophoresis), separating the molecules by size.
Smaller fragments move through the chip faster than larger fragments.
The dye molecules intercalate into the DNA, which is then detected by laser-induced fluorescence.
The software then determines the concentration of the samples.
1) B cells secrete antibodies into the body's fluids.

2) Antibodies circulate and bind to foreign antigens in the bloodstream; the B cells are antigen-presenting cells (APCs).

3) Each B cell is programmed to make one specific antibody. After activation by antigen interaction, B cells develop into memory B cells.
noninvasively monitor the volume of a tumor and the patient's response to therapy
track how tumor mutations evolve over time by sequencing specific regions of the DNA
cancer cells continuously divide and die, releasing DNA into the bloodstream
Why is ctDNA analyzed?
Diffuse Large B-Cell Lymphoma
Diffuse Large B-Cell Lymphoma (DLBCL) is a fast-growing and fast-spreading cancer that affects B-Cell lymphocytes, causing them to become large.
60% of patients are not diagnosed with DLBCL until the disease is advanced (stage III or IV)
Symptoms of DLBCL:
a quickly growing, non-painful mass that is typically an enlarged lymph node in the neck, groin, or abdomen
fever
weight loss
drenching night sweats
64 years is the average age at time of diagnosis
DLBCL is NOT an inherited disease
Men are slightly more like to develop the disease than women
Small Lymphocytic Lymphoma (SLL) is an indolent (slow-growing) cancer characterized by an excess of white blood cells in the lymph nodes, but not in the blood.
Symptoms of SLL:
painless swelling in the neck, armpit or groin
loss of appetite and tiredness
night sweats
fever
weight loss
Chronic Lymphocytic Leukemia (CLL) is very similar to SLL. Patients with CLL have an increased number of white blood cells (in the blood, unlike patients with SLL). Both are low-grade, slow-growing cancers.

1) Fragments of DNA are sheared or sonicated (sound energy that vibrates particles) in order to produce blunt edges.

2) An "A" (Adenine) nucleotide base is added to one end of the DNA fragment.

3) An adapter ("DNA handle") is then attached to the overhanging A. The adapter has an overhanging "T" (Thymine) nucleotide base, which corresponds to A.
Step 6: Polymerase Chain
Reaction (PCR)
PCR is a biochemical technology used to amplify copies of a piece of DNA, generating thousands to millions of copies of a specific DNA sequence.
PCR relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of DNA primers (short DNA fragments) containing sequences complementary to the target region.
DNA polymerase enzymatically assembles a new DNA strand by attaching DNA building-blocks called nucleotides (A, C, T, G) to the end of a primer.
The generated DNA itself is a template used for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified until a particular substance runs out.

Step 8: Analysis
The DNA is sequenced and then analyzed, and the mutations present in the DNA of each patient are recorded over time.
This large-scale bioinformatic meta-analysis of human tumors allows us to build prognostic and predictive models for clinical and therapeutic outcomes of diverse human malignancies.
The information gathered allows the lab to gain a better understanding of the initiation, maintenance, and progression of tumors, and their response to current therapies toward improving future treatment strategies.
We are applying this knowledge toward the design of clinical trials in the treatment of patients with various malignancies.
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