Mutation FFPE for Research and Discovery

FFPEMUTATION

Breast Cancer 2

Table of Contents

Lung Cancer 8

Colorectal Cancer 4

Melanoma 12

CBio's COA 14 FAQS 17

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Sources 19

CBIO Contact 20

Breast Cancer Mutations

HER2 belongs to a family of receptor tyrosine kinases (RTKs) that

includes EGFR/ERBB1, HER2/ERBB2/NEU, HER3/ERBB3, and

HER4/ERBB4. The gene for HER2 is located on chromosome 17 and

has been found to be amplified with an increased copy number in several

cancers. Activated HER2 leads to activation of multiple downstream

pathways within the cell, including the PI3KAKTmTOR pathway, which

is involved in cell survival, and the RASRAFMEKERK pathway, which

is involved in cell proliferation.

HER2 overexpression occurs in 18–20% of breast cancer patients and

arises from multiple mechanisms, with gene amplification being the most

common. Overexpression in general is measured by IHC and gene

amplification is measured by FISH methods. Activating mutations in

HER2, detected by PCR or NGS, are estimated to occur at a frequency

of 1.6–2.0% in breast cancer.

Her2 Overview

Her2 (ERBB2) in Breast Cancer

Implications for Patient Care HER2 overexpression in breast cancer carries prognostic and predictive

significance. In the adjuvant setting, HER2 status is prognostic for

outcomes and predictive for outcomes with HER2targeting therapies

such as trastuzumab based therapy and with anthracyclinebased

therapies. In the metastatic setting, HER2 status predicts outcomes with

trastuzumab and HER2targeting agents. Preclinical studies have

indicated that some HER2 mutations may result in sensitivity or

resistance to trastuzumab, neratinib, or lapatinib, depending on the

specific mutation.

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Phosphatidyl 3kinases (PIK3) are a family of lipid kinases involved in

many cellular processes, including cell growth, proliferation,

differentiation, motility, and survival. PI(3,4,5)P3 recruits important

downstream signaling proteins, such as AKT, to the cell membrane

resulting in increased activity of these proteins. Mutant PIK3CA has been

implicated in the pathogenesis of several cancers, including colon

cancer, gliomas, gastric cancer, breast cancer, endometrial cancer, and

lung cancer.

Somatic mutations in PIK3CA have been found in a substantial fraction of

breast cancers, including 26% of Invasive Breast Cancer, 35% of ER+

and/or PR+ Breast Cancer, 31% of Her2+ Breast Cancer, and 8% of

ER/PR/Her2 Breast Cancer.

PIK3CA Overview

PIK3CA in Breast Cancer

Implications for Patient CarePIK3CA mutations are positive prognostic factors in breast cancer.

PIK3CA mutations are also associated with decreased benefit to

neoadjuvant EGFR targeted therapies in breast cancer, and have also

been shown to blunt benefit of antiHer2 drugs in breast cancer treatment.

PTEN (phosphatase and tensin homolog deleted on chromosome ten) is

a lipid/protein phosphatase that plays a role in multiple cell processes,

including growth, proliferation, survival, and maintenance of genomic

integrity. PTEN acts as a tumor suppressor. inactivation and thus

increased activity of the PI3KAKT pathway. Somatic mutations of PTEN

occur in multiple malignancies, including gliomas, melanoma, prostate,

endometrial, breast, ovarian, renal, and lung cancers.

PTEN Overview

PTEN continued...

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Somatic mutations in PTEN have been found in a fraction of breast

cancers, including 7% of Invasive Breast Cancer, 4% of ER+ or PR+

Breast Cancer, 5% of Her2+ Breast Cancer, and <1% of ER/PR/Her2

Breast Cancers.

PTEN in Breast Cancer

Implications for Patient CareIn vitro studies have shown that inactivating mutations in the PTEN gene

confer sensitivity to PI3KAKT inhibitors as well as FRAP/mTOR

inhibitors. These findings are targets for clinical trials.

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Colorectal Cancer Mutations

Three different human RAS genes have been identified: KRAS

(homologous to the oncogene from the Kirsten rat sarcoma virus), HRAS

(homologous to the oncogene from the Harvey rat sarcoma virus), and

NRAS (first isolated from a human neuroblastoma). RAS proteins are

central mediators downstream of growth factor receptor signaling

and therefore are critical for cell proliferation, survival, and differentiation.

KRAS mutations are particularly common in colon cancer, lung cancer,

and pancreatic cancer. ¹

KRAS Overview

KRAS in Colorectal CancerApproximately 36–40% of patients with colorectal cancer have tumor

associated KRAS mutations. The concordance between primary tumor

and metastases is high, with only 3–7% of the tumors discordant.² The

majority of the mutations occur at codons 12, 13, and 61 of the KRAS

gene.

Implications for Patient CareMultiple studies have now shown that patients with tumors harboring

mutations in KRAS are unlikely to benefit from antiEGFR antibody

therapy, such as cetuximab and panitumumab. This means while EGFR

inhibitors are an option for a majority of the Colorectal Cancer patient

population, nearly 40% of patients will require an alternative therapy.

Researchers continue to try and develop a KRAS inhibitor therapy, which

to this point has been largely unsuccessful. There are currently over 50

US and international KRAS Associated Colorectal Cancer clinical trials.

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Phosphatidyl 3kinases (PI3K) are a family of lipid kinases involved in

many cellular processes, including cell growth, proliferation,

differentiation, motility, and survival. PI3K is a heterodimer composed of

2 subunits. The PIK3CA gene encodes p110a, one of the catalytic

subunits. Mutant PIK3CA has been implicated in the pathogenesis of

several cancers, including colon cancer, gliomas, gastric cancer, breast

cancer, endometrial cancer, and lung cancer.¹

PIK3CA Overview

PIK3CA in Colorectal CancerSomatic mutations in PIK3CA have been found in 1030% of colorectal

cancer cases. These mutations usually occur within two “hotspot” areas

within exon 9 (the helical domain) and exon 20 (the kinase domain). ¹

The concordance between primary tumor and metastases is high, with

only 3–6% of the tumors discordant.²

Implications for Patient CarePIK3CA mutations may be a potential biomarker in identifying colorectal

cancer patients with an increased risk for local recurrences. PIK3CA

mutations may also be associated with clinical resistance to EGFR

targeted therapies, but there have been conflicting clinical results. ¹ Pi3K

inhibitors are being investigated in phase IB and II clinical trials.

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BRAF belongs to a family of serinethreonine protein kinases that

includes ARAF, BRAF, and CRAF (RAF1). RAF kinases are central

mediators in the MAP kinase signaling cascade. As part of the MAP

kinase pathway, RAF is involved in many cellular processes, including

cell proliferation, differentiation, and transcriptional regulation.¹

BRAF Overview

BRAF in Colorectal CancerApproximately 8–15% of colorectal cancer (CRC) tumors harbor BRAF

mutations. The concordance between primary tumor and metastases is

high, with only 14% of the tumors discordant.² The presence of BRAF

mutation is significantly associated with rightsided colon cancers and is

associated with decreased overall survival.

Implications for Patient CareSeveral studies have reported that patients with metastatic CRC (mCRC)

that harbor BRAF mutations do not respond to antiEGFR antibody

agents cetuximab or panitumumab in the chemotherapyrefractory

setting. Based on these findings, BRAF mutations were suggested to be

a negative predictor of response to antiEGFR therapy. While BRAF

V600mutated melanomas are sensitive to vemurafenib (Sosman et al.

2012), BRAF V600mutated CRCs may not be as sensitive.

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central mediators downstream of growth factor receptor signaling and

therefore are critical for cell proliferation, survival, and differentiation.

NRAS mutations are particularly common in melanoma, hepatocellular

carcinoma, myeloid leukemia, and thyroid carcinoma. ¹

NRAS Overview

NRAS in Colorectal CancerNRAS mutations occur in ~1–6% of colorectal cancers. The concordance

between primary tumor and metastases is high, with only 3–7% of the

tumors discordant. ²

Implications for Patient CareWild type NRAS, together with wild type BRAF and KRAS, is associated

response to EGFR antibody. Several studies have shown that patients

with NRASmutated tumors are less likely to respond to cetuximab or

panitumumab, but this may not have an effect on PFS or overall survival.

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Lung Cancer Mutations







KRAS mutations are particularly common in colon cancer, lung cancer,

and pancreatic cancer.

KRAS Overview

KRAS in Lung CancerApproximately 15–25% of patients with lung adenocarcinoma have tumor

associated KRAS mutations. KRAS mutations are uncommon in lung

squamous cell carcinoma. In the vast majority of cases, KRAS mutations

are found in tumors wild type for EGFR or ALK ; in other words, they are

nonoverlapping with other oncogenic mutations found in NSCLC. KRAS

mutations are found in tumors from both former/current smokers and

never smokers. They are rarer in never smokers and are less common in

East Asian vs. US/European patients.

Implications for Patient CareThe role of KRAS as either a prognostic or predictive factor in NSCLC is

unknown at this time. Very few prospective randomized trials have been

completed using KRAS as a biomarker to stratify therapeutic options in

the metastatic setting. Unlike in colon cancer, KRAS mutations have not

yet been shown in NSCLC to be negative predictors of benefit to anti

EGFR antibodies. However, KRAS mutations are negative predictors of

radiographic response to the EGFR tyrosine kinase inhibitors, erlotinib

and gefitinib. Currently there are no direct antiKRAS therapies available.

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Epidermal growth factor receptor (EGFR) belongs to a family of receptor

tyrosine kinases (RTKs) that include EGFR/ERBB1, HER2/ERBB2/NEU,

HER3/ERBB3, and HER4/ERBB4. The binding of ligands, such as

epidermal growth factor (EGF), induces a conformational change that

facilitates receptor homo or heterodimer formation, thereby resulting in

activation of EGFR tyrosine kinase activity. Activated EGFR results in

activation of multiple downstream pathways within the cell, which is

involved in cell survival and proliferation.

EGFR Overview

EGFR in Lung CancerApproximately 10% of patients with NSCLC in the US and 35% in East

Asia have tumor associated EGFR mutations. Regardless of ethnicity,

EGFR mutations are more often found in tumors from female never

smokers (defined as less than 100 cigarettes in a patient's lifetime) with

adenocarcinoma histology. In the vast majority of cases, EGFR

mutations are nonoverlapping with other oncogenic mutations found in

NSCLC (e.g., KRAS mutations, ALK rearrangements, etc.).

Implications for Patient CarePatients with nonsmallcell lung cancer sometimes show a dramatic

clinical response to gefitinib or erlotinib, smallmolecule tyrosine

kinase inhibitors (TKI) specific for the epidermal growth factor

receptor (EGFR).

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The anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that

is aberrant in a variety of malignancies. All ALK fusions contain the entire

ALK tyrosine kinase domain. Signaling downstream of ALK fusions

results in activation of cellular pathways known to be involved in cell

growth and cell proliferation.

ALK Overivew

ALK in Lung CancerApproximately 3–7% of lung tumors harbor ALK fusions. ALK fusions are

more commonly found in light smokers (< 10 pack years) and/or never

smokers. ALK fusions are also associated with younger age and

adenocarcinomas with acinar histology or signetring cells. In the vast

majority of cases, ALK rearrangements are nonoverlapping with other

oncogenic mutations found in NSCLC.

Implications for Patient CareClinically, the presence of ALK fusions is associated with EGFR tyrosine

kinase inhibitor (TKI) resistance.

MET in Lung CancerMET amplification or protein expression may also be abnormal in tumors.

MET amplification or overexpression in tumor tissue relative to adjacent

normal tissues occurs in 13% of NSCLC cases and is associated with

poor prognosis. In a recent phase II study in which patients with NSCLC

were randomized to MetMab (an antiMET antibody) plus erlotinib vs

erlotinib alone, increased expression of MET protein was associated with

improved progression free survival and overall survival in patients who

received MetMAb and erlotinib.

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Melanoma Mutations

BRAF belongs to a family of serinethreonine protein kinases that

includes ARAF, BRAF, and CRAF (RAF1). RAF is involved in many

cellular processes, including cell proliferation, differentiation, and

transcriptional regulation. Mutant BRAF has been implicated in the

pathogenesis of several cancers, including melanoma, nonsmall cell

lung cancer, colorectal cancer, papillary thyroid cancer and ovarian

cancer.

BRAF Overview

BRAF in MelanomaSomatic mutations in BRAF have been found in 3750% of all malignant

melanomas. BRAF mutations are found in all melanoma subtypes but are

the most common in melanomas derived from skin without chronic sun

induced damage. In the vast majority of cases, BRAF mutations are non

overlapping with other oncogenic mutations found in melanoma (e.g.,

NRAS mutations, KIT mutations, etc.).

Implications for Patient CareWhile BRAF inhibitor therapy is associated with clinical benefit in the

majority of patients with BRAF V600E mutated melanoma, resistance to

treatment and tumor progression occurs in nearly all patients, usually in

the first year. A variety of mechanisms have been implicated in primary

and acquired resistance to BRAF inhibitors. Firstline combination

therapy with BRAF and MEK inhibitor therapy may delay or prevent some

of the mechanisms. Additionally, BRAF inhibitors have been investigated

in combination with MEK inhibitors in subsets of patients with BRAF

V600Emutated melanoma previously resistant to BRAF inhibitors.

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Melanoma Mutations







NRAS Overview

NRAS in MelanomaSomatic mutations in NRAS have been found in ~13–25% of all

malignant melanomas. The result of these mutations is constitutive

activation of NRAS signaling pathways. NRAS mutations are found in

all melanoma subtypes, but may be slightly more common in melanomas

derived from chronic sundamaged (CSD) skin. In the vast majority of

cases, NRAS mutations are nonoverlapping with other oncogenic

mutations found in melanoma (e.g., BRAF mutations, KIT mutations,

etc.).

Implications for Patient CareCurrently, there are no direct antiNRAS therapies available.

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Certificate of AuthenticityTesting ProcessAll blocks were from USsourced malignant Colorectal Cancer, Non

Small Cell Lung Cancer, Melanoma, and Breast Cancer patient cases

confirmed by 2 US Board Certified Pathologists. Six 5micron unstained

tissue slides per case were supplied. One slide was H&E stained and

reviewed by a CLIA Lab pathologist. The pathologist circled the most

appropriate area of tumor tissue for testing and estimated the tumor

density within the circled region. Tissue from unstained slides was

microdissected using the H&E stained slide as a guide. DNA was

extracted from the dissected tissue using one of two chemistries,

depending on the quantity of tumor tissue available for testing.

Spectrophotometric analysis of the optical density (O.D.) at 260 and 280

nm was performed to determine the quality and quantity of the extracted

nucleic acids.

KRAS codons 12 and 13This test was performed by PCR followed by \pyrosequencing. The

method detects and differentiates virtually all mutations at these codon

positions and has a superior limit of detection (5%) compared to Sanger

sequencing (20%). Results were reported as either mutation not detected

or mutation detected, with a description of both the DNA and protein

alteration [e.g.,mutation detected G12D (c.35G>A)].

BRAF V600This test was performed by PCR followed by pyrosequencing. The

method detects and differentiates virtually all mutations at codons 600

and 601.This method has a superior ability to differentiate mutations

compared to allelespecific PCR, and has a superior limit of detection

(5%) compared to Sanger sequencing (20%). Results were reported as

either mutation not detected or mutation detected, with a description of

both the DNA and protein alteration [e.g., mutation detected V600E

(c.1799T>A)].

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Certificate of Authenticity

NRAS codons 12 and 61This test was performed by PCR followed by pyrosequencing. The

method detects and differentiates virtually all mutations at codons 12 and

61. This method has a superior limit of detection (5%) compared to

Sanger sequencing (20%). Results were reported as either mutation not

detected or mutation detected, with a description of both the DNA and

protein alteration [e.g.,mutation detected Q61H (c.183A>T)].

EGFR Exons 18, 19, 20 and 21This test used two different methodologies to detect mutations in EGFR

exons 18, 19, 20, and 21. One part of the assay involves multiplex PCR

of exons 19 and 20, followed by sizing of the PCR products by capillary

electrophoresis. This method detects deletions and insertions in exons 19

(primarily deletions) and exon 20 (primarily insertions). The second part

of the assay involves multiplex PCR amplification of EGFR exons 18, 20,

and 21, followed by single nucleotide primer extension (minisequencing)

to detect mutations at amino acids G719 in exon 18, T790 in exon 20,

and L858 and L861 in exon 21. The limit of detection of this assay (5%) is

superior to Sanger sequencing (20%). Results are reported as either

mutation not detected or mutation detected. If the mutation is a single

base substitution, results included a description of both the DNA and

protein alteration [e.g., mutation detected L828R (c.2573T>G)].

PI3KCAThis test was performed by PCR followed by pyrosequencing. The

method detects and mutations in exon 9 (amino acids 542, 545, and 546)

and in exon 20 (amino acid 1047) of the PI3KCA gene. Results were

reported as either mutation notdetected or mutation detected, with a

description of both the DNA and protein alteration [e.g., mutation

detected H1047R (c.3140A>G)].

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Certificate of Authenticity

ALKFISH was performed on unstained TMA slides. One unstained TMA slide

is H&E stained and each tissue on the slide is reviewed by a pathologist

to ensure that the tissue is appropriate and adequate for the test(s)

requested. ALK is performed by a lab developed method with two probes

targeting the ALK gene and using a breakapart probe with a microgap

design. The 5’ALK (centromeric) probe is labeled in green and 3’ALK

(telomeric) probe is labeled in red. Fifty cells are analyzed. Results were

reported as: Negative, No evidence of an ALK gene rearrangement, or

Positive, Evidence of an ALK gene rearrangement (>50% positive) or

Negative, Polysomy for chromosome 2 or amplification of the ALK gene

locus (quantified). A sample was considered negative if <5 cells out of 50

(<10%) are positive. A sample is considered positive if >25 cells out of 50

(>50%) are positive. A sample is considered equivocal if 5 to 25 cells out

of 50 (1050%) are positive.

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Frequently Asked Questions

Were the assays bench-validated?Assays were developed and performed in a CLIA and CAP certified

laboratory under the guidance of a fellowship trained molecular

pathologist, utilizing a panel of benchvalidated probes.

What controls are used for the assay?All PCRbased tests were performed using at least three controls on

each run. A notemplate control (NTC) was used to monitor for

contamination of reagents and equipment. Positive and negative controls

were also included on each run. For all tests, the results demonstrated

the presence of the wildtype (nonmutated, negative) target and/or the

presence of a mutated (positive) target. Thus, a failed reaction was easily

distinguished from a negative (wildtype) result.

What is the data analysis method used for thesomatic mutations?Data was analyzed using Ctvalue cutoffs established for each assay. A

control reaction was performed to assess the amount of amplifiable DNA

in the sample and to calculate the difference between the reaction and

the control reaction from the same sample.

What is the limit of detection in the assays?This method has a superior ability to differentiate mutations compared to

allelespecific PCR, and has a superior limit of detection (5%) compared

to Sanger sequencing (20%).

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Frequently Asked Questions

Is the mutation result represented across the entireblock?Tissue blocks are collected from the primary malignant resection as fixed

and embedded by the pathology lab at the clinical site. Tissues represent

heterogeneity expected within the tumor, with a range of tumor cellularity

of 30% 70% for most cases. Mutations have been confirm expected

based on tumor cellularity.

What sub mutations are available?

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Sources

Additional information gathered from My Cancer Genome:

http://www.mycancergenome.org/content/disease/breastcancer/ unless

otherwise noted.

¹ My Cancer Genome:

http://www.mycancergenome.org/content/disease/colorectalcancer/

² Nature:

www.nature.com/srep/2015/150202/srep08065/full/srep08065.html

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Mutation FFPE for Research and Discovery

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