THE FIINAL EDIT--Strategies and Molecular Techniques for.edited-3

Strategies and Molecular Techniques in Breast Cancer Cases

Strategies and Molecular Techniques for

Diagnostics, Classification, and Treatment of Breast Cancer Cases

Alyssa R. Lanza

Dominican University of California

December 2, 2015

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Abstract

The traditional approaches to treating breast cancer including surgical, chemical,

and radiation therapies all have been shown to prevent recurrence. However, like most

other therapies, their negative effects directly affect the patient's overall quality of life.

These treatments have high toxicity, and it is common for the patient to show treatment

resistance or no clinical benefit from the therapies.

Molecular diagnostic tests provide prognostic information regarding the particular

cancer case in its early stages. The diagnostics allow for possible immediate early

management of the case, thereby providing the patients with a more personalized

diagnostic approach to their case as well as provide a personalized treatment that ceases

resistance, nonresponse, and unnecessary toxicity.

Introduction

Breast cancer persists to be the major health issue throughout the world. In 2015

and still counting, there were 231,849 invasive cases, 60,290 in Situ cases, and nearly

40,290 breast cancer deaths in the United States alone (Parkin, 2002). Breast cancer occurs

in one out of every eight women (Wilson, 2012). This complex disease comes in various

forms as well as the several treatment options available. The different therapies all have

the same objective to diminish cancer entirely, avoid cell metastasis, and to inhibit future

reproduction. The traditional clinical methods of treatment for breast cancer includes

surgical, chemical and radiation focused therapies (Wilson, 2012).

Several other molecular techniques and treatment options are available to scientists

and patients fighting breast cancer. These various new methods of molecular diagnostics

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and patient care are further researched for them to be successfully utilized as today’s

standard medical care of breast cancer. Despite the recent scientific discovery of

oncogenes, tumor-suppressor genes, and natural alternative treatments, the standard

treatment of breast cancer remains.

Personalized medicine and current comprehensive molecular diagnostics have been

discovered and are creating great excitement among clinicians, yet they have yet to fully

emerged into the standard care of breast cancer.

Researchers have been running new tests every year that can supplement or

possibly even exceed the current methods of diagnosis including, methods using selective

estrogen receptor modulators (SERMs) and gene expression analysis (Grann, 2005). These

findings include protein and gene expression analysis and the use of SERMs, estrogen

receptor modulators. These selective modulators, tamoxifen, and anastrozole are hormonal

therapies. The methods of treatment and therapies have shown success in the past and

continue to make victims of the disease survivors. However, the reproducibility, expense,

toxicity, and proof of validity beyond the protocols serve as a setback in the standard

treatment of breast cancer (Hortobagyi, 2005).

This review will focus on and evaluate the current and studied molecular diagnostic

methods, specifically regarding breast cancer. Also, the molecular diagnostic practices will

be assessed on whether they demonstrate features as a foretelling marker for breast

cancer.

Hormone Receptors

The first molecular marker to be evaluated for prognosis and therapy for breast

cancer was found in the 1950’s (Grann, 2005). Today the presence of estrogen receptors

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and progesterone receptors are still under experimentation with breast cancer cases. The

estrogen receptors have continuously shown a significant predictive response on tumors

during the hormone therapy (Buzdar, 1998). Progesterone receptors are also currently

undergoing tests and the response of tumors to the therapy remains unclear. However, in

some tests the progesterone receptor appears to be promising soon, as certain cases

validate the positive response to treatment (Layfield, 2000).

Status tests for the estrogen and progesterone hormones are standard for all types

of breast cancers as well as for recurring cancers (Layfield, 2000). This test is to predict the

patient’s potential cancer outcome as it serves as a prognostic marker and ultimately

determines if the hormone therapy will be a beneficial treatment. The patient's average

prognosis and cancer are likely to respond to the hormone therapies if the patient's cancer

is ER-positive and PR-positive. However, as the cancer is ER-negative and PR-positive, and

vice versa, they will still benefit from the treatment yet they may have a lower response

(Van’t Veer, 2002). The patient will not respond well or are not likely to take advantage

from the hormone therapy if both the ER and PR are negative (Grann, 2005). The case in

which the tumor is a double positive ER/PR does show to respond better than the single

positive tumors in hormone therapy.

The patient’s reaction to the endocrine therapy does depend on various factors, yet

the positive vs. negative ER and PR do serve as typically favorable responses to hormone

therapy. Whether the grades of ER and PR serve as a substantial prognosis value for cancer

patients has been studied extensively, and they remain under critical question and debate.

The status of the hormone is tested by immunohistochemical staining (IHC) of the

tissue sections or by a ligand binding assay (McGuire, 2007). The ligand-binding assay

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depicts the competitive binding of the radiolabeled steroid to the desired hormone

receptor. It shows measurable quantification of the estrogen and progesterone receptors,

(femtomole) of the receptor protein per milligram of the total cytosol protein (Budzar,

1998).

The immunohistochemical staining approach to the diagnostics of breast cancer is

the more fundamentally standard method as a hormone receptor analysis. This method

stains a tissue of interest removed during biopsy, by using antibodies and enzymes

including horseradish peroxidase, for evaluation (Layfield 2000). It is used to prove

whether or not the breast cancer cells have HER 2 receptors or the ER and PR receptors on

their surface (Layfield, 2000). This method of evaluation has a greater advantage as it can

show the intensity of the staining of individual positive nuclei as well as determine the

percentage of positive nuclei (Dowsett, 2008).

However, as this method does have its greater advantages it also has minor

setbacks. The technique and results are performed individually by pathologists, which

increases the chance of variability of analysis; as pathologists tend to use slightly different

criteria when deciding if the results are positive or negative (Hortobagyi, 2005). The

inaccurate HER2 test results may lead to the woman receiving less than the most beneficial

care for her case. If the breast cancer is HER2 negative and the test results come back

classifying the case as HER2 positive, doctors may give the patient treatment that is anti-

HER2 (Layfield, 2000). This case may be problematic for the patient as they will be unlikely

to benefit from the treatment and may also be at risk of the medicine's effect (Grann, 2005).

As with the cases where HER2 positive is present but the results classify the test as HER 2

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negative, doctors are highly unlikely to give the patient the treatment, regardless to if she

could potentially benefit from the medicines (Grann, 2005).

Even with the troubleshooting, the immunohistochemical staining method is the

most common and beneficial approach when determining the status of hormones in breast

cancer (Van’t Veer, 2002). The results of the staining provide vital information in treatment

planning for the patient.

HER2

As mentioned before, human epidermal growth factor receptor 2 (HER2) is another

prognostic marker that is considered for the assessment of primary invasive breast cancer.

This receptor is an oncogene that belongs to the EGF receptor family (Wolff AC, 2007). The

HER2 gene produces HER2 proteins that exist as receptors on breast cells. As the amount of

HER2 gene increases by gene amplification, the extra HER2 genes increase the number of

HER2 receptors the breast cell makes, known as HER2 protein overexpression (Wolff AC,

2007). The gene amplification of HER2 are reported to have occurred in 10-40% of primary

tumors, and the HER2 protein overexpression is found to occur in nearly 25% of all breast

cancers (Sotiriou, 2006). Cases as such are called HER2-positive reports. Compared to

HER2 negative cases, the HER2 positive breast cancers often grow rapidly and have more

chances of spreading and returning after treatment (Wolff, 2007). The HER2 expression is

a vital prognostic marker. Studies have shown that the HER2 positive cases with protein

overexpression have a higher mortality rate compared to women who have HER2 negative

expression (Wolff, 2007).

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To evaluate the HER2 status, IHC tests for the protein expression while a FISH

identifies the gene expression (Paik, 2004). Compared to ER and PR, the staining of HER2

for expression is a percentage of the stained versus the unstained tumor cells in the breast

tissue. As these methods to determine the status of HER2 gene expression and the protein

overexpression are easily reproducible and to perform, there is a downfall of

standardization and varying interpretation as mentioned earlier. However, the

determination of HER2 status is not a recommended first determination of a patient's

prognosis. A patient's HER2 status allows them to receive various therapies. These

treatments make it difficult to prove if the HER2 status is the sole factor that estimates the

disease progression or if HER2 is just a component paired with the therapies (Paik, 2004).

HER2/neu Analysis

The breast cancer assay to improve the current methods of HER2/neu analysis is

the HERmark™ assay (HER2 Status | Breastcancer.org ). It accurately measures the amount

of functional HER2 homodimer HER2 protein present on the surface of the breast cancer

cells, as well as measure the total amount of HER2 protein present (National Institute for

Health and Clinical Excellence, 2009). This assay is used primarily as an assistant test for

IHC and FISH analysis. The testing provides further testing when IHC and FISH tests seem

to give conflicting or questionable results. The HERmark assay is also used when the

clinical picture of the patient is different from the HER2 status reported (National Institute

for Health and Clinical Excellence, 2009).

The HERmark runs on a dual antibody system, where the second antibody containing the

photo-activated molecule splits the fluorescent tag on the antibody. After the cleavage, the

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tags are measured with capillary electrophoresis (Paik, 2004). The levels of the quantified

HER2 protein from the HERmark report determines whether the patient is indeed HER2

positive or negative.

A Recent investigation on the HERmark assay indicates that this assay successfully

identifies the breast cancer cases that are more likely to benefit from trastuzumab therapy

(Van't Veer, 2002). When determining the predictor of breast cancer progression, reports

suggest the measurements of the activated form of HER2 serve as a more useful

measurement of the total HER2 expression (Buyse, 2006). Therefore, regardless of the

HERmark assay’s ability to be used as a foretelling indicator of the patients’ response to the

anti-HER2 therapy, further experimental studies are required to validate the findings and

to prove whether the HER2 activation measurement does serve a prime prognosticator of

the patients’ medical outcome.

However, there is considerable controversy over the accuracy, reliability, and

analysis variability of the assay methods (Buyse, 2006). About 20% of the HER2 testing

done in the field are proven to be inaccurate when tested against “expert” laboratories

(National Institute for Health and Clinical Excellence, 2009). To improve the consistency

and overall accuracy with the HER2 testing, the American Society of Clinical Oncologists

and the College of American Pathologists created and proposed a parameter of

recommendations for the testing with the IHC and FISH (HER2 Status | Breastcancer.org).

Theros H/I^SM and MGI^SM

Several RNA-based molecular diagnostic tools are utilized and readily available for

breast cancer diagnostics. These tests focus on measurable reverse transcription PCR

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analysis or gene expression microarrays (Ma XJ, 2004 ). These RNA-based molecular

diagnostic tools include the Theros H/I^SM and MGI^SM and Oncotype DX.

Theros H/I^SM and MGI^SM, which describes the combination of two gene indexes,

predicts the clinical outcome of cancer patients who were treated with tamoxifen (Paik,

2004). This test measures the ratio of HOXB13: IL17BR gene expressions (Buyse, 2006).

The MGI is the additional test that reclassifies the tumors in ER-positive breast cancer

patients and provides information regarding the tumor’s features such as; the tumor grade,

proliferation status, resistance to chemotherapy, and it’s sensitivity. This test separates ER-

positive patients into the high or low risk of relapse groups (Ma XJ, 2004). The likelihood of

recurrence reclassifies a grade 2 tumor into a grade 1 or a grade 3 product, depending on

the results.

This test takes a biopsy sample of the breast tumor tissue, and the mRNA is

extracted from the sample. This part of the sample is then further used to measure the gene

expression through the qRT-PCR (Ma XJ, 2004). The results of the test express the potential

advantages over the presently used methods. This test removes the uncertainty of the

tumor grading and reclassifies a once grade 2 (intermediate proliferative) tumor as a grade

1 or 3 tumor. The reclassification allows for the patients to receive the most beneficial

therapy for their breast cancer case. The qRT-PCR assay is standardized in the laboratory.

Therefore, it eliminates the variability connected with the individual grading by the

pathologist (Paik, 2004).

As this molecular diagnostic test indeed shows predictive values for patients treated

with hormonal therapy, the American Society of Clinical Oncology (ASCO) is currently

investigating its clinical utility and necessary application. ASCO has found there to be no

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analysis proving that it provides a superior classification of high-risk patients and their

recurrence outcomes when this test is compared with the standard methods (Harris,

2007). However, the Theros H/Ism and MGIsm has proved to show predictive value for the

patients treated with hormone therapy and further examination of the ability of the gene

ratio expression HOXB13: IL17BR is studied in its ability to predict the patients' possible

chemotherapy benefits (Harris, 2007).

Oncotype DX

Ocotype DX is genomic test analyzing the activity of genes that have the potential to

affect how cancer is likely to perform and react to a certain treatment. This test allows

clinicians to figure out the patient's risk of early-stage, ER-positive breast cancer for

relapsing and how likely it is that the patient will benefit from chemotherapy after surgery

(Cronin, 2007). The Oncotype DX test has been the best out of the four genomic tests for

breast cancer (Cronin, 2007) to have significant and valid results.

The Oncotype DX genomic test analyzes 21 genes’ activity that influences the

likelihood of cancer's possible growth and response to the treatment. The results are based

on the activity, and a calculation of a recurrence scored between 0 and 100 is made

(Sparano et al., 2015). The higher the score means, the greater chance of recurrence. The

test serves as both a prognostic and a predictive test since it predicts the possibility of

benefit from the chemotherapy or radiation therapy as well as how likely the cancer is to

return to the patient (Cronin, 2007). This test is crucial for determining if the patient with

early-stage breast cancer can get a variety of treatment options narrowed down and

tailored for their personal case based on their score.

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The Oncotype DX has been part of the ASCO guidelines, and they specifically

highlight the assay's ability to predict the patient's risk of recurrence and how they will

benefit from chemotherapy (Sparano et. al, 2015). The ASCO guidelines point out that the

patients treated with tamoxifen and then were classified by the Oncotype DX assay into the

low-risk recurrence group, can avoid adjuvant chemotherapy (Cronin, 2007). The

guidelines in the 2008 NCCN breast cancer treatment also suggests this gene expression in

the methodic adjuvant treatment pathway for the patients who have hormone receptor-

positive, node-negative, HER2-negative tumors that measure less than 1cm, is recognizably

significant compared to the adverse features of the tumors over 1cm (Paik, 2004).

As this test has made considerable progress in the management and breast cancer

diagnosis, scientists are still studying for a method that will be a universal, and

reproducible test to foretell precisely the patients' therapeutic response and overall

outcome.

Currently, more than 170,000 breast cancer patients have changed their treatment

decision based on their Oncotype DX test as their scores varied compared to other tests

prior (Parkin, 2005). The women who had the high scores selected chemotherapy as their

potentially life-changing treatment, as the women who had the low scores were able to

carry on and pursue hormonal therapy alone. The women with the low scores were able to

avoid altogether the chemotherapy treatment and the side effects that come with that

treatment.

Discussion

The standards for assessing disease status and treatment options for breast cancer

patients is enhancing as today's readily available molecular diagnostic tests are showing

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great promise for the future. It is evident that the tests still need to overcome some key

obstacles to reach the front lines of breast cancer diagnostics and treatment. However, with

more of the latest tests being limited to more specific divisions of breast cancer patients,

the parameters restrict the wide-scale clinical effectiveness. When it comes to tissue

amount and the methodology in which the tests are run, their value becomes smaller as the

exact reproducibility becomes more difficult. As these assays become more precise and

completed, their chances of equaling or even exceeding the current ER PR and HER2

detection methods, when considering the value of prognostics and predictions. As

technology advances and these assays validate, they will become fully emerged into the

clinical assessment of each breast cancer patient on a personal level.

The enduring objective of molecular diagnostics will always be to serve as the

enhancement and fine-tuning of all oncologic decisions for various treatment options for

patients. In the circumstance for breast cancer, the evolution of molecular diagnostics will

maximize the overall clinical benefit and decrease unnecessary toxicity and nonresponses

from breast cancer patients.

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