Pathology Assessment of Tumor Tissue

Introduction

This module reviews the importance of pathologic evaluation of tissue in establishing a diagnosis of cancer and provides a basic summary of the processes and tests used in the pathology laboratory culminating in the final pathology report. Topics include the workings of the pathology laboratory; tumor sampling techniques, including liquid biopsies; handling of tissue in the pathology lab; and common laboratory testing methodologies performed on tumors used to establish diagnosis, provide prognostic information, guide therapy and monitor response to therapy.

Introduction to the Pathology Laboratory

Health care providers may be unfamiliar with the workings of the pathology laboratory. The delivery of a specimen to the pathology laboratory initiates a complex series of events resulting in a pathologic diagnosis/interpretation. The following section reviews the importance and key objectives in the pathologic evaluation of tissue and provides information on the types and members of the pathology laboratory.

Importance of Pathologic Examination

The diagnosis of cancer is not conclusively established, nor safely assumed, in the absence of a tissue diagnosis, nor should definitive therapy for cancer, with rare exception, be undertaken. Policies supporting this practice are written into the bylaws of most hospitals and are regularly monitored by hospital tissue committees and accrediting agencies.

The goal of pathology examination of tissue is to provide accurate, specific and sufficiently comprehensive diagnoses to enable the treating physician to develop an optimal plan of treatment. There are hundreds of varieties tumors, most with characteristic biology, that require accurate diagnosis by pathologists. Data on markers with prognostic and predictive significance are also routinely incorporated into pathology reports, allowing individualized treatment plans for patients. It is not only important to obtain sufficient tissue for a specific diagnosis of malignancy, but for many malignancies, additional tissue is required for prognostic and predictive ancillary studies.

While some have postulated that we are moving toward a gene/mutation driven categorization of tumors replacing disease site clinics and treatment planning (e.g., PIK3CA mutated carcinomas instead of “ovarian” cancer or “breast” cancer), data is accumulating that histology, morphology, disease site location and microenvironment in addition to genomic changes are still important factors in understanding the disease biology for treatment planning.

Types of Pathology Labs

• Hospital labs
  Almost all hospitals contain a laboratory to support the clinical services offered at the hospital. The specific pathology services would include both anatomic (surgical pathology, cytopathology, autopsy) and clinical (laboratory medicine) pathology at most hospitals. Most, if not all, inpatient and many outpatients seen by hospital-affiliated physicians require tests performed by hospital labs.
• Reference labs
  Reference labs are usually private, commercial facilities that do both high volume and specialty (high complexity and/or rare) laboratory testing. Most of these tests are referred from physician’s offices, hospital facilities and other patient care facilities such as nursing homes. Reference labs, typically located at a site other than the healthcare facilities, are often used for specialized tests that are ordered only occasionally or require special equipment for analysis.
• Public health labs
  Public health laboratories are typically run by state and local health departments to diagnosis and protect the public from health threats such as outbreaks of infectious disease. These labs perform tests to monitor the prevalence of certain diseases in the community which are a public health concern, such as outbreaks of foodborne or waterborne illnesses or detection of unique infectious agents.

Members of the Pathology Lab

The staff of most clinical laboratories is diverse. A non-comprehensive summary of the major types of individuals found in these laboratories is provided below.

Anatomic pathology which encompasses surgical pathology, cytopathology and autopsy pathology includes the following:

• Pathologists: Physicians with special training in the diagnosis and detection of disease. Practicing pathologists may be subspecialty or general pathologists, depending on the types of cases they review on a daily basis. Some pathologists may perform a subspecialty fellowship in a specific area of pathology such as cytopathology, hematopathology, dermatopathology, nephropathology, neuropathology, etc.
• Pathologists' assistants (PAs): These individuals assist with the gross description and dissection of surgical cases and biopsies, working closely with supervising pathologists. PAs may also assist in the technical aspects of intraoperative assessment such as frozen section and selection of tissue for research and clinical trials (tissue procurement).
• Cytotechnologists: These individuals assist in screening specimens that are composed of small samples of cells rather than whole sections of tissue, e.g., Pap smear specimens. After screening and marking diagnostic cells in slides, a cytotechnologist refers cases with abnormal cells to pathologists for review. Other common cytologic specimens include fine needle aspirations (FNAs), washings or scrapings of cells and other body fluids.
• Histotechnologists: These individuals manage the processing of tissue in the laboratory and perform the technical components of making slides from tissue for evaluation by a pathologist. These components include the process of fixing the tissue, embedding it in paraffin, sectioning tissue onto slides and staining of the tissue on slides.

Clinical pathology which encompasses laboratory medicine includes the following:

• Pathologists/PhD scientists: These professionals provide direction of clinical labs to ensure accurate and timely reporting of lab tests and serve as a resource for result interpretation to clinicians. Individuals often have specific training in one or more of the following areas: clinical chemistry, microbiology, molecular pathology, hematology, immunology and blood banking
• Medical laboratory technicians: These health care professionals perform laboratory testing and analysis on body fluids and other specimens to help determine the presence or absence of disease.
• Phlebotomists: These health care professionals are trained to draw blood from a patient for clinical testing, transfusions, donations or research.

Specimen Preparation

Obtaining sufficient tissue and practicing proper specimen handling (which begins even before the specimen arrives in the pathology laboratory) are essential components for accurate pathologic diagnoses. The following section reviews the various types of biopsies, including liquid biopsies, used to sample tumors and important aspects of tissue fixation.

Tumor Sampling

Although many types of tests may be used to make assessments that are suggestive of cancer, only a biopsy can be used to confirm a cancer diagnosis.

Tissue Biopsy

A biopsy is the removal of a small amount of tissue for pathology assessment. The goal of tissue biopsy is to obtain diagnostic tissue while minimizing morbidity, limiting potential tumor spread and avoiding interference with future treatments.

Needle Biopsies

• If the tumor is palpable near the surface, the needle is guided by palpation.
• If the tumor is deeper in the body, then the needle is guided by imaging (typically ultrasound or CT scan).
• Uses a thin (typically 22 gauge or smaller), hollow needle attached to a syringe. The needle used is even smaller than the ones used for blood tests.
• Removes individual cells, extremely small pieces of tumor tissue or fluid for pathologic evaluation.
• Multiple FNA passes can be made of a single large tumor in order to sample different sections.
• In cellular samples, material can be concentrated into a cell block for ancillary studies such as immunohistochemistry.
• Advantages: simple, rapid, minimally-invasive method that can be performed as an outpatient procedure. It is well tolerated with low risk for complications.
• Limitations: sampling errors due to low cellularity or inadequate tumor sampling.
• A pathologist may be present during image guided FNA procedures. If this is the case, the FNA aspirate is immediately processed on a slide and stained to preview the cells to ensure an adequate specimen was obtained. This is termed rapid on-site adequacy assessment.

• Uses a hollow needle that is slightly larger than the one used in FNA.
• Removes a small cylinder of tissue (about 1/16 inch in diameter and about 1/2 inch in length).
• Less invasive than surgery, but often requires local anesthesia.
• Advantages: In most cases, more tissue is obtained as compared to FNA, allowing more detailed ancillary studies to be performed. Histologic architecture is preserved as compared to FNA.
• Limitations: Limited sampling and inaccessibility of some masses (secondary to size, depth, density or location).

Surgical Biopsies

• Either local or general anesthesia is required.
• More invasive than needle biopsies.
• Recovery time is required, increased morbidity and cost as compared to needle biopsy.

Incisional biopsy

• A portion of a large tumor is removed.
• Typically only performed if the tumor is too large or too invasive to be removed in its entirety, or attempts at needle biopsy were non-diagnostic.
• Excisional biopsy - The entire tumor or suspicious area is removed.

Excisional biopsy

• The entire tumor or suspicious area is removed.
• Typically some of the surrounding normal tissue is removed as well (termed the surgical margin).
• If an excisional biopsy specimen is found to be cancerous, the pathologist will examine the surgical margin to ensure that the tumor was removed in its entirety. This is determined based on whether there is a wide enough rim of normal tissue around the tumor.

Other Types of Biopsies

Scrape or brush cytology

• A small spatula or brush is used to scrape cells from the tissue being tested.
• Most common example is a Pap test.
• Other tissues commonly sampled in this way include the esophagus, the stomach, the bronchi, and the mouth.

Endoscopic biopsy

• An endoscope is a thin, flexible, lighted tube that has a lens or camera on the end.
• Forceps may also be attached to the end of the tube and used to remove a small tissue sample of a suspicious area identified via the camera.
• An endoscope is used to visualize and biopsy different parts of the body, including the nose, sinuses, throat, esophagus, stomach and upper intestine.
• Some endoscopes are called a different name when they are used on a particular anatomic area. A bronchoscope is used to visualize and biopsy the lungs and bronchi. A colonoscope is used to visualize and biopsy the colon and rectum. A laparascope is used to visualize and biopsy the interior of the abdomen.

Bone marrow aspiration and biopsy

• Used to diagnose hematologic cancers including lymphoma, leukemia and multiple myeloma.
• Typically performed at the same time to examine the bone marrow.
• Bone marrow aspiration is used to sample a small amount of the liquid component of bone marrow. Bone marrow biopsy is used to remove a small amount of the solid tissue component of bone marrow.
• A wide needle is pushed into the bone. A sample of the liquid portion is removed using a syringe attached to the needle. The needle is then rotated to remove a sample of the bone.
• Most frequently performed on the pelvic bone.

Sentinel Lymph Node Mapping and Biopsy

• Termed sentinel lymph nodes because they “stand watch” over the tumor. They are lymph nodes that drain lymph fluid from the tumor tissue. A sentinel lymph node is defined as the first node or group of nodes to which cancer cells are most likely to spread from a primary tumor. Sentinel lymph node biopsy is most commonly used to help stage breast cancer and malignant melanoma, but it has been used for a variety of cancer types.
• Mapping involves using a colored dye and/or a radioactive material to trace the routes of lymph drainage from the tumor to identify the sentinel node(s).
• The sentinel node(s) are then removed and examined microscopically to determine if they contain cancerous cells. A negative sentinel lymph node biopsy result suggests that the cancer has not spread to regional lymph nodes or other organs. If the sentinel lymph node(s) are negative, then no additional regional lymph nodes are removed at surgery because the tumor has not yet metastasized to the lymph nodes. If cancerous cells are found, then the remaining lymph nodes in the area may be removed in a process termed lymph node dissection.

Liquid Biopsy

Liquid biopsy technology is a rapidly emerging field. The terminology liquid biopsy came about because we are rapidly moving to an era where some of the traditional assessments done with a tissue biopsy (e.g., molecular marker testing) can now be done in blood, urine or other bodily fluid that is less invasive than a tissue biopsy. Currently, about 40 companies have developed assays to detect cell-free circulating DNA (cfDNA), circulating tumor DNA (ctDNA), or circulating tumor cells (CTCs).

Tumors shed cells (CTCs) into the bloodstream, which can be isolated for analysis. The challenge is that there are very few tumor cells, but there are a number of advantages in isolating them for analysis. The most obvious advantage is that the patient would not need to undergo an invasive biopsy procedure and only would have a blood sample drawn. This permits easier serial analysis over time to monitor a tumor’s changes to better guide therapy changes as the tumor progresses. Additionally, tumors are heterogeneous, making it challenging to obtain a molecular representation of the tumor from a small biopsy sample (such as those from fine needle aspiration or core needle biopsy). Treating a patient with a tumor based on the analysis of a small biopsy may result in only a portion of the cells being effectively targeted. Using CTCs, the heterogeneity of a tumor is better represented, as the cells can come from multiple locations within a tumor as well as from multiple tumors in the case of metastatic disease. Research has shown that the number of CTCs reflects the state of disease — having more CTCs corresponds with more disease. Circulating tumor cells that have been isolated can also be sequenced individually or as a group to identify actionable targets for treatment. Several companies, e.g., CellSearch, Biocept, etc., have commercialized CTC analysis. A challenge in utilizing CTCs for diagnostics is the low numbers found in the blood at any time. While the numbers increase with metastatic state, they are still few compared to the number of red and white blood cells. Companies have developed proprietary collection methods to stabilize the CTCS in the samples sent to them overnight for isolation upon arrival in the lab. Further research is needed to improve this approach.

Dying cells release DNA into the bloodstream (cfDNA). Tumor cells do this as well (ctDNA), but typically, only a small portion of the total DNA is found in the blood. The amount of tumor DNA found in the blood typically correlates with stage, increasing with stage and number of metastases. Multiple approaches are used by different companies to analyze the ctDNA molecularly, but the most common utilize polymerase chain reaction or next-generation sequencing to identify molecular alterations in the DNA. The same advantages of isolating CTCs for analysis vs. biopsies apply to ctDNA, but it is often possible to obtain higher quantities of ctDNA than CTCs.

Currently, a few companies are expanding the concept of liquid biopsies, by using other bodily fluids, such as urine and cerebral spinal fluid (for brain tumors and metastases). Urine provides an interesting option if proven successful because the sample can be collected at home and shipped by the patient, providing the most convenient and least invasive option of all. For more information, see the section "Liquid Biopsies."

Video 1-5

Video 1-5: Liquid Biopsies vs. Tissue Biopsies. Wafik El-Deiry MD, discusses the pros and cons of both tissue and liquid biopsies.

Comparison of Tissue and Liquid Biopsies

Tissue Fixation

Tissue fixation serves several purposes during the pathologic evaluation of specimens. Fixation preserves tissue by preventing autolysis by cellular enzymes, helps prevent decomposition of tissue by bacteria and molds, hardens tissue to facilitate sectioning, inactivates infectious agents and enhances tissue avidity for dyes. Fixation also has undesirable effects on tissue such as alteration of protein structure (loss of antigenicity), loss of soluble tissue components and degradation of DNA and RNA.

Types of Fixatives

• Formalin: The standard fixative used in the pathology laboratory is 10% phosphate-buffered formalin. It fixes most tissues well and is compatible with most ancillary testing such as immunohistochemistry and molecular tests.
• Bouin solution (picric acid, formaldehyde and acetic acid): Fixation in Bouin solution results in sharp hematoxylin and eosin staining and is preferred by some pathologists. Disadvantages include decreased sensitivity of immunohistochemical tests and increased degradation of DNA and RNA by picric acid.
• B5 (mercuric chloride, sodium acetate and formalin): B5 is often used for routine fixation of lymph nodes, spleens and other tissue if a lymphoproliferative process is suspected. B5 provides rapid fixation with excellent cytologic details and antigen preservation for lymphoid markers. Tissue may become brittle if over-fixation occurs with B5, and special procedures for disposal are needed due to the presence of mercury.
• Glutaraldehyde: The fixative glutaraldehyde is used for tissue that is to be evaluated by electron microscopy.

Creating Formalin-Fixed Paraffin-Embedded Tissue Blocks
After appropriate fixation, tissue, in blocks, is placed into a processor that dehydrates tissue through a series of graded alcohol baths and infiltrates the tissue with paraffin wax, resulting in a formalin-fixed paraffin-embedded tissue block. Tissue from these blocks is then sectioned thinly (0.4 µm to 0.5 µm) using a microtome and placed onto a glass slide. Tissue on the slides is stained with hematoxylin and eosin and covered with a coverslip before examination by a pathologist.

Effect of Time on Fixation
Several factors related to tissue fixation may affect the results of ancillary studies such as immunohistochemical and molecular testing. Autolysis begins immediately after tissue is removed from a patient. Although autolysis can be reduced by refrigeration, delays before fixation can adversely affect the diagnostic quality of tissue. The time between when a specimen is removed from a patient to when it is in contact with formalin is called the cold ischemic time. Extended cold ischemic times (greater than 1 hour) may result in false-negative testing for markers such as estrogen receptor, progesterone receptor and HER-2. It is important that specimens are transported to the lab in a timely fashion to avoid extended cold ischemic times. An adequate amount of fixative in the specimen container is usually considered to be 15 to 20 times the volume of the tissue. However, even in this short time, changes in phosphorylation of important proteins occur (both increase and decrease at specific sites has been noted).

The process of fixation is a chemical reaction that usually requires a minimum of 6 hours (even for small biopsy specimens) to reach sufficient tissue fixation. Certain tissue types, such as those containing a high content of adipose tissue, and larger specimens require longer fixation times. Larger tissues from resections may also require opening up to enable the fixative to enter all areas and provide even fixation. Both under-fixation and over-fixation of tissue may result in loss of antigenicity and degradation of RNA and DNA. Specific ASCO/College of American Pathologists guidelines for fixation of breast specimens exist to preserve antigenicity of tissue for hormone receptor and HER-2 testing. Breast specimens are to be fixed for a minimum of 6 hours and no more than 72 hours in 10% neutral buffered formalin. These guidelines may be applied to other specimen types in an attempt to standardize pre-analytical variables for ancillary testing.

Initial Pathology Assessment

Visual inspection of the gross specimen and tissue staining are two important aspects of assessing tumors in the pathology lab and are briefly summarized in the following section.

Gross Examination

Gross examination is the visual macroscopic inspection of the tumor, without the use of a microscope. All anatomic structures present, and the tissue specimen’s size, color and consistency, are recorded. Gross examination helps the pathologist determine the size of the specimens to dissect and assess. Histologic sections that best demonstrate the features seen at gross description, including assessment of margins (if applicable) are taken during gross examination. Inking of margins also is performed at gross examination. The process provides important diagnostic information used for staging and prognosis, and a picture may be taken as part of the record.

Histology Staining

Histology is the microscopic appearance of stained cell and tissue structures of a specimen. The characteristic histology of cells/tissue is used to identify all of the pathologic processes involving a specimen.

Commonly used histologic stains include:

• Hematoxylin (nuclei) and eosin (cytoplasm) staining (H&E)
  • H&E is the standard stain performed for routine examination of tissue under the microscope to form the cornerstone of pathologic diagnoses. Hematoxylin is a dark blue or violet stain that binds to DNA and RNA in the nucleus of cells. Eosin is a red or pink stain that binds to cytoplasmic proteins.
• A wide variety of special stains are available to evaluate pathologic processes, a few of which are quickly summarized below:
  • Alcian blue
    • Identification of acid mucins within cells (may be used to facilitate identification of Barrett’s mucosa in biopsy specimens).
  • Congo red
    • Detection of amyloid within tissue.
  • Mucicarmine
    • Detection of mucin within neoplasms, supporting classification as adenocarcinoma (e.g. non-small cell lung carcinomas).
  • Periodic acid-Schiff
    • Detection of glycogen or mucin within neoplasms.
  • Trichome stain
    • Primarily used to demonstrate collagen and muscle in normal tissue (e.g. detection of increased fibrosis in the liver).

Overview of Biomarker Testing

Biomarkers for risk, diagnosis, prognosis, prediction and response are used throughout the continuum of care in oncology.

Risk Markers

The field of cancer genetics is focused on the evaluation of important risk markers and inherited disease. Most well-known are markers such as BRCA1 and BRCA2 for inherited risk of breast/ovarian cancer. Other examples include markers for Lynch syndrome (hereditary nonpolyposis colorectal cancer) and Li-Fraumeni syndrome. Individuals with Lynch syndrome have mutations in genes typically involved in repair of DNA (MLH1, MSH2, MLH3, MSH6, PMS1, PMS, and TGFBR2) giving them a much higher likelihood of developing colon cancer as well as other cancers (eg, endometrial, ovarian, pancreatic) at an earlier age. Mutations in the tumor suppressor gene TP53 and CHEK2 may indicate the patient has Li-Fraumeni syndrome, which often affects children or young adults and leads to development of multiple types of tumors in their lifetime.

Diagnostic Markers

Whereas histology and morphology are important to determine if a tumor is benign or cancerous, molecular markers can help confirm diagnosis. An example of this is the use of BCR-ABL fusion marker to confirm the diagnosis of leukemia. This marker is also useful for the prediction of response to treatments and to monitor disease. The CA-125 marker is often used to help determine if a mass in the ovaries is potentially cancerous.

Prognostic Markers

Prognostic markers are used to help a physician assess the potential outcome for a patient regardless of treatment. They can help assess the aggressiveness of disease. An example of a prognostic marker is CA19-9 in pancreatic cancer, which can help assess the operability of the tumor and provide insight into potential survival. The expression of CD44 is often associated with a poor prognosis in bladder cancer, whereas expression of cyclin D1 is associated with a better prognosis with lower odds of recurrence.

Predictive Markers

Predictive markers are used to determine potential for response to a specific treatment. Targeted therapies often use companion diagnostics to direct treatment decisions. These tests use predictive markers to identify which drugs may provide a favorable response for a patient. Examples include the EML4-ALK fusion gene for treatment with crizotinib (Xalkori, Pfizer) in non–small cell lung cancer and BRAF V600E mutation for treatment of melanoma with vemurafenib (Zelboraf, Genentech).

Response Markers

Markers of response are often considered the same as predictive markers. However, with the introduction of circulating tumor DNA tests, it is now possible to monitor response over time, not just before treatment.

Clinical trials assessing utility of biomarkers
Most trials now incorporate assessment of biomarkers. The markers may be used to guide selection of treatment in the study or may be added as correlatives for analysis after the study is completed to learn if there are associations of specific markers to response to treatment. Examples of large multidrug studies using markers to drive treatment choices include:

• ASCO Targeted Agent and Profiling Utilization Registry (TAPUR) study: This prospective, nonrandomized trial will collect information on the antitumor activity and toxicity of commercially available targeted cancer drugs in multiple tumor types (including advanced solid tumors, multiple myeloma, and B-cell non-Hodgkin’s lymphoma) with a genomic variation known to be a drug target. The study will evaluate 18 drugs targeting 44 genes contributed by seven pharmaceutical companies, with cohorts of up to 35 patients defined by tumor type, genomic abnormality and drug. This study, which opened in cancer center networks in Michigan and at Carolinas Healthcare System’s Levine Cancer Institute in March 2016, has the potential to identify new applications of current drugs. Additional drugs/targets are expected to be added as the study continues.
• NCI-Molecular Analysis for Therapy Choice (MATCH) trial: This phase 2 trial will evaluate more than 20 different study drugs or drug combinations, each targeting a specific gene mutation, to match each patient in the trial with a therapy that targets a molecular abnormality in his or her tumor.

Biomarkers and Their FDA-Approved Companion Diagnostic Test

Others (listed at FDA, List of Cleared or Approved Companion Diagnostic Devices [In Vitro and Imaging Tools])

Common Pathology Tests Performed in Oncology

A wide variety of tests are performed in the laboratory for accurate pathologic diagnosis and assessment of tumor biomarkers. These tests range from immunohistochemistry to determine expression of proteins within cells to a whole host of molecular tests evaluating tumor DNA and RNA characteristics. Methodology and examples of utility for the most commonly used tests are summarized in the following section.

Immunohistochemistry

Immnohistochemistry (IHC) is performed routinely in pathology laboratories to determine the expression of proteins within a tissue specimen.

Method

• Tissue is fixed and prepared on a microscope slide.
• The tissue is first exposed to the primary antibody. This is the antigen-specific antibody.
• The tissue is next exposed to the secondary antibody. The secondary antibody is typically developed to bind to the first one by using a general species-specific antibody created against the immunoglobulin G of the species in which the primary antibody was made. For example, if an antibody was made in a mouse or in mouse cells in culture, then the secondary antibody would be anti-mouse. This antibody binds to the primary antibody and serves two purposes:
  • Signal amplification: Often, a number of secondary antibodies bind to a single primary antibody; thus, for every single antigen-primary antibody binding event, multiple secondary antibody-primary antibody binding events occur, thus amplifying the initial binding event.
  • Signal detection: Commonly, a reporter is conjugated to the secondary antibody. This reporter molecule is used to monitor the IHC reaction.
    • For example, the peroxidase enzyme may be conjugated to the secondary antibody. Upon addition of the substrate diaminobenzidine (DAB), the peroxidase enzyme catalyzes its oxidation, resulting in the production of a brownish colored product that can be visualized under an ordinary microscope.
    • As another example, a fluorophore such as fluorescein may be conjugated to the secondary antibody, which may be visualized using a fluorescent microscope.
    • By using a secondary antibody — termed indirect detection — the cost is reduced by avoiding having to conjugate every antibody to an enzyme for detection (known as direct detection); only general secondary antibodies need this done.
• Immunohistochemistry can give semi-quantitative results as well as cellular location (eg, cytoplasm, nucleus, membrane) of the protein of interest. Typically, IHC is quantified by percent of cells that are positive for the stain and intensity on a scale of +1 to +3. If a protein is found by this method, then it is termed IHC positive, and the protein is considered over-expressed.

Considerations

• Tissue processing and specimen fixation (pre-analytical variables) can significantly affect the results. Standardization of pre-analytical variables helps ensure accurate and reproducible results.
  • For example, over-fixation can destroy the epitope on some antigens, rendering it unrecognizable by the antibody.
  • Decalcification agents (eg, hydrochloric acid) used on bone or other calcified tissues may result in decreased antigenicity of tissue.
• Antibodies can be characterized by their specificity, sensitivity and affinity.
• Visual interpretation can be subjective and differs between pathologists. Computerized image analysis of IHC staining (eg, scoring of HER-2 in breast cancer) has removed some of the subjectivity from IHC.

Example Applications in Evaluation of Tumors

• Categorization of malignant tumor type (eg, carcinoma, lymphoma, germ cell, sarcoma, melanoma): It may not be possible to classify certain tumors based solely on their histologic appearance.
• Determining site of origin for metastatic tumors.
• Subclassification of tumor in various organ systems (eg, specific subtype of lymphoma).

Example Applications in Oncology Biomarkers

• Estrogen receptor/progesterone receptor staining (breast cancer): used to determine if a patient with breast cancer is hormone receptor positive; these patients will be candidates for treatment with tamoxifen or aromatase inhibitors.
• HER-2 (breast cancer): used to determine if a patient with breast cancer will be a candidate for HER-2–directed therapy, most notably trastuzumab (Herceptin, Genentech), lapatinib (Tykerb, Novartis), pertuzumab (Perjeta, Genentech) and trastuzumab emtansine (Kadcyla, Genentech).
• PD-L1 (non–small cell lung cancer): used to determine if a patient with metastatic NSCLC may be a candidate for pembrolizumab (Keytruda, Merck).

In situ hybridization

In situ hybridization (ISH) is a laboratory test that allows for the precise localization of a specific region of nucleic acid on a chromosome or in tissue.

• Uses a sequence-specific DNA, RNA or oligonucleotide probe to bind to a complementary section of the DNA or RNA.
• Visualization of a reporter molecule on the probe allows for the detection and localization of a particular nucleic acid target within a heterogenous cell population, such as a tissue sample.
• Used to evaluate genetic abnormalities, including amplifications (as opposed to over-expression by changes in translational or post-translational events), deletions, translocations and aneuploidy.

Method

• Most ISH procedures use a fluorescently-labeled probe. This is referred to as fluorescent in situ hybridization (FISH). FISH studies are evaluated under a fluorescent microscope. Alternatively, when a chromogenic probe is used, it is referred to as chromogenic in situ hybridization (CISH). CISH studies are evaluated using a bright-field microscope.
• Within the sample, the target DNA is first denatured with either heat and/or chemicals. This step is necessary to create a single-stranded template that will hybridize to the probe.
• The labeled probe is then added to the sample. The probe hybridizes, or binds, to the target sequence in a sequence-dependent fashion.
• Probe-bound DNA is visualized using a fluorescent microscope (or a bright-field microscope in the case of CISH).

Considerations

• FISH is amenable to multiplexing when multiple targets are visualized in the same sample by using different fluorophores to label the probe.

Example Applications in Oncology Biomarkers

• Anaplastic lymphoma kinase gene fusions: FISH tests can be used to determine if a fusion of the ALK gene (most commonly to the EML-4 gene) is present in patients with metastatic non–small cell lung cancer . If so, then these patients are candidates for treatment with ALK-targeted therapies, including crizotinib (Xalkori, Pfizer), alectinib (Alecensa, Genentech), and ceritinib (Zykadia, Novartis). The most common FISH test for ALK rearrangement includes two differently colored (red and green) fluorescent probes that flank the highly conserved translocation breakpoint within ALK. In non-rearranged cells, the red and green probes are in close proximity, resulting in a yellow (fused) signal. In patients with an ALK rearrangement, these probes are separated, resulting in a space between the red and green signals, which then fluoresce independently in color.
• Epidermal growth factor receptor amplification: EGFR gene amplification can result in overexpression of the EGFR cell surface receptor, a target for inhibition by either small molecule inhibitors (including erlotinib [Tarceva; Genentech, Astellas Oncology] and gefitinib [Iressa, AstraZeneca]) or by anti-EGFR antibodies (such as cetuximab [Erbitux, Eli Lilly], panitumumab [Vectibix, Amgen] and necitumumab [Portrazza, Eli Lilly]). EGFR amplification has been associated with a favorable clinical benefit to targeted EGFR inhibition in both NSCLC and colorectal cancer.
• HER-2 amplification: HER-2 status of breast and gastric/gastroesophageal junction carcinomas may be assessed by in situ hybridization. These studies typically use two separately colored probes, one for the HER-2 gene and one as the control probe, to assess for HER-2 gene amplification.

Polymerase Chain Reaction

Polymerase chain reaction (PCR) is a laboratory method, developed by Kary B. Mullis, PhD, in which a target DNA template sequence is amplified several orders of magnitude. This technique is used in many fields in addition to oncology, including cloning, forensics and infectious disease. For more information on PCR as it relates to immuno-oncology.

• Original versions of PCR, known as end-point PCR, required the user to separate the amplified DNA products by size on an agarose gel electrophoresis.
• Current clinical versions of PCR are real-time PCR, which include the use of a fluorescently labeled sequence-specific probe to monitor the production of a specific amplified product in real time during the reaction. This can be used quantitatively (termed quantitative PCR or qualitatively. Digital PCR is another quantitative approach to PCR using a very diluted DNA template, and many PCRs run in parallel with the proportion of negative results used to calculate the DNA concentration.
• RNA may be used as the template in the reaction, when it is first reverse transcribed to a complementary DNA sequence. This form of PCR is termed reverse transcription PCR.

• Typically, a purified DNA sample is required. This means that the DNA must first be extracted from the tumor specimen.
• The PCR process is a thermocyclic process, meaning that it proceeds through changes in temperature in a repeated fashion. Many real-time PCR assays use a three-step cycling method.
• In the first step, denaturation, the DNA is heated to a high temperature (at or near 98°C). This step separates the DNA strands, turning the double-stranded DNA sample into a single-stranded DNA template.
• In the second step, annealing, the temperature is lowered to below the melting temperature of the sequence. This allows two primers (referred to as the forward and reverse primers), to bind to the complementary sequence within the target DNA. Usually, these primers are within 100 bp and 500 bp apart.
• In the third step, elongation, a DNA polymerase (often, Taq DNA polymerase) is used to synthesize new DNA sequence. The polymerase sits down on the DNA at the 3’ end of each primer and travels along the template strand, adding new DNA nucleotides in a sequence-specific manner.
• The product of each cycle is the generation of a new double-stranded DNA amplicon that is identical to the target sequence within the template strand.
• This process is repeated many (35 to 55) times.

Considerations

• Often, the second and third step are combined to occur at a single temperature, which may speed up the overall PCR reaction.
• In real-time PCR, a sequence-specific probe is included in the reaction. The probe is labeled with a fluorophore on one end and a quencher on the other. When the probe is not bound to the target sequence, the fluorescence of the fluorophore is effectively quenched by the quencher. When the probe is bound to the target sequence, the exonuclease activity of the polymerase cleaves the fluorophore off the probe as it travels along the DNA template. This increases the distance of the fluorophore from the quencher, thereby causing a measurable increase in the fluorescent signal.
• An important consideration in any PCR reaction is the DNA polymerase used. High-fidelity DNA polymerases have lower error rates but are also more expensive. Because of the amplification, errors can also be greatly amplified using PCR.
• Contamination is also an issue because of the ability to amplify a single DNA molecule and detect it. Consequently, many labs have clean areas to separate DNA isolation and PCR amplification or the use of special laminar flow hoods to protect the sample from contamination.
• Finally, the design of the oligonucleotide primers is a critical step to ensure specificity and amplification of the correct region of DNA.

Example Applications in Oncology Biomarkers

• Epidermal growth factor receptor mutation testing: Real-time PCR is used to monitor for the presence or absence of certain mutations within the EGFR gene in patients with NSCLC. These mutations are known to sensitize the tumor to treatment with EGFR tyrosine kinase inhibitors such as erlotinib (Tarceva; Genentech, Astellas Oncology) and gefitinib (Iressa, AstraZeneca).

Sequencing

Sequencing is a method to determine the order and type of nucleotide within a DNA molecule. It is an alternative way to determine genetic alterations.

• Advantageous over quantitative polymerase chain reaction (PCR) because it detects all mutations present, not just known mutations.
• Provides a high-resolution, base-by-base view of the entire genome, exomes, targeted genes or hotspots, depending on the sample preparation.
• Captures both large and small abnormalities that might otherwise be missed including point mutations, small insertions or deletions of nucleotides (called indels), frameshifts, fusions between genic regions and amplification or loss of genes or regions of genes.
• Creating personalized plans to treat disease may be possible based not only on the mutant genes causing a disease, but also on other genes in the patient’s genome.
• Genotyping cancer cells allows physicians to select the best treatment and potentially expose the patient to less toxic treatment because the therapy is tailored (also known as personalized, individualized or precision medicine).
• Previously unknown genes may be identified as contributing to a disease state; whereas traditional genetic testing looks only at the common “troublemaker” genes.
• Disadvantages are cost and time; however, the price continues to decrease, and the time to results continues to shorten.
• The role of most genes in the human genome is still unknown or incompletely understood; therefore, a significant amount of the information found in a human genome sequence is currently unusable.
• Most physicians are not trained in how to interpret genomic data. The rate of new findings is rapid, and it is challenging stay current.
• An individual’s genome may contain information that they do not want to know. For example, a patient may have genome sequencing performed to determine the most effective treatment plan for high cholesterol. In the process, researchers discover an unrelated allele that assures a terminal disease with no effective treatment. Thus, it is important to inform the patient of the potential results before testing.
• The volume of information contained in a genome sequence is vast. Policies and security measures to maintain the privacy and safety of this information are still new.

Sanger Sequencing

• Traditional chain-termination sequencing methodology that was developed in 1977, also known as dideoxy sequencing.
• Method has been updated and is conducted by automated sequencers using a modification of the original method, in which a different color dye is used for each nucleotide and capillary electrophoresis is used in place of the gel electrophoresis of the original method.
• Although Sanger sequencing was widely used for more than 25 years, it is rarely used now due to the increased sensitivity and reduced costs of next-generation methods.

Pyrosequencing

• Target sequence-based method that allows rapid quantification of sequence variation using detection of pyrophosphate release as nucleotides are incorporated in a sequencing-by-synthesis mode.
• Target section of DNA is first synthesized by hybridizing the template to a primer and incubating with necessary reagents releasing a pyrophosphate. This is converted to a luciferase-catalyzed reaction, which is detected by a camera creating a pyrogram that is interpreted to determine the sequence.
• A limitation is the shorter length of the individual reads obtained as compared with Sanger sequencing, leading to more complicated assembly.

Next-Generation Sequencing

• Next-generation sequencing is performed by a process similar to capillary electrophoresis, but instead of sequencing a single DNA sequence, next-generation sequencing extends the process across millions of fragments of DNA in a parallel fashion, greatly speeding up the sequencing and decreasing the cost. This approach requires complicated reassembly, alignment and bioinformatics analysis to obtain the final sequence. Multiple technology platforms are considered next-generation sequencing.
• Next-generation sequencing requires three steps: sample preparation, sequencing and data analysis.
  • Sample preparation requires two steps:
    • Library preparation: Sample preparation requires creation of a library. Many nuances and approaches to library preparation are available, depending on the next-generation sequencing platform used, but the goal is the same requiring random fragmentation and tagging of DNA with adaptors. Tags with specific known sequences, known as barcodes, may be used to enable sample pooling, decreasing the cost, increasing efficiency and aiding in assembly after sequencing. Targets may be enriched using multiplexed PCR or hybridization to isolate the regions of interest before sequencing (eg, hotspots [specific base-pairs], targeted exons of genes, whole exome).
    • Cluster amplification is used to create clonal clusters of a library. After denaturing and hybridization to immobilize within the next-generation sequencing flow cell, the DNA is amplified. Repeating this many times creates the cluster.
  • Sequencing: There are several major next-generation sequencing platforms for sequencing.
    • Sequencing by synthesis: Advances in sequencing by synthesis transformed pyrosequencing to a next-generation method. DNA samples are cleaved into short fragments (about 100 bp to 150 bp), and then adapters are ligated to these fragments for PCR to be carried out, creating many copies of the same read in a single spot. Taking an image after flooding with fluorescently-labeled nucleotides, DNA polymerase and a terminator to add a single base at a time, enables the sequence to be obtained. An advance to this method is called paired-end sequencing, which incorporates sequencing from both ends of the DNA fragment and aligning both the forward and reverse reads, improving accuracy. Illumina, a company that manufactures next-generation sequencing equipment, uses this type of technology.
    • Ion semiconductor sequencing: Another method of sequencing by synthesis uses detection of hydrogen ions released during polymerization on a semiconductor chip to obtain the sequence directly. A single nucleotide is released at a time, and a signal is detected if it is incorporated. The signal is proportional to the number of same nucleotides in a row (eg, three T nucleotides corresponds with three times a single T). This method also uses DNA fragments, but the size is a little larger (about 200 bp). Thermo Fisher Scientific’s Ion Torrent platform and Ion Proton system use this approach.
    • Single-molecule real-time sequencing: Pacific Biosciences uses a different technology, parallelized single-molecule, real-time technology, to perform next-generation sequencing. In this approach, the DNA polymerase is immobilized with a single DNA molecule template. Fluorescent-labels are cleaved off of the nucleotides as they are incorporated, providing a signal for detection and determination of the sequence. This approach has much longer read lengths making alignment and assembly much easier.
  • Data analysis: Data analysis for next-generation sequencing typically has multiple steps in the process often referred to as a "pipeline," as it is standardized for each analysis type. Here are the major steps typically found in most analysis pipelines:
    • After the bases are called in each of the reads, the reads are aligned to a reference genome to assemble the gene sequence. Duplications are removed, leaving unique reads only.
    • Single-nucleotide polymorphisms (SNPs) must be identified before calling variants to remove population heterogeneity.
    • Variants, including small insertions/deletions (indels), are called subsequently by comparison to the reference genome after identifying SNPs. The accuracy of variant calling depends on the depth of coverage at each base. Depth is how many times a base is sequenced from unique reads (after removal of duplications).
    • Annotation of the variants called typically relies on a number of databases to note whether the variant is a known pathogenic or benign variation or one whose function is still unknown (variant of unknown significance). A number of companies have created extensive custom data systems to annotate the alterations identified. Publically available databases are also often used (eg, the Short Genetic Variations database [dbSNP], HapMap (decommissioned and archived), 1000 Genomes Project, Catalogue of Somatic Mutations in Cancer [COSMIC], ClinVar, Online Mendelian Inheritance in Man [OMIM]).

Next-generation sequencing technology can also be used with RNA to look at the transcriptome or expression of genes. Often this is referred to as RNA-Seq, or RNA sequencing. Other modifications enable analysis of the methylome.