www.MedLabMag.com

FISH

2 MedicalLab Management • October 2015

� By Kathryn R. Perkinson, BS, HTL(ASCP)

Value of FISH Testing in Cancer Care

tool to determine appropriateness of targeted therapy in cancer patients. There are several benefits of paraffin FISH testing, in-cluding the ability to perform retrospective studies, and the abil-ity to identify the cells of interest morphologically and score the area of interest. However, as compared to FISH testing on con-ventional suspension samples, paraffin FISH can be labor inten-sive and variable due to differing fixation times between samples and referring histology labs, and the interpretation must be care-fully reviewed and the assay vigilantly controlled due to trunca-tion of signal and overlapping cells. Despite these challenges, paraffin FISH is advantageous to cancer testing given the multi-tude of targeted therapies that are now available.

Paraffin FISH must be considered separately from the con-ventional suspension FISH method, and it must be noted that due to the use of interphase nuclei, a prior knowledge of the anomaly of interest is required [see FIGURE 1].

Within the clinical practice of molecular pathol-ogy, our understanding of deoxyribonucleic acid (DNA) and its functions is one of the most important discoveries of the last century. Our knowledge of DNA structure and function aids

in understanding hereditary factors within disease, assessing risk factors in advance of disease development, and developing medications to treat several genetic diseases, including cancer.

Among the numerous, medically beneficial technical tools available to decipher DNA changes is fluorescence in situ hybrid-ization (FISH). FISH is a powerful technique used in diagnostic, prognostic, and predictive cancer testing (see SIDEBAR). The key element of this technology is the utilization of fluorescent probes to detect DNA sequences of interest. A cancer diagnosis is most often based on pathologic interpretation of the hematoxylin and eosin (H&E) stained slide and immunohistochemical stains. Once the determination of malignancy has been made, FISH testing is utilized to determine if a targeted therapeutic drug is applicable.

Paraffin FISHPathologists use paraffin FISH testing—the application of the FISH methodology to formalin-fixed, paraffin-embedded (FFPE) sections—to confirm or exclude a histologic diagnosis, or as a

Pretreatment

Hybridization

Post Hybridization Wash

Denaturation

Paraffin Cytospin/Fresh Prep

Probe Denaturation

Overnight

FIGURE 1

How does FISH work?

SIDEBAR

Cancer Diagnosis, Prognosis, and Predictive Therapy DiagnosisSome cancers develop as a result of chromosomal changes, such as translocations, and these changes are linked to specific types of cancer. Translocations occur when a section of DNA moves from one chromosome to another chromosome. The resulting DNA sequence may cause a chimeric protein to be produced, which in turn could be the genetic event that causes carcinogen-esis. Translocation FISH probes can be used in the diagnosis of this type of cancer.

PrognosisOther cancers may develop as part of a multiple-hit theory of carcinogenesis. In the multiple-hit theory, more than one genetic mutation is required for the development of cancer. In these types of cancer, multiple FISH probes may be used.

Predictive or Targeted Therapy FISH testing for solid tumor oncology is often performed after the patient has been diagnosed with cancer. For example, following a breast cancer diagnosis based on an H&E examination, FISH studies can be ordered to determine if amplification of the HER2/neu gene is present. If amplification is present, the patient may be eligible for the drug Herceptin.

www.MedLabMag.com

FISH

4 MedicalLab Management • October 2015

Methods and Tissue PreparationThe two FISH methods commonly utilized in clinical laboratories differ in their provenance. The sample type determines which FISH method will be used. The initial method was developed in cytoge-netics laboratories that first performed FISH on samples, such as blood and bone marrow. This is known as metaphase FISH be-cause the cells are arrested during the metaphase portion of the cell cycle. The more recently developed method is the previously mentioned paraffin method, which is referred to as the histologic method due to its derivation from methods typically used in immu-nohistochemistry (IHC) laboratories. This method is also known as interphase FISH because the cells are in their natural cell cycle state within the tissue and have not been arrested in metaphase. Regardless of the method utilized, superior technical skill and strict quality control are necessary for the procedure to be reproducible in the clinical lab (see FIGURE 2).

Metaphase and Interphase FISHMetaphase FISH—most commonly used with blood for hemato-logic oncology cases—is performed in cells that have been arrest-ed in the metaphase portion of the cell cycle when DNA is coiled into recognizable chromosomes. Interphase FISH is used when the DNA is unwound and is most often performed using FFPE tis-sues for solid tumor oncology cases, such as breast cancer, lung cancer, and brain cancer.

Whether FISH is conducted in metaphase or interphase DNA, the assay is based on the same principles. Briefly, the DNA probe is fluoresceinated and has a specific sequence that binds to the DNA within the nuclei of the cell creating a signal within the cell. A technologist analyzes the slide to determine the pattern of FISH signals within each cell and records the information. The data are analyzed statistically, depending upon the probe type, and reported according to strict standard operating procedures. For the purpose of this article, we will discuss interphase FISH performed in FFPE tissue.

For FFPE tissue, a tissue sample is obtained surgically and is placed in 10% neutral-buffered formalin (NBF) for fixation. This process requires at least 6 hours, but should not exceed 48 hours, and the precise time to fixation and fixation time should be

documented. Next, the tissue is embedded in paraffin and placed in a tissue mold. Tissue can then be sectioned at 5 microns using a microtome and placed onto silanized slides. The first slide will always be the H&E for diagnostic purposes. Most often, the next slides will be used for immunohistochemical stains. After the deter-mination of malignancy is made, FISH is performed on subsequent slides for determination of targeted therapy. The tumor area is marked on the H&E slide by the pathologist, and this area is then etched on the underside of the FISH slide with a diamond-tip pen.

Pretreatment and Digestion During all phases of the FISH assay, timing and temperature are critically important. With over 40 steps, the FISH assay is lengthy; as such, failure to adhere to timing and temperature requirements can result in lower quality, higher background, or complete loss of tissue. One of the most critical steps in the FISH assay for FFPE tissue is the pretreatment step, which is required due to fixation. The purpose of the pretreatment step is to break chemical bonds that have formed during formalin fixation. Subsequent to pre-treatment is the protease digestion step. Both the pretreatment and protease digestion steps are necessary to allow penetration of fluorescent probes. Digestion is essential to the FISH assay and must be well controlled. Tissues vary in their degree of connective tissue and, thus, the time of digestion needed for optimal probe binding varies by tissue types. Many clinical labs receive requests for metastatic breast cancer lesions, so it is important to consider the tissue to which the breast cancer has metastasized when de-termining how much time to digest the tissue. For example, breast tissue tends to have more connective tissue than liver tissue, and therefore, breast tissue requires a longer time period for diges-tion than liver tissue. It should be noted that bone core biopsies that have been decalcified are not amenable to FISH testing. The strong acid used in decalcification of bone is detrimental to DNA.

Denaturation and Hybridization DNA denaturation, also called DNA melting, is the process by which double-stranded DNA is essentially unzipped, or opened, using a combination of heat and chemical treatment. Both the DNA in the tissue and the DNA probe must be denatured so that

FIGURE 2

Paraffin vs. Suspension Method

Slide aging

Wax removal

Tissue rehydration

Heat pretreatment

Enzyme pretreatment

Tissue dehydration Add probes Co-denature Overnight

hybridization Postwash Counterstaining and visualisation

Enzyme pretreatment

Tissue dehydration Add probes Co-denature Overnight

hybridization Postwash Counterstaining and visualisation

Paraffin pretreatment steps

Suspension pretreatment stepsAdditional steps for paraffin pretreatment

(Continues on page 17)

www.MedLabMag.com October 2015 • MedicalLab Management 17

FISH�

www.MedLabMag.com/requestwww.MedLabMag.com/requestwww.MedLabMag.com/request

Request Your FREE Subscription! THE CLEAR SOLUTION

FOR THE CHALLENGES IN YOUR LAB!

Necessary Equipment Cytogenetics laboratories usually perform FISH on metaphase slides and, therefore, they typically have the necessary equipment to perform FISH assays with the possible exception of the micro-tome. IHC laboratories should have the necessary equipment to perform FISH assays as well, with the possible exception of a fluorescent microscope. If the IHC laboratory performs immu-nofluorescence assays, a fluorescent microscope would be avail-able. Beyond this, basic equipment necessary for FISH include a pH meter, balance, stir plate, microcentrifuge, refrigerator/freezer, waterbath, and slide drying oven.

Value of Accurate TestingThe basic premise of FISH testing involves establishing the area of interest on the H&E slide, then transferring this area to an un-stained paraffin slide, which is then pretreated, probed, and co-denatured using FISH methodology. Crucial to paraffin-based analysis is the assessment of signal patterns in the cells. For this reason, robust internal and external quality control slides are re-quired for diagnostic paraffin FISH testing in order to decrease the likelihood of an incorrect result due to an analysis error.

There are a number of variations to the basic FISH method that can be used in cancer treatment depending on the nature and number of samples being processed and the type of technology in use. On a related note, reimbursement for FISH testing has a good history, and new CPT codes are developed frequently to account for new types of FISH being performed. ■

Kathryn R. Perkinson, BS, HTL(ASCP), is the manager of molecular pathology at Duke University Health System.

when favorable conditions are applied, the probe can hybridize to the tissue. Under favorable conditions, hybridization occurs when the fluorescently labeled probe binds to the DNA in the tissue ac-cording to the specific sequence.

Probes in Use Genetic aberrations are detected by FISH based on the type of probe correlated to the type of aberration. For example, amplifi-cation probes are used to determine if a gene is amplified within a cell. Normally, a cell has two copies of a particular gene, but amplification indicates that more than two copies per cell are present. HER2/neu is an example of an amplification probe.

Alternatively, deletion probes are used to determine if a gene has been deleted and whether the deletion is homozygous (both copies of the gene are deleted) or hemizygous (one copy of the gene is deleted). Examples of deletion probes include PTEN and 9p21. A third type, the translocation probe, is used to determine if a gene or portion of a gene has translocated to another chromosome. This type of probe is usually a break-apart probe, which has a fluorescent signal on both ends. If the DNA is separated due to a translocation, the signals will appear separated (hence the breakapart terminology). Examples of this type of probe include EWSR1 found in Ewing sarcoma and SYT found in synovial sarcoma.

Keep in mind that FISH probes can be expensive, and specialty equipment and experienced technologists and pathologists are required for proper testing to be performed effectively. As such, FISH testing is not inexpensive; nevertheless, it is significantly less expensive than an errant course of chemotherapy. Using the FISH results, clinicians can be confident in their chemotherapy selection.

(Continued from page 4)