In our previous article, we described about the molecular genetic technique of FISH and enumerated its many advantages in cancer diagnostics. Please refer to FISH in molecular cancer diagnostics: the whats and whys?
Application in different cancers – towards personalized medicine!
Cancer is particularly suitable to FISH evaluation and analysis as the genetic defects uncovered by FISH represents early genetic triggers or events responsible for cancer at stem-cell level. FISH is amenable to detecting specific genomic aberrations for cancer diagnosis and therapeutic management and prognostics. Particularly - in cancer diagnostics - FISH is the gold standard for detecting already established and validated biomarkers like Bcr/abl1, Her2 and Alk rearrangements in specific known cancers.
Leukemias – are a cluster of heterogeneous clonal disorders of hematopoietic stem and progenitor cells associated with multiple genetic aberrations. These chromosomal rearrangements result in aberrant fusion proteins leading to abnormal cellular functions.
Chronic myeloid leukemia (CML) – is characterized by t (9:22) chromosomal translocation resulting in oncogenic fusion protein Bcr/abl1 that leads to dysregulated tyrosinase activity. This has been therapeutically harnessed by developing tyrosine kinase inhibitor Imatinib.
Promyelocytic leukemia (APML) has the chromosomal translocation t (15: 17) that generates the novel fusion protein PML/RARa, and ATRA (all-trans retinoic acid) offers an effective therapy for APML by specifically suppressing oncogenic activities of the PML/RARa fusion protein.
The FISH assay is considered the gold standard for detecting these chromosomal translocations and it therefore plays a crucial role in selecting a targeted therapy for various leukemias.
Chronic lymphocytic leukemia (CLL) is another type of leukemia that can take an aggressive form in some patients due to multiple genetic aberrations. The commonly known chromosomal aberrations are trisomy 12, del(13q), del(11q), del(17p) and del(6q) [19-21]. FISH has been a superior technology in detecting these chromosomal anomalies (80% patients has benefited from the technology in contrast to 40-50% patients who have benefited from conventional cytogenetic techniques). Additionally, FISH tests can distinguish between and hence aid in diagnosis between CLL and other types of small B-cell lymphoma/leukemia.
FISH can also guide critical therapeutic management decisions and help in stratification of patients according to prognostic outcomes. It has been demonstrated that chemoimmunotherapy using fludarabine, cyclophosphamide and rituximab (FCR) receives a better treatment response from patients with trisomy 12 or del(11q), whereas patients with 17p deletions do not benefit from FCR treatment at all.
Multiple myeloma is characterized by primary genomic aberrations mainly translocations in the early stage followed by secondary aberrations as the cancer progresses. The secondary genomic aberrations are responsible for an aggressive phenotype in the advanced stage of multiple myeloma (MM). Because proliferation is slow during initial stage of MM, conventional cytogenetic analysis of non-dividing cells cannot detect the primary genomic aberrations in such cells. Additionally, small chromosomal rearrangements in MM cannot be detected by low-resolution to chromosome banding analysis. Here FISH can be used to circumvent these shortcomings.
Myelodysplastic syndrome (MDS) - is a mix of clonal hematopoietic disorders resulting in improper hematopoiesis leading to blood cytopenias. About 20-30% of such patients progress to Acute Myeloid Leukemia (AML) in a short span of time. FISH - being more sensitive than conventional metaphase chromosomal banding assay - can be utilized to detect transient and minor abnormal clones and cryptic chromosomal aberrations. It can also relay novel information for those patients with an apparently normal karyotype or unsuccessful culture.
Anaplastic lymphoma kinase (ALK) rearrangements are stringently associated with a large number of non-smoking female patients with pulmonary adenocarcinomas - this occurs in approximately 5% of patients with non-small cell lung cancer (NSCLC). ALK rearrangements are fusion of the echinoderm microtubule-associated protein-like 4 (EML4) or other fusion partners such as TFG or KIF5B with ALK at chromosome 2p23 - leading to constitutive activation of ALK kinase. Patients with EML4-ALK fusion-positive NSCLC are treated with the FDA-approved small-molecule kinase inhibitor Crizotinib – it showed a response rate of 50-60%. FDA has also approved companion diagnostic FISH detection kit (ALK FISH probe kit) – this demonstrates a critical role of the FISH assay in guiding ALK-targeted therapy.
Breast cancer is a heterogeneous cancer that perpetuates through various inherited as well as acquired genomic aberrations. Her-2 (human epidermal growth factor receptor-2) – also called c-erbB-2 – on chromosome 17q12-21.32 encodes a trans-membrane tyrosine kinase protein that plays an important role in normal cell growth and differentiation. Her-2 gene amplification occurs in 20-30% of breast cancer patients and denotes a short survival time and more aggressive phenotype. Her-2 overexpressed breast cancer is sensitive to Herceptin treatment and exhibits more responsiveness to paclitaxel and anthracycline. Hence the prognostication of Her-2 gene amplification is of paramount importance. FISH assay is considered the gold standard for evaluating Her-2 status. US-FDA have approved FISH assay for HER2 evaluation in formalin-fixed paraffin-embedded samples.
Melanoma is a heterogeneous group of melanin-producing skin cancers with acquired genetic aberrations. FISH has been utilized in detecting a variety of recurrent chromosomal aberrations in malignant melanoma. Melanocytic lesions possess uncertain morphologic features that are more discernible with a genetic test like FISH. Most primary melanomas exhibit either structural or chromosomal abnormalities, such as deletions in 9p, 10, 6q and 8p and copy-number increase in 7, 8, 6p, 1q, 20, 17, and 2. For such cases, quantitative multicolor FISH approach has been instrumental.
Prostate cancer is majorly characterized by genetic rearrangements of androgen-regulated TMPRSS2 and ETS family members (ERG, RTV1, and ETV4). This has been harnessed as a predictive biomarker for prostate cancer. These rearrangements have been successfully detected by FISH to stratify patients into subtypes responding to specific therapies. Initially, dual-color ERG break-apart probes were developed that can only detect rearrangement of the ERG gene. Subsequently a tricolor FISH incorporating the red/green break-apart probes for TMPRSS2 with an orange-labeled fusion probe for the 3’ region of ERG were developed.
Bladder cancer (urothelial cancer)
Bladder cancer comprises of several different subtypes – 90% is transitional cell carcinoma. A large proportion of transitional cell carcinoma patients are considered in situ carcinoma. These patients have considerable chances of recurrence or advancement to metastasis. Cytology/cytoscopy – the gold standard - is constrained in its sensitivity that significantly depends on tumor grade, size, and stage. Bladder cancer have been demonstrated to have correlation with various chromosomal aneuploidies. Transitional cell carcinoma is diagnosed with extra copies of two or more chromosomes and/or loss of 9p21. One such FISH assay provides probes that attach to centromeric regions for chromosomes 3, 7, 9 (p16 gene) and 17.
Due to the critical need for predictive biomarker in cancer therapy and outcome – FDA has approved several nucleic acid-based test kits encompassing both DNA and RNA–based assays including quantitative PCR and mutation assays, transcriptomics and arrays and FISH and related ISH methods.
In a nutshell, FDA-approved test kits for FISH diagnostics/prognostics:
- 3 assays for AML
- 1 assay for MDS
- 2 assays for B-cell – CLL
- 1 assay for bladder cancer
- 5 for breast cancer
- 3 for general chromosome abnormalities
- 11 - Hematological malignancies
- 1 – non-small cell lung cancer
Advances in FISH technology – how it has helped in cancer diagnostics
1) FISH automation
Automation of labor-intensive and time-consuming FISH would help in routine clinical diagnostics to evaluate known and validated gene markers. Both Bcr/abl1 gene rearrangements in CML patients as well as Her2 gene amplification in breast cancer patients have been studied through automation. Additionally subjective scoring can be mitigated using a fully automated setup comprising of image analysis and data calculation.
Single labeled probe or dual labeled probes have been conventionally used to detect a single genetic event like deletion or amplification of a locus or chromosomal translocation. However with the advent of multigeneic diseases including cancer, simultaneous detection of such genes by using multiple probes have been devised (quantitative multicolor FISH). Examples of multi-color probes:
- A four-color FISH assay detecting chromosomes 1, 2, 6, 9, 7, 17, the loci 3p24pter, and 3p13p14 has been used for the early diagnosis of renal carcinoma.
- A four-color FISH kit for simultaneously detecting chromosome 6 and the 5p15, 7p12 (EGFR gene), and 8q24 (MYC gene) loci was developed to assist in the differential diagnosis of ambiguous lung cancers.
- Simultaneous assay using multiple probes have has been implemented in many hematological cancers as well.
Readers are referred to a good discussion on multicolor FISH probe sets and their application