The growing momentum of molecular analysis has enormous promise for diagnostics and numerous research applications. The vast arsenal of newer modalities in the field of molecular pathology available today is bewildering and ever expanding. They provide opportunities to study etiopathogenesis, to aid diagnosis, to prognosticate tumours and to devise targeted therapy.
Molecular diagnostics
Immunohistochemistry helps classify soft tissue and bone tumors, but a proportion of pediatric tumors defy such attempts at classification. Specific translocation based molecular “signatures” of these tumors have the potential to resolve such diagnostic dilemmas.
Immunohistochemistry helps classify soft tissue and bone tumors, but a proportion of pediatric tumors defy such attempts at classification. Specific translocation based molecular “signatures” of these tumors have the potential to resolve such diagnostic dilemmas.
Ewing sarcoma family of tumors (ESFTs) are being characterized by non-random gene rearrangements between the EWS gene on chromosome 22q12 and various members of the ETS gene family such as FLI1 (chromosome 11), ERG (chromosome 21), ETV1 (chromosome 7), E1AF (chromosome 17) and FEV (chromosome 2). Accurate diagnosis of ESFT is crucial for the most appropriate clinical management of patients. Desmoplastic small round cell tumor is characterized by t(11;22) (p13;q12) resulting in EWS-WT1 fusion. Alveolar rhabdomyosarcoma demonstrates PAX3-FKHR and PAX7-FKHR fusion. t(X;18) (p11;q11) is associated with > 90% of both monophasic and biphasic synovial sarcomas; SYT-SSX1 and SYT-SSX2 fusion transcripts correlate with biphasic and monophasic morphology respectively.
Sensitive technique like RT-PCR has made it possible to evaluate tiny biopsies, archived paraffin blocks and needle aspiration cytology material for the presence of specific translocations and arrive at a definitive diagnosis. The use of Fluorescence in Situ Hybridization, FISH, on interphase nuclei, allows the detection of nucleic acid targets while preserving the morphology of the tissue. Many chromosomal abnormalities such as duplication, deletion or translocation, are diagnosable using FISH. This technique requires less tissue, can be performed on archival tissue, and there is visual control of the tumour. Investigating aneuploidy at chromosomes 3, 7, 17, and 9p21 (e.g. loss at 9p21) using a multitarget FISH performed on urine specimens is a promising approach and useful adjunct to urine cytology and cystoscopy for the non-invasive detection of recurrent urothelial tumours.
Molecular diagnostics, however, are generally viewed as expensive and labor intensive, which means that advances to improve automation, throughput, scalability, and reliability are critical.
Prognostic uses
– The fusion transcript type in the evaluation of ESFT may be prognostically useful with the presence of EWS-FLI1 type1 transcript being associated with improved outcome compared with that in patients with other fusion transcript types.
– The fusion transcript type in the evaluation of ESFT may be prognostically useful with the presence of EWS-FLI1 type1 transcript being associated with improved outcome compared with that in patients with other fusion transcript types.
– Alveolar rhabdomyosarcoma with PAX3-FKHR fusion is associated with poor prognosis as compared to those with PAX7-FKHR translocation.
– SYT/SSX1 variant of synovial sarcoma might be less favorable, associated with higher tumor proliferating rate and reduced overall survival as compared to SYT/SSX2.
– N-myc amplification is a major prognostic factor connoting poor prognosis in neuroblastoma.
– HER-2/neu, ERBB2, a proto-oncogene located on chromosome 17 has become an important prognostic indicator in breast cancer. FISH based assay uses probes for chromosome 17 centromere and HER-2 gene simultaneously, and HER-2 gene amplification is defined as a ratio of HER-2 gene copies per chromosome 17 copy equal to or greater than 2.0. Amplification and /or overexpression of HER-2 occurs in 15% to 25% of human breast cancers and is associated with a poor clinical outcome. Over expression of the HER-2 protein on FISH is closely correlated with amplification of the HER-2 gene in tumours scored immunohistochemically as 3+, but not in tumours scored as 2+. It has been documented in the literature that HER-2 gene amplification determined by FISH is a better predictor of response to trastuzumab-based therapy than HER-2 overexpression determined by immunohistochemical scores of 2+ to 3+. The accurate assessment of HER-2 gene amplification is critical in the management of these tumours.
Diagnosis of Recurrent Disease
Urinary cytology has been the reference test for the evaluation of symptomatic patients, for the detection of lesions in high-risk patients, and for the follow-up of patients with a prior history of urothelial carcinoma. Although routine cytology has a relatively high level of specificity for the detection of high-grade in situ and invasive lesions, the sensitivity for patients with low-grade TCC is low. Urine cytology is further limited by poor specificity for clinically significant lesions in cases that are classified as atypical on the basis of cells with equivocal cytologic features. Therefore, the clinical outcomes for individual cases cannot be accurately determined by cytologic examination alone. As a result, many patients undergo unnecessary procedures to rule out malignancy based on limited specificity for clinically significant lesions. The U.S. Food and Drug Administration (FDA) recently approved the UroVysionTM Bladder Recurrence Cancer Test (Vysis; Downers Grove, IL) for the detection of urothelial carcinoma in urine. The UroVysionTM test has been designed for interphase cell quantification of chromosomes 3, 7, 17, and 9p21 region (p16/CDKN2A gene), which are recognized as frequently involved in numerical anomalies in bladder cancer. Interestingly, the UroVysionTM test also was proven to be effective in detecting tumor recurrence before evidence of urothelial carcinoma was found on biopsy, likely due to the ability of voided urine to represent the entire urothelium versus the regional evaluation of the biopsy. Patients with atypical/suspicious findings on urine cytology and negative FISH may be subjected to longer intervals of cystoscopic surveillance. Thus negative FISH is also considered a valuable negative finding. The assay also holds hope for a group of patients with hematuria and dysuria with negative cystoscopic findings and urine cytologic findings. Patients with positive FISH but negative biopsy should be carefully followed, as they are at risk of harboring occult disease. Anticipatory positive cases (FISH pos/CYSTO neg/HISTO neg) recur at twice the rate and in half the time as true negative cases.
– SYT/SSX1 variant of synovial sarcoma might be less favorable, associated with higher tumor proliferating rate and reduced overall survival as compared to SYT/SSX2.
– N-myc amplification is a major prognostic factor connoting poor prognosis in neuroblastoma.
– HER-2/neu, ERBB2, a proto-oncogene located on chromosome 17 has become an important prognostic indicator in breast cancer. FISH based assay uses probes for chromosome 17 centromere and HER-2 gene simultaneously, and HER-2 gene amplification is defined as a ratio of HER-2 gene copies per chromosome 17 copy equal to or greater than 2.0. Amplification and /or overexpression of HER-2 occurs in 15% to 25% of human breast cancers and is associated with a poor clinical outcome. Over expression of the HER-2 protein on FISH is closely correlated with amplification of the HER-2 gene in tumours scored immunohistochemically as 3+, but not in tumours scored as 2+. It has been documented in the literature that HER-2 gene amplification determined by FISH is a better predictor of response to trastuzumab-based therapy than HER-2 overexpression determined by immunohistochemical scores of 2+ to 3+. The accurate assessment of HER-2 gene amplification is critical in the management of these tumours.
Diagnosis of Recurrent Disease
Urinary cytology has been the reference test for the evaluation of symptomatic patients, for the detection of lesions in high-risk patients, and for the follow-up of patients with a prior history of urothelial carcinoma. Although routine cytology has a relatively high level of specificity for the detection of high-grade in situ and invasive lesions, the sensitivity for patients with low-grade TCC is low. Urine cytology is further limited by poor specificity for clinically significant lesions in cases that are classified as atypical on the basis of cells with equivocal cytologic features. Therefore, the clinical outcomes for individual cases cannot be accurately determined by cytologic examination alone. As a result, many patients undergo unnecessary procedures to rule out malignancy based on limited specificity for clinically significant lesions. The U.S. Food and Drug Administration (FDA) recently approved the UroVysionTM Bladder Recurrence Cancer Test (Vysis; Downers Grove, IL) for the detection of urothelial carcinoma in urine. The UroVysionTM test has been designed for interphase cell quantification of chromosomes 3, 7, 17, and 9p21 region (p16/CDKN2A gene), which are recognized as frequently involved in numerical anomalies in bladder cancer. Interestingly, the UroVysionTM test also was proven to be effective in detecting tumor recurrence before evidence of urothelial carcinoma was found on biopsy, likely due to the ability of voided urine to represent the entire urothelium versus the regional evaluation of the biopsy. Patients with atypical/suspicious findings on urine cytology and negative FISH may be subjected to longer intervals of cystoscopic surveillance. Thus negative FISH is also considered a valuable negative finding. The assay also holds hope for a group of patients with hematuria and dysuria with negative cystoscopic findings and urine cytologic findings. Patients with positive FISH but negative biopsy should be carefully followed, as they are at risk of harboring occult disease. Anticipatory positive cases (FISH pos/CYSTO neg/HISTO neg) recur at twice the rate and in half the time as true negative cases.
Molecular Therapeutics
The emergence of molecularly targeted therapeutics in oncology has placed increased importance on molecular diagnostic tests in guiding therapeutic intervention.
The emergence of molecularly targeted therapeutics in oncology has placed increased importance on molecular diagnostic tests in guiding therapeutic intervention.
EGFR Activity in Cancer
The EGFR is a member of a family of four receptors [EGFR (HER1 or ErbB1), ErbB2 (HER2/neu), ErbB3 (HER3), and ErbB4 (HER4)].
These receptors are large proteins which reside in the cell membrane. When a ligand, for example EGF or TGF-a, binds to the EGFR, it causes receptor dimerisation and autophosphorylation of the internal receptor domain.
This initiates a cascade of cellular reactions that result in increased cell division and influences other aspects of malignant progression of the tumor - angiogenesis, metastasis and an inhibition of apoptosis.
Inhibition of the EGFR thus provides a rational target for anticancer therapy. Currently, two approaches are in clinical development: monoclonal antibodies and small molecule inhibitors of the EGFR tyrosine kinase enzyme.
A monoclonal antibody to HER2/neu (trastuzumab) has proved successful in limiting the growth of tumors and has been licensed for the treatment of advanced breast cancers that overexpress this receptor. The measurement of p185HER-2/neu expression has gained new importance based on the approval of trastuzumab for the treatment of patients with metastatic breast cancer. Treatment with Trastuzumab alone or in combination with chemotherapy is indicated only for patients whose tumours over express p185HER-2/neu or demonstrate HER-2/neu gene amplification based on FISH.
The combination of trastuzumab with cytotoxic chemotherapy is also considered to improve other clinical endpoints, including response duration, time to tumour progression, and overall survival, compared with the chemotherapy-only treatment option.
Recent studies have shown promising results with tyrosine kinase inhibitors of EGFR (ZD1839 and OSI-774) in patients with esophageal and lung carcinomas. In non-small-cell lung carcinomas, ZD1839 (Iressa®; AstraZeneca; London, UK) produced objective responses in about 15%-20% of patients with advanced disease who had failed one or two chemotherapy regimens (at least one platinum based) and in 10% of patients who had failed two or more prior chemotherapy regimens containing platinum and docetaxel, with a favorable adverse event profile. These findings raised considerable excitement regarding the potential role of such therapies for lung cancer patients with less advanced disease and their potential to improve survival.
Genomics
A major leap in functional genomic investigations would be the ability to perform global gene expression analysis with in vivo-derived genetic material originating from morphologically distinct cellular populations that constitute the various stages of cancer progression. The recent advent of high-density DNA microarray technology, with its capacity for simultaneous monitoring of thousands of genes, provides a unique opportunity for high-throughput genetic analysis of tumours.
The EGFR is a member of a family of four receptors [EGFR (HER1 or ErbB1), ErbB2 (HER2/neu), ErbB3 (HER3), and ErbB4 (HER4)].
These receptors are large proteins which reside in the cell membrane. When a ligand, for example EGF or TGF-a, binds to the EGFR, it causes receptor dimerisation and autophosphorylation of the internal receptor domain.
This initiates a cascade of cellular reactions that result in increased cell division and influences other aspects of malignant progression of the tumor - angiogenesis, metastasis and an inhibition of apoptosis.
Inhibition of the EGFR thus provides a rational target for anticancer therapy. Currently, two approaches are in clinical development: monoclonal antibodies and small molecule inhibitors of the EGFR tyrosine kinase enzyme.
A monoclonal antibody to HER2/neu (trastuzumab) has proved successful in limiting the growth of tumors and has been licensed for the treatment of advanced breast cancers that overexpress this receptor. The measurement of p185HER-2/neu expression has gained new importance based on the approval of trastuzumab for the treatment of patients with metastatic breast cancer. Treatment with Trastuzumab alone or in combination with chemotherapy is indicated only for patients whose tumours over express p185HER-2/neu or demonstrate HER-2/neu gene amplification based on FISH.
The combination of trastuzumab with cytotoxic chemotherapy is also considered to improve other clinical endpoints, including response duration, time to tumour progression, and overall survival, compared with the chemotherapy-only treatment option.
Recent studies have shown promising results with tyrosine kinase inhibitors of EGFR (ZD1839 and OSI-774) in patients with esophageal and lung carcinomas. In non-small-cell lung carcinomas, ZD1839 (Iressa®; AstraZeneca; London, UK) produced objective responses in about 15%-20% of patients with advanced disease who had failed one or two chemotherapy regimens (at least one platinum based) and in 10% of patients who had failed two or more prior chemotherapy regimens containing platinum and docetaxel, with a favorable adverse event profile. These findings raised considerable excitement regarding the potential role of such therapies for lung cancer patients with less advanced disease and their potential to improve survival.
Genomics
A major leap in functional genomic investigations would be the ability to perform global gene expression analysis with in vivo-derived genetic material originating from morphologically distinct cellular populations that constitute the various stages of cancer progression. The recent advent of high-density DNA microarray technology, with its capacity for simultaneous monitoring of thousands of genes, provides a unique opportunity for high-throughput genetic analysis of tumours.
Proteomics
Molecular diagnostics, however, are generally viewed as expensive and labor intensive, which means that advances to improve automation, throughput, scalability, and reliability are critical.
Molecular diagnostics, however, are generally viewed as expensive and labor intensive, which means that advances to improve automation, throughput, scalability, and reliability are critical.
Prognostic uses
– The fusion transcript type in the evaluation of ESFT may be prognostically useful with the presence of EWS-FLI1 type1 transcript being associated with improved outcome compared with that in patients with other fusion transcript types.
– The fusion transcript type in the evaluation of ESFT may be prognostically useful with the presence of EWS-FLI1 type1 transcript being associated with improved outcome compared with that in patients with other fusion transcript types.
– Alveolar rhabdomyosarcoma with PAX3-FKHR fusion is associated with poor prognosis as compared to those with PAX7-FKHR translocation.
– SYT/SSX1 variant of synovial sarcoma might be less favorable, associated with higher tumor proliferating rate and reduced overall survival as compared to SYT/SSX2.
– N-myc amplification is a major prognostic factor connoting poor prognosis in neuroblastoma.
– HER-2/neu, ERBB2, a proto-oncogene located on chromosome 17 has become an important prognostic indicator in breast cancer. FISH based assay uses probes for chromosome 17 centromere and HER-2 gene simultaneously, and HER-2 gene amplification is defined as a ratio of HER-2 gene copies per chromosome 17 copy equal to or greater than 2.0. Amplification and /or overexpression of HER-2 occurs in 15% to 25% of human breast cancers and is associated with a poor clinical outcome. Over expression of the HER-2 protein on FISH is closely correlated with amplification of the HER-2 gene in tumours scored immunohistochemically as 3+, but not in tumours scored as 2+. It has been documented in the literature that HER-2 gene amplification determined by FISH is a better predictor of response to trastuzumab-based therapy than HER-2 overexpression determined by immunohistochemical scores of 2+ to 3+. The accurate assessment of HER-2 gene amplification is critical in the management of these tumours.
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