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Tests offered Molecular Oncology Laboratory at Amrita

EGFR (Tyrosine Kinase Domain) Mutational Analysis

Indication: Advanced Stage of adenocarcinoma of the lung, diagnosed by histopathological examination currently have a better treatment in terms of both survival rate and quality of life index by selective Tyrosine Kinase Inhibitors which have specific activating mutations in the Tyrosine Kinase Domain of the EGFR gene. About 30% subjects with advanced stage of lung adenocarcinoma (Stage IV) has been shown to have this type of mutations from Asian populations. We are doing targeted mutational scanning for the EGFR gene in histopathologically identified tumor tissue samples.
Test Description: The test should be able to identify any point mutation in the four exonic regions (from 18 to 21) including the splice site regions of the introns.  While other tests could detect only certain selective mutations in the gene, our test should be able to detect any/all mutations in the regions so that any novel mutations in the region will also be reported. Also, our test should be able to detect deletions/duplications of the gene region. Also, this can detect the novel chemo-resistance mutation of EGFR (T790M).
Test Method: Real-time PCR & High-resolution melting analysis for the pre-selection of amplicons followed by Sanger sequencing.
Sample: Histopathological examination followed by identification of selective tumor tissue samples in Formalin Fixed Paraffin Embedded (FFPE) tumor tissue samples. 
Reporting time: 3-4 weeks

IDH 1 & 2 (Targeted) Mutational Analysis 

Indication: IDH mutations has been identified as one of the best molecular marker for grading and predicting the prognosis of astrocytoma. Along with 1p19q deletion cytogenetic analysis by Fluorescent in situ Hybridization analysis, IDH 1 & 2 (Exon 4) targeted mutational scanning is also routinely done for all glioma cases. Being an oncogene, IDH mutations have less variance and Exon 4 is considered to be the most common region of mutation. Our method could identify any mutation occurring in Exon 4 region of both IDH 1 and IDH 2 genes. This includes the most common R132H and other specific mutations as well.
Test Description: The test should be able to identify any point mutation in the Exon 4 region including the splice site regions of the introns.  While other tests could detect only certain selective mutations in the gene, our test should be able to detect any/all mutations in the regions so that any novel mutations in the region will also be reported.
Test Method: Real-time PCR & High-resolution melting analysis for the pre-selection of amplicons followed by Sanger sequencing.
Sample: Histopathological examination followed by identification of selective tumor tissue samples in Formalin Fixed Paraffin Embedded (FFPE) tumor tissue samples. 
Reporting time: 3-4 weeks

 

BRCA 1 and BRCA 2 Genetic Screening

Indication: Hereditary Breast and Ovarian Cancer syndrome (HBOC) are now on the rise in Indian population. There has been a recent study which showed that the HBOC common in young (<50 years) breast cancer patients or if young patient with multiple breast cancer incidence, or associated with other factors according to the National Cancer Care Network (NCCN) Guidelines. In order to identify, early detection and proper preventative care it is important to do HBOC screening. It has been identified that more than 90% of the cases of HBOCs are associated with mutations of BRCA 1 and 2 genes. Subjects who have mutations in BRCA1 or BRCA 2 genes are reported to have about 80% of recurrence of breast or ovarian cancer and significantly reduce the survival rate.
Test Description: The test should be able to identify any point mutation in the entire gene of BRCA 1 and BRCA2 exonic regions and the splice site regions of the introns. 
Test Method: Next Generation Sequencing
Sample: Whole Blood (in EDTA containing tube- Lavender Colored Vacutainer): 3-5ml
Reporting time: 4-6 weeks

NF1 Deletion/Duplication Analysis

Indication: Neurofibromatosis type 1 (NF1) is a congenital autosomal dominant neuro-cutaneous syndrome (OMIM# 162200) characterized by occurrence of multiple typed brain tumors (meningioma, schwannoma, optic glioma), with patchy skin pigmentation and tumors, skeletal and soft tissue abnormalities. 
Test Description: The test should be able to identify any deletions/duplications of the sixty exonic regions and the splice site regions of the introns.  
Test Method: Multiple Ligation Probe Amplification (MLPA) and fragment analysis.
Sample: Whole Blood (in EDTA containing tube- Lavender Colored Vacutainer): 3-5ml
Reporting time: 3-6 weeks

NF2 Deletion/Duplication Analysis

Indication: Neurofibromatosis type 2 (NF2) is an autosomal dominant hereditary neurocutaneous syndrome (OMIM# 162200) characterized by occurrence of recurrent brain and spinal tumors (meningioma, schwannoma, ependymoma).
Test Description: The test should be able to identify any deletions/duplications of the sixty exonic regions and the splice site regions of the introns.  
Test Method: Multiple Ligation Probe Amplification (MLPA) and fragment analysis.
Sample: Whole Blood (in EDTA containing tube- Lavender Colored Vacutainer): 3 - 5ml
Reporting time: 3-6 weeks

VHL Deletion Duplication Analysis

Indication: Clinically suspected cases of von Hippel-Lindau syndrome (OMIM #193300). Majority of the patients with the syndrome would have non-synonymous point mutation of the gene leading on to abnormal or absence of the VHL protein which leads to the tumor syndrome. There can be mutations such as deletions/indels, frame-shift mutations, exonic deletions/insertions can happen with the VHL gene for the disorder. 
Test Description: The test should be able to identify any deletions/duplications of the three exonic regions and the splice site regions of the introns.  
Test Method: Multiple Ligation Probe Amplification (MLPA) and fragment analysis
Sample: Whole Blood (in EDTA containing tube- Lavender Colored Vacutainer): 5ml
Reporting time: 1-2 week(s)

VHL sequence analysis

Indication: Clinically suspected cases of von Hippel-Lindau syndrome (OMIM #193300) in recurrent hemangioblastoma, renal cell carcinoma, pheochromocytoma, multi-cystic pancreas, liver or kidney, or endolymphatic sac tumor of inner ear. The human VHL is a gene located in 3p25.3 and the mutations of that gene is highly associated with the syndrome. Majority of the patients with the syndrome would have non-synonymous point mutation of the gene leading on to abnormal or absence of the VHL protein which leads to the tumor syndrome. 
Test Description: The test should be able to identify any point mutation in the three exonic regions and the splice site regions of the introns. 
Test Method: Sanger sequencing
Sample: Whole Blood (in EDTA containing tube- Lavender Colored Vacutainer): 5ml
Reporting time: 3-4 weeks

 

Microsatellite-instability (MSI) analysis PCR

Test Indication: MSI is a unique mechanism in tumour development especially for colon and endometrial cancers. The derangement in DNA mis-match repair mechanism or the “mutator phenotype” is an extensively characterized molecular pathological change in those cancers in which MSI has definite therapeutic and prognostic roles. The test directly inspect five different DNA repeat markers selected according to widely accepted criteria for Hereditary Non-Polyposis Colorectal Cancer or Lynch Syndrome (Revised Bethesda Criteria). MSI can occur in somatic and germline cells according to the condition.

Test Description: Our assay is custom developed with previously designed and characterized primers and probes. The markers are D2S123, D5S346, D17S250, BAT-25, BAT-26. The test is done on DNA extracted from Formalin Fixed Paraffin Embedded (FFPE) tumour or control tissue samples. The tissue must be first histo-pathologically screened and triaged by the trained pathologist. Our excellent pathology team is coordinating with us for the test. MSI should be checked and compared between tumour and normal tissue from each individual since the markers can be unique to each individual.

Test method: Amplification of short-tandem repeat markers and fragment analysis in ABI 3500® Genetic analyzer.

Sample: Histopathological examination followed by identification of selective tumor tissue samples in Formalin Fixed Paraffin Embedded (FFPE) tumor tissue samples.

Order Names:

  • “Histopath Comprehensive MSI Testing – IHC and PCR” for combined MMR Immunohistochemistry & MSI-PCR
  • “Microsatellite Instability Analysis (MSI-PCR) for single test.

Reporting-time: 2-3 days

Extended RAS Sequencing

Test Indication: Extended RAS genes, which includes exon 2, 3, and 4 of KRAS and exon 2, 3, and 4 of NRAS, are sequenced for various cancers such as Lung, colorectal, gasto-intestinal, genito-urinary cancers. These three exons comprise almost all the mutations described in these two genes.

Test method: Somatic gene mutation analysis by gene sequencing. Specific primers are custom synthesized, and sanger sequenced using ABI 3500® Genetic Analyzer.

Sample: Histopathological examination followed by identification of selective tumor tissue samples in Formalin Fixed Paraffin Embedded (FFPE) tumor tissue samples.

Order Name: “Extended RAS Sequencing”

Reporting-time: 2-3 days once sample is received.

BRAF (Exon 15) sequencing

Test Indication: BRAF mutations are detected to prognosticate many cancers (methylator phenotype colorectal cancers, melanoma, thyroid cancers etc). There are newer anti-cancer agents which are specifically targeted against cancers with mutations in this gene (e.g. Dabrafinib).

Test Description: BRAF Exon 15 sequencing comprises the somatic mutation analysis by sequencing. This identifies any novel mutation in this region, over and above the hotspot mutation such as BRAF V600E.

Test Method: Sanger Sequencing using ABI 3500® Genetic Analyzer.

Sample: We require tumour tissue FFPE samples. No need for control tissue for this test. The tissue must be first histo-pathologically screened by a pathologist. Specific primers are synthesized and optimized to cover the entire Exon 15 region. Sequencing is done using ABI-3500 ® Genetic Analyzer.

Order Name: “BRAF (Exon 15) Sequencing”

Reporting-time: 2-3 days once sample is received.

Paediatric Glioma BRAF fusion panel

Test Indication: BRAF-KIAA 1549 translocation, IDH1&2 hotspots, and a set of other genes (CDKN2A/B, FGFR1, etc) are included in this panel which are specifically purposed to prognosticate high grade intractable paediatric glioma.

Test Method: The test uses a kit based on Multiplex Ligation Probe Amplification (MLPA) assay, with fragment analysis done in the ABI 3500 Genetic analyser. The list of probes which are included in the assay can be provided upon request.

Sample: This is a somatic gene mutation analysis. We require tumor and control tissue FFPE samples. Since MLPA fragments can be unique to individual person. The tissue must be first histo-pathologically screened by a pathologist.

Order name: “Paediatric Glioma panel (BRAF fusion & IDH Mutation Analysis)”