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Hello, my name is Deepika Sirohi. I am a Molecular Genetic Pathology Fellow at University of California, San Francisco. Welcome to this Pearl of Laboratory Medicine on “Introduction to Cancer Genetics.”
Molecular genetics is increasingly being applied to solid tumors for establishing definitive diagnosis; providing a tool to inform clinical management decisions by serving as prognostic and predictive markers as well as guide treatment decisions to initiate or monitor therapy.
Some of the more common tumor types that are received for molecular testing are listed here and we will discuss the testing strategies and guidelines for some of these in this talk. Many sarcomas have specific molecular alterations that can be targeted to confirm diagnosis, however these are too vast and for the sake of brevity are not being covered in this module.
One of the most frequently tested tumors are lung adenocarcinomas that can have specific molecular alterations that can be used to inform treatment strategies. EGFR and KRAS mutations are the most frequent alterations in lung adenocarcinomas.
Mutations in EGFR are known to occur in East Asian women with no history of smoking and commonly have a bronchoalveolar histologic pattern. KRAS on the other hand, occurs more often in non-Asians and smokers in poorly differentiated adenocarcinomas with mucinous and bronchoalveolar features. While Tyrosine kinase inhibitors (TKIs) are used to target lung carcinomas with EGFR mutations, presence of KRAS mutations is a negative predictor of response to TKIs. Other alterations of therapeutic significance are EML-ALK4 and ROS1 fusions that are amenable to Crizotinib therapy and BRAF mutations, which confer resistance to EGFR inhibition but respond to MEK inhibitors.
EGFR mutations are seen in up to 25-30% of non- small cell lung carcinomas. Clinically significant mutations occur in the tyrosine kinase domains and are the most reliable predictors of response to TKIs. Mutations such as exon 19 deletion and L858R in exon 21 predict sensitivity to TKIs, whereas insertions in exon 20 suggest resistance. A missense mutation T790M is known to confer secondary resistance in patients being treated with TKIs.
Codon 12 and 13 are mutation hotspots in the KRAS gene that are mutually exclusive of EGFR mutations. They are predictive of reduced sensitivity to Cisplatin and better response to Paclitaxel and Pemetrexed. ALK rearrangement in lung carcinomas usually involves fusion with EML4, but can involve other partner genes. The rearrangement can be tested by FISH break apart testing. A small proportion of lung carcinomas show BRAF mutations and ROS1 gene rearrangements.
The American Society of Clinical Oncology recommends EGFR as the first line test in limited specimens. For biopsy specimens where adenocarcinomas cannot be excluded, EGFR and ALK testing is still recommended. On the contrary, in resected specimens where no adenocarcinoma component is identifiable, EGFR and ALK testing is not recommended.
In the context of colorectal carcinomas (CRC), oncogenesis follows one of the 3 pathways: Familial adenomatosis polyposis (FAP) pathway associated with chromosomal instability and causes downstream activation of APC/WNT signaling; the serrated pathway with aberrant CpG methylation, a subset of which can have epigenetic silencing of the MLH1 gene and consequent microsatellite instability; and the hereditary microsatellite instability (MSI) or Lynch syndrome caused by germline mutations in one of the DNA mismatch repair genes.
Testing for mismatch repair (or MMR) gene alterations is best achieved by an algorithmic approach that starts with immunohistochemical testing for the 4 mismatch repair proteins. The absence of one or more of the MMR proteins by immunohistochemistry (IHC) suggests a MSI carcinoma that could be either sporadic or inherited. These can be differentiated by testing for MLH1 promoter hypermethylation seen in most cases and BRAF mutations that are present in 3/4th of these. If these are positive, this supports a sporadic microsatellite instability and if these are negative, germline testing for alterations in the MMR gene is indicated. Lynch syndrome is caused by germline mutations in one of the four mismatch repair genes which are present in a third of the cases with microsatellite instability
The clinical significance for MSI testing is substantiated by the better prognosis of MSI- H carcinomas and their resistance to 5-fluorouracil, cisplatin and alkylating agents.
Instead they show better response to irinotecan based therapies with early data suggestive of better response to anti- PD-1 /PD-L1 immune checkpoint inhibitor therapy.
Single gene testing is important in colorectal cancers to guide treatment decisions. Even though EGFR mutations are very rare in colorectal cancers, they can be sensitive to EGFR-directed therapy. Therapeutic decisions can be guided by testing for KRAS mutations that when present are indicative of resistance to anti- EGFR antibodies.
Common KRAS mutations differ among smokers and never smokers. Other less frequently encountered genetic alterations occur in BRAF, PIK3CA, NRAS, MEK1 and AKT genes.
In line with this, the recent ASCO/CAP recommendation for CRCs are to test for KRAS codons 12, 13, 61, and 146 and NRAS mutations which together are also called an extended RAS panel. Though mutations in BRAF (i.e. V600E) and PIK3CA have been shown in some studies to correlate with poor outcomes and decreased response to EGFR antibodies, there is insufficient evidence to recommend testing for these genes under current standard of care guidelines.
Mutations in melanomas show a somewhat site specific pattern with BRAF mutations being more frequent in melanomas of intermittently sun damaged skin, NRAS in nodular melanomas of sun-damaged skin, KIT in acral and mucosal melanomas and GNAQ/GNA11 in uveal melanomas. BRAF V600E is the most frequent alteration seen in melanomas and can be targeted with a specific antibody, Vemurafenib, that has changed the management and outcomes of many advanced melanomas.
Another tumor type in which molecular testing is frequently requested is glial neoplasms of the brain. The 2016 WHO Classification of Tumors of the Central Nervous System now requires both an IDH gene family mutation and a 1p/19q-co-deletion to establish the definitive diagnosis of grade II and grade III oligodendroglioma These two alterations confer a better prognosis as well as predict response to chemotherapy and radiation therapy. A 1p/19q co-deletion pattern has been shown to have a high association with oligodendroglial morphology and is thought to be an early event in tumor development, although they can also be seen in up to 5-10% of Glioblastoma multiforme with unclear significance.
EGFR amplifications or mutations are seen in up to half of high grade gliomas and the amplification is essentially pathognomonic of glioblastoma multiforme (or GBM). The amplification is more often seen in primary GBMs and in older patients and is not predictive of response to EGFR therapy. Somatic mutations of IDH1 or IDH2 can be seen in up to 75% of grade II and III gliomas (including oligodendrogliomas) and can co- occur with TP53 alterations or 1p/19q deletions. Mutations in IDH1 and IDH2 are highly specific for gliomas (although they may be seen in other tumor types including acute myeloid leukemia, cartilaginous tumors, and thyroid cancer) and are predictive of a favorable prognosis.
HER2 or ERBB2 is amplified in approximately 15 to 20% of primary breast cancers and targetable with specific therapy with trastuzumab or lapatinib. Per ASCO/CAP recommendations HER2 testing should be done in all invasive primary breast cancers and metastatic tumors if specimen is available by IHC and/or FISH. ASCO/CAP guidelines also include specific recommendations regarding tissue fixation and scoring to ensure accuracy of testing and utility as a predictive marker. Additionally, there are germline changes in certain genes that can predispose to breast carcinoma.
Identification of these in familial cancers can help guide the health care of family members, including and take preventative measures, if required.
Another testing modality for predicting prognosis and guiding therapy is gene expression profiling using an array of biomarkers. Oncotype DX uses 21 genes and is recommended for women with early stage (I-IIIa) hormone-receptor-positive, HER2- negative, invasive breast cancer. The recurrence risk is stratified into 3 categories of low, intermediate, and high risk. MammaPrint, in contrast, uses a 70-gene array and is FDA approved for node-negative, treatment naive breast cancer patients with stage I or II disease. Patients are stratified into low or high recurrence risk categories. Other expression profiling tests for breast carcinomas include PAM50 and Theros.
Gastrointestinal stromal tumors (or GIST) are the most common soft tissue tumors of the GI tract that show KIT mutations in up to 95% of spindled lesions that makes them amenable to treatment with tyrosine kinase inhibitors such as imatinib. Exon 11 mutations are the most frequent and have been shown to predict worse outcomes. Exon 9 mutations in KIT are more often seen in GISTs of the small intestine and colon. 5% of GISTs have mutations in PDGFRA and these tumors typically have epithelioid morphology. Certain mutations in KIT and PDGFRA confer resistance to imatinib. Some of these mutations, such as those in exon 9, can be managed with a higher dose or alternative TKIs.
Mutations in thyroid cancers are commonly either point mutations or translocations. The BRAF V600E mutation is seen in more than half of papillary thyroid carcinomas (or PTC) and is especially frequent in the classic and tall cell variants. BRAF mutation is an early event and is associated with worse outcomes. Translocations involving RET/PTC occur in PTC, commonly in radiation induced cancers while RAS mutations are seen in papillary or follicular carcinomas and PAX8/PPARγ rearrangements typically are identified in follicular carcinoma in younger individuals. RET mutations on the other hand are seen in Medullary Thyroid carcinomas.
Slide 20: References
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- Sepulveda AR, Hamilton SR, Allegra CJ et al. Molecular biomarkers for the evaluation of colorectal cancer.Guideline from the American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and American Society of Clinical Oncology. J Mol Diagn 2017;19(2):187-225
- Igbokwe A, Lopez-Terrada D. H. Molecular testing of solid tumors. Arch Pathol Lab Med 2011;135:67–82)
- Perry A, Fuller CE, Banerjee R, et al. Ancillary FISH analysis for 1p and 19q status: preliminary observations in 287 gliomas and oligodendroglioma mimics. Front Biosci. 2003 Jan 1;8:a1-9.
- Banin Hirata BK, Oda JM, Losi Guembarovski R et al. Molecular markers for breast cancer: prediction on tumor behavior. Dis Markers. 2014:513158. Epub 2014 Jan 28.
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- Grossmann AH, Grossmann KF, Wallander ML. Molecular testing in malignant melanoma. Diagn Cytopathol. 2012;40(6):503-10.
- Nikiforov YE. Molecular analysis of thyroid tumors. Mod Pathol 2011 24, S34–S43
Slide 21: Disclosures
I have no disclosures or conflicts of interest.
Thank you for joining me on this Pearl of Laboratory Medicine on “Introduction to Cancer Genetics.”