Development
of a Comprehensive Mutational Pannel as an Effective Tool for Personalized
Diagnostic of Medullary Thyroid Carcinomas
Medullary
Thyroid Carcinoma (MTC) originates from mutations in calcitonin-producing
parafollicular C cells of the thyroid, is a rare malignancy, accounting for
3-4% of all thyroid carcinomas. It occurs in a hereditary form (HMTC, 25%) or
in a sporadic form (SMTC, 75%). The prognosis for patients with MTC is poor, as
the tumor metastasizes at early stages; and the only curative therapeutic
option so far is radical surgery. Genetic analysis helps identify inherited
cases at a stage where prophylactic surgery can be offered to carriers of such
mutations to prevent the disease. This approach may also be used to determine
better treatment options for patients who are already diagnosed with MTC.
The
goal of this project was to develop a comprehensive mutational panel for the
detection of clinically relevant mutations in MTC samples. A total of 143
mutations (nucleic acid variations) in 8 human genes were selected from
numerous papers and public databases and included into the MTC mutational
panel. The selection criteria were based on the coding mutations (mutations
that occur in the coding area of the genes) that were reported to occur in MTC
patients and considered functionally relevant. Some of the mutations included
into the MTC mutational panel (such as BRAF gene V600E mutation) were also
described in other types of thyroid cancer, but most of the mutations were
unique to MTC (such as all RET gene mutations). The entire assay design was
carried out using Sequenom's online design tools (ProxSNP and PreXTEND
(https://www.mysequenom.com/Tools) and Assay Design software (v. 3.1)).
The
final file comprised from 115 assays corresponding to all 143 mutations
included into the MTC panel. This file will be further processed using the
SEQUENOM® Mass-ARRAY iPLEX® platform for DNA genotyping of clinical samples by
the cancer research scientists at the Abramson Cancer Center of the University
of Pennsylvania.
References:
1. Millis,
M. (2011, Summer). Medium-Throughput SNP Genotyping Using Mass Spectrometry:
Multiplex SNP Genotyping Using the iPLEX® Gold Assay. Springer Protocols, 700.
Retrieved August 20, 2012, from http://link.springer.com/protocol/10.1007%2F978-1-61737-954-3_5
This paper discusses and explains
the basics of genotyping with the using MALDI-TOF Mass Spectrometry. This was
the first paper that I have read right after being given the project. After
fully interpreting this paper, I realized that I am going to be able to finish
the project on my own. This paper served as a guide to me throughout the
process of doing this project, as well as while writing the entire research
report. In addition, this paper was used to make sure that I am not saying
something that is factually incorrect.
2. Gabriel, S., Ziaugra, L., &
Tabbaa, D. (2009, January 1). UNIT 2.12 SNP Genotyping Using the Sequenom
MassARRAY iPLEX Platform. Current Protocols in Human Genetics.
This paper describes in details the
SNP genotyping method based on the Sequenom MassARRAY platform. It includes two
step protocol (initial locus-specific PCR reaction, followed by single base
extension using mass-modified dideoxynucleotide terminators) an assay structure
and how using MALDI-TOF mass spectrometry identify the SNP allele. The paper is
mentioned in the corresponded section of the Introduction.
3. Ricarte-Filho, J., Ryder, M.,
Ghossein, R., Fagin, J., Chitale, D., Rivera, M., et al. (2009, June 1).
Mutational Profile of Advanced Primary and Metastatic Radioactive
Iodine-Refractory Thyroid Cancers Reveals Distinct Pathogenetic Roles for BRAF,
PIK3CA, and AKT1. CANCER RESEARCH. Retrieved July 1, 2012, from
http://cancerres.aacrjournals.org/content/69
The paper describes profiling of
111 mutations in RET, BRAF, NRAS, HRAS, KRAS, PIK3CA, AKT1 genes in clinical
poorly differentiated, anaplastic and radioactive iodine-refractory
differentiated thyroid cancers. The genotyping method is based on the Sequenom
MassARRAY platform. It was shown that RAS mutations were prevalent in primary
PDTC, whereas BRAF was more common in metastatic PDTC and ATC. PIK3CA or AKT1
mutations were rare. The paper is mentioned in the corresponded section of the
Introduction.
4. MEN2
Database. (n.d.). AURP Scientific Resource for Research and Education..
Retrieved July 12, 2012, from http://arup.utah.edu/database/MEN2/MEN2_display.php
This database was only used for its
mutations list during the collection phase of the project.
5. Human
BLAT Search. (n.d.). UCSC Genome Browser. Retrieved August 17, 2012, from http://genome.ucsc.edu/cgi-bin/hgBlat
The Human BLAT database was used
for alignment of nucleic sequences. The sequences retrieved from COSMIC were
inputted in the BLAT Database to be aligned with the rest of the nucleic sequence,
as only a small part of it could be gathered from COSMIC.
6. Catalogue
of Somatic Mutations in Cancer - COSMIC. (n.d.). Wellcome Trust Sanger
Institute. Retrieved July 10, 2012, from http://www.sanger.ac.uk/genetics/CGP/cosmic/
This database was used to retrieve
the nucleic sequences that were used as the base to be inputted in the BLAT
database. As it is impossible to use BLAT database with only knowing the
position of the mutation, COSMIC was used to retrieve the minimal part of the
sequence required to find the full nucleic sequence for any particular gene.
7. Genetics
of Endocrine and Neuroendocrine Neoplasias (PDQ®). (n.d.). National Cancer
Institute. Retrieved July 12, 2012, from http://www.cancer.gov/cancertopics/pdq/genetics/medullarythyroid/HealthProfessional/Table4
This database was only used for its
mutations list during the collection phase of the project.
8. OMIM
Entry - # 171400 - MULTIPLE ENDOCRINE NEOPLASIA, TYPE IIA; MEN2A . (n.d.). OMIM
- Online Mendelian Inheritance in Man . Retrieved July 19, 2012, from http://omim.org/entry/171400
This entry was used as the basis of
understanding of the MEN 2A syndrome. It talks about the relationship between
MTC and diseases such as pheochromocytoma, and about the basics of what MEN 2A
syndrome actually is. In addition, it talks about what mutations are associated
with the syndrome. It was specifically used for comparison with the MEN 2B and
FMTC.
9. Jimenez,
C., Hu, M. I., & Gagel, R. (2008, Spring). Management of Medullary Thyroid
Carcinoma. Elsevier Saunders, ?, 15.
This MTC review was the first of
many that I have read in the duration of this project. This review provided me
with the basic information about MTC without which any attempt at actually
finishing this project would have been obsolete. Many parts of the introduction
are referred to this paper, as it was very influential. Similarly to many other
sources, mutations were taken from this publication during the first phase of
the project.
10. MacConaill, L Profiling Critical Cancer Gene Mutations in Clinical
Tumor Samples. PLoS ONE (2009). http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007887
This publication was one of many
that were used only for its mutations. No part, except for the abstract, which
contributed to the overall idea of the research report, has been read.
11. Ehsan Alvandi, Seyed Mohammad Akrami, Mohsen
Chiani, Mehdi Hedayati, Babak Noori Nayer, Mohammad Reza Mohajeri Tehrani,et
al. (2011, April 5). Molecular Analysis of the RET Proto-Oncogene Key Exons in
Patients with Medullary Thyroid Carcinoma: A Comprehensive Study of the Iranian
Population. Thyroid, 1. Retrieved September 1, 2012, from http://online.liebertpub.com/doi/abs/10.10
This publication was one of many
that were used only for its mutations, and or one small piece of information.
No part, except for the abstract, which contributed to the overall idea of the
research report, has been read.
12.
Moura, M., Cavaco, B., Pinto, A., & Leite, V. (2011, February 16). High
Prevalence of RAS Mutations in RET-Negative Sporadic Medullary Thyroid
Carcinomas. JCEM ONLINE, 95, 6.
This paper has shown a study where
64% of the patients that had Sporadic MTC were found to have a BRAF mutation in
position 600. This is very unusual as this mutation is considered to be PTC
specific. In addition, this publication has given additional context to this
research report. Similarly to many other sources, mutations were taken from
this publication during the first phase of the project.
13. OMIM Entry - # 162300 - MULTIPLE ENDOCRINE
NEOPLASIA, TYPE IIB; MEN2B . (n.d.). OMIM - Online Mendelian Inheritance in Man
. Retrieved June 19, 2012, from http://omim.org/entry/162300
This entry was used as the basis of
understanding of the MEN 2B syndrome. It talks about the relationship between
MTC and diseases such as pheochromocytoma, and about the basics of what MEN 2B
syndrome actually is. In addition, this entry talks about what mutations are
associated with the syndrome. It was specifically used for comparison with the
MEN 2A and FMTC.
14. hybridization, f. i., & (1989), I. e.
(n.d.). OMIM Entry - + 164761 - REARRANGED DURING TRANSFECTION PROTOONCOGENE;
RET . OMIM - Online Mendelian Inheritance in Man . Retrieved July 19, 2012,
from http://omim.org/entry/164761
This entry was used as the basis of
understanding MTC, and why the mutations RET gene are found in so many cases of
MTC. Unfortunately, as with many other publications, no definitive answer was
given. This entry was also used as a guide, to make sure that what I say about
mutations in RET gene is factually correct.
15. RT-PCR., & (2003), M. e. (n.d.). OMIM
Entry - # 155240 - THYROID CARCINOMA, FAMILIAL MEDULLARY; MTC . OMIM - Online
Mendelian Inheritance in Man. Retrieved July 19, 2012, from http://omim.org/entry/155240
This entry was used as the basis of
understanding of the FMTC syndrome. It talks about the relationship between MTC
and diseases such as pheochromocytoma, and about the basics of what FMTC
syndrome actually is. In addition, this entry talks about what mutations are
associated with the syndrome. It was specifically used for comparison with the
MEN 2B and MEN 2A.
16. Hazard, J., Hawk, W., & Crile, G. (1959,
January 1). MEDULLARY (SOLID) CARCINOMA OF THE THYROID—A CLINICOPATHOLOGIC ENTITY.
JCEM, 19. Retrieved June 26, 2012, from http://jcem.endojournals.org/content/19/1/152
This publication was the first time
MTC was classified. The paper itself was not read by this applicant, however,
due to its historic relevance, it was referred to in the first paragraph of the
introduction.
17. Cakir, M., & Grossman, A. (2009, May 25).
Medullary Thyroid Cancer: Molecular Biology and Novel Molecular Therapies.
Neuro Endocrinology, 25.
This
publication, alongside many MTC reviews, was used as the guideline for this
research report. Many facts, such as information about Sporadic and Hereditary
MTC were confirmed by this publication. Similarly to many other sources,
mutations were taken from this publication during the first phase of the
project.
