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“We are witnessing a revolution in the way we practice medicine—from what we understand about pathogenesis to how we predict, diagnose, and treat diseases. The challenge is to find that right balance between the molecular information we can collect against the information that is most beneficial to our individual patients.” –Jon Heusel, MD, PhD, Molecular Pathologist. This is the guiding principle that drives new test development in the Molecular Pathology Laboratory.
Along with the recent addition of Dr. Deqin Ma to the Molecular Pathology (and Surgical Pathology) faculty, Molecular Pathology has also hired a full-time Research Scientist, Natalya Guseva, PhD, to provide assistance in molecular test development.
Dr. Guseva has over 20 years experience in molecular oncology and immunology research, and has been instrumental in the validations of many molecular tests that have arrived on the Molecular Pathology testing menu over the last year. The Molecular Pathology Laboratory Director, Dr. Aaron Bossler, credits the entire laboratory staff led by Jon Pruessner, Lead Scientist, with a ‘unified, focused effort to expand our testing while maintaining a very high degree of daily clinical testing service. These people are incredibly dedicated.’
The Molecular Pathology Laboratory has emphasized expansion of testing for targeted mutations in oncology (e.g., BRAF and KRAS mutation detection in colorectal cancer, papillary thyroid cancer and melanoma, quantitative BCR-ABL transcript detection for CML, CEBPA sequencing in AML, KIT sequencing for AML, melanoma and GIST, and NRAS mutation detection in melanoma) in part through ongoing dialogue with the Holden Comprehensive Cancer Center clinicians and investigators. The idea is to provide the tests that University of Iowa Hospitals and Clinics and regional Iowa physicians need most, and to assist in directing reference laboratory testing for those tests that are not provided on the current Molecular Pathology testing menu.
The lab also develops new tests in the areas of genetics and infectious disease, such as ISPD and DYSF gene sequencing for patients with suspected inherited neuromuscular disease, and a qualitative PCR test for hepatitis E virus (HEV). Details about these and other molecular tests now available are provided below.
Lung Cancer, ALK FISH
The Vysis ALK Break Apart FISH is a qualitative test to detect rearrangements involving the ALK gene via fluorescence in situ hybridization (FISH) in non-small cell lung cancer tissue specimens to aid in identifying those patients eligible for treatment with XALKORI® (crizotinib).
BCR/ABL1 (T(9;22)) RNA Quantitative with Interpretation, Blood
The BCR/ABL gene rearrangement is observed in CML, ALL, and, rarely, AML. A positive result indicates the presence of the Philadelphia chromosome, but the diagnosis of CML or ALL should be based on the presence of characteristic cellular abnormalities in bone marrow. In patients with ALL, the BCR/ABL rearrangement is associated with poor prognosis. Serial monitoring of assay values may provide a quantitative measure of tumor burden and response to therapy. Increasing levels of BCR/ABL are associated with clinical progression. Testing is performed on RNA from peripheral blood or bone marrow specimens using real time PCR. Results are calibrated and reported relative to the WHO International Standard as the percent international standard or %IS.
CEBPA Full Gene Sequence with Interpretation, Blood
Somatic mutations of CEBPA are among the most common in acute myelogenous leukemia ranging from 5-14% of cases particularly in the former FAB M1 and M2 subtypes. Along with having a normal karyotype, the majority of CEBPA-mutated cases have two distinct CEPBA mutations affecting different functional domains of the protein. In the absence of other unfavorable mutations that mitigate CEBPA double-mutations, there is an associated favorable prognosis as measured by overall and disease-free survival.
Dysferlin (DYSF) Full Gene Sequence with Interpretation
Mutations in the dysferlin gene (DYSF) on chromosome 2p13, are at the origin of dysferlinopathies, a heterogeneous group of rare autosomal recessive inherited neuromuscular disorders. The main clinical presentations are the distal-onset muscular dystrophy called Miyoshi myopathy and the proximal-onset form LGMD2B, both characterized by progressive muscle weakness, usually appearing in the second decade, and highly elevated serum creatine kinase (CK) levels. Mutational analysis of DYSF is complicated by the large mutational spectrum and a high proportion of "private" mutations, with the recurrent difficulty of interpreting novel DYSF sequence variants, in particular putative splicing and missense variants.
The 55 coding exons and the flanking intronic regions of the DYSF gene are amplified by PCR and sequenced in both directions. The reference sequence with which patient sequences are compared is NM_003494.3 (in some case with alternative exons HGVS recommended).
Epstein Barr Virus (EBV) Quantitative PCR
This test is intended for use in conjunction with clinical presentation and other laboratory markers as an indication of an EBV-related tumor. This test is also used as an aid in accessing viral response to treatment as measured by changes in EBV DNA levels. Testing is performed using real time PCR. Results are calibrated to the WHO International Standard and will be reported in international units (IU)/ml and the log transformed values. EBV PCR should not be used to diagnose or confirm primary or reactivated mononucleosis; serology should be requested for those situations.
Hepatitis E Virus PCR
Hepatitis E virus (HEV) causes sporadic and epidemic forms of acute hepatitis. It is responsible for waterborne hepatitis epidemics in developing countries, while in the United States, it is usually diagnosed in recent travelers to endemic areas (India, Asia, Africa, and Central America). It can infect animals, and pigs are an important reservoir. One clinical concern is the development of chronic HEV infection in solid organ transplant patients. Hepatitis E IgG antibodies can be detected particularly during the acute phase, but in most patients IgG antibodies are short lived. Molecular testing for the viral RNA may be used if there is concern for chronic HEV infection.
KIT (GIST) Targeted Gene Analysis Exons 9, 11, 13, 17 with Interpretation
The principle use of this test is to detect mutations in patients with gastrointestinal stromal tumor (GIST) and melanoma. For acute myeloid leukemia (AML) testing, please order KITAML Mutation Analysis (LAB7659). This test is not intended to detect D816 mutation in bone marrow for mast cell disease or minimal residual disease, see KITMAST Mast Cell Disease (LAB7567).
KIT (AML) Targeted Gene Analysis Exons 8, 17 with Interpretation
KIT (also known as c-KIT) belongs to a family of type III receptor tyrosine kinases. Activating KIT mutations are seen in 70-80% of gastrointestinal stromal tumors (GISTs), ~95% mastocytosis, 25% germ cell tumor, 2% melanoma and a subset of acute myeloid leukemia (AML). The location and type of KIT mutations are associated with prognosis and response to imatinib therapy.
Testing of KIT mutations can be utilized:
1. To assist in diagnosis of GIST, melanoma and AML.
2. To assist in therapy selection for GIST and melanoma.
3. To assist in prognosis of GIST and AML.
KIT AML Mutation Analysis detected exons related to patients with AML. If patient has GIST or melanoma, order KIT Mutation Analysis (LAB7660). This is a sequence-based assay. This test is not intended to detect D816 mutation in bone marrow for mast cell disease or minimal residual disease (see KIT Mutation (D816V) Mast Cell Disease (LAB7567).
KIT Mutation (D816V) MAST Cell Disease
Activating point mutations at codon 816 in the KIT receptor tyrosine kinase are seen in the majority of patients with systemic mast cell disease and can help in diagnosis of this disorder.
ISPD Full Gene Sequence with Interpretation
Mutations in the isoprenoid synthase domain containing (ISPD) gene (HGNC 37276) have been shown recently to cause WalkerWarburg syndrome (WWS). WWS is characterized by congenital muscular dystrophy, hydrocephalus, agyria, retinal dysplasia, with or without encephalocele.
MatBA®- CLLArray- CGH Assay
Loss of specific chromosomal loci is associated with prognostic information for CLL patients. MatBa-CLL Array-CGH Assay provides for a more comprehensive panel of chromosomal loci information not available in a single test.
NRAS Mutation Analysis with Interpretation
The presence of an oncogenic mutation in codons 12, 13, or 61 of NRAS is indicative of a tumor that may respond to drugs targeted at genes downstream of NRAS in the mitogen activating protein kinase (MAPK) signaling cascade, as in malignant melanoma cases. In contrast, mutations in NRAS can inhibit therapeutic response to EGFR-targeted therapies in patients with metastatic colorectal cancer. NRAS mutations are found in the more aggressive variant of chronic myelogenous leukemia (CMML) and may predict response to farnesyl transferase inhibitors therapy in acute myeloid leukemia (AML).