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Test Code TFH FH Mutation Analysis, Next-Generation Sequencing, Tumor


Ordering Guidance


Multiple oncology (cancer) gene panels are available. For more information see Hematology, Oncology, and Hereditary Test Selection Guide.



Necessary Information


A pathology report (final or preliminary), at minimum containing the following information, must accompany specimen for testing to be performed:

1. Patient name

2. Block number-must be on all blocks, slides, and paperwork (can be handwritten on the paperwork)

3. Tissue collection date

4. Source of the tissue



Specimen Required


This assay requires at least 20% tumor nuclei.

-Preferred amount of tumor area with sufficient percent tumor nuclei: tissue 216 mm(2)
-Minimum amount of tumor area: tissue 36 mm(2)
-These amounts are cumulative over up to 10 unstained slides and must have adequate percent tumor nuclei.
-Tissue fixation: 10% neutral buffered formalin, not decalcified
-For specimen preparation guidance, see Tissue Requirement for Solid Tumor Next-Generation Sequencing. In this document, the sizes are given as 4 mm x 4 mm x 10 slides as preferred: approximate/equivalent to 144 mm(2) and the minimum as 3 mm x 1 mm x 10 slides: approximate/equivalent to 36 mm(2).

 

Preferred:

Specimen Type: Tissue block

Collection Instructions: Submit a formalin-fixed, paraffin-embedded tissue block with acceptable amount of tumor tissue.

 

Acceptable:

Specimen Type: Tissue slide

Slides: 1 Stained and 10 unstained

Collection Instructions: Submit 1 slide stained with hematoxylin and eosin and 10 unstained, nonbaked slides with 5-micron thick sections of the tumor tissue.

Note: The total amount of required tumor nuclei can be obtained by scraping up to 10 slides from the same block.

Additional Information: Unused unstained slides will not be returned.

 

Specimen Type: Cytology slide (direct smears or ThinPrep)

Slides: 1 to 3 Slides

Collection Instructions: Submit 1 to 3 slides stained and coverslipped with a preferred total of 5000 nucleated cells or a minimum of at least 3000 nucleated cells.

Note: Glass coverslips are preferred; plastic coverslips are acceptable but will result in longer turnaround times.

Additional Information: Cytology slides will not be returned.


Secondary ID

619658

Useful For

Identifying specific mutations within the FH gene to assist in tumor diagnosis/classification, including renal cell carcinoma, uterine/cutaneous leiomyoma, and pheochromocytoma/paraganglioma

Additional Tests

Test ID Reporting Name Available Separately Always Performed
SLIRV Slide Review in MG No, (Bill Only) Yes

Testing Algorithm

When this test is ordered, slide review will always be performed at an additional charge.

Method Name

Sequence Capture Next-Generation Sequencing (NGS)

Reporting Name

FH Mutation Analysis, Tumor

Specimen Type

Varies

Specimen Minimum Volume

See Specimen Required

Specimen Stability Information

Specimen Type Temperature Time Special Container
Varies Ambient (preferred)
  Refrigerated 

Reject Due To

Specimens that have been decalcified (all methods)
Specimens that have not been formalin-fixed, paraffin-embedded, except for cytology slides
Extracted nucleic acid (DNA/RNA)		 Reject

Clinical Information

Disease-causing alterations of the fumarate hydratase (FH) gene have been implicated in renal cell carcinoma, uterine/cutaneous leiomyoma, and pheochromocytoma/paraganglioma. While these tumors can occur in the sporadic setting, germline alterations of the FH gene have been associated with renal cell carcinoma, and uterine/cutaneous leiomyoma in hereditary leiomyomatosis and renal cell carcinoma (HLRCC) syndrome.

 

The 5th edition of the World Health Organization Classification of Tumours recognizes fumarate hydratase-deficient renal cell carcinoma as a molecularly defined entity.(1) This single gene assay, performed using formalin-fixed paraffin-embedded tissue or cytology material, is therefore helpful in documenting an underlying disease-causing alteration of the FH gene and is diagnostically significant. It is important to note that this assay does not distinguish between germline and somatic alterations.

Reference Values

An interpretive report will be provided.

Interpretation

The interpretation of molecular biomarker analysis includes an overview of the results and the associated diagnostic, prognostic, and therapeutic implications.

Cautions

This test cannot differentiate between somatic and germline alterations. Additional testing may be necessary to clarify the significance of results if there is a potential hereditary risk.

 

DNA variants of uncertain significance may be identified.

 

A negative result does not rule out the presence of a variant that may be present below the limits of detection of this assay. In a specimen with 20% or more tumor content, the analytical sensitivity of this assay for sequence reportable alterations is 5% mutant allele frequency with a minimum coverage of 500X.

 

Point mutations and small deletion-insertion mutations will be detected in FH gene only. This test may detect single exon deletions but does not detect multi-exon deletions, duplications, or genomic copy number variants.

 

Variant allele frequency (VAF) is the percentage of sequencing reads supporting a specific variant divided by the total sequencing reads at that position. In somatic testing, VAF should be interpreted in the context of several factors, including, but not limited to, tumor purity/heterogeneity/copy number status (ploidy, gains/losses, loss of heterozygosity) and sequencing artifact/misalignment.(2,3)

 

Rare alterations (ie, polymorphisms) may be present that could lead to false-negative or false-positive results.

 

Test results should be interpreted in the context of clinical, tumor sampling, histopathological, and other laboratory data. If results obtained do not match other clinical or laboratory findings, contact the laboratory for discussion. Misinterpretation of results may occur if the information provided is inaccurate or incomplete.

 

Reliable results are dependent on adequate specimen collection and processing. This test has been validated on cytology slides and formalin-fixed, paraffin-embedded tissues; other types of fixatives are discouraged. Improper treatment of tissues, such as decalcification, may cause polymerase chain reaction failure.

Supportive Data

Performance Characteristics:

The limit of detection for calling a somatic variant (single nucleotide variants [SNV] and deletions-insertions [delins, formerly indel]) is 5% variant allele frequency (VAF) and having at least 500x deduplicated coverage.

 

Verification studies demonstrated concordance between this test and the reference method for detection of SNV and delins is 98.5% (673/683) and 98.4% (122/124) of variants, respectively. Concordance for the detection of delins was 99.0% (100/101) in variants 1 to 10 base pairs (bp) in size, 93.3% (14/15) in variants 11 to 50 bp in size, and 100% (8/8) in variants over 50 bp in size.

 

To ensure accuracy, this test will be performed on cases that are estimated by a pathologist to have at least 20% tumor cells.

Clinical Reference

1. WHO Classification of Tumours Editorial Board, eds. Urinary and male genital tumours. 5th ed. World Health Organization; 2022. WHO Classification of Tumours. Vol 8

2. Strom SP. Current practices and guidelines for clinical next-generation sequencing oncology testing. Cancer Biol Med. 2016;13(1):3-11. doi:10.28092/j.issn.2095-3941.2016.0004

3. Spurr L, Li M, Alomran N, et al. Systematic pan-cancer analysis of somatic allele frequency. Sci Rep. 2018;8(1):7735. Published 2018 May 16. doi:10.1038/s41598-018-25462-0

4. Trpkov K, Hes O, Williamson SR, et al: New developments in existing WHO entities and evolving molecular concepts: The Genitourinary Pathology Society (GUPS) update on renal neoplasia. Mod Pathol. 2021;34(7):1392-1424

5. Forde C, Lim DHK, Alwan Y, et al: Hereditary leiomyomatosis and renal cell cancer: Clinical, molecular, and screening features in a cohort of 185 affected individuals. Eur Urol Oncol. 2020;3(6):764-772

6. Gupta S, Swanson AA, Chen YB, et al. Incidence of succinate dehydrogenase and fumarate hydratase-deficient renal cell carcinoma based on immunohistochemical screening with SDHA/SDHB and FH/2SC. Hum Pathol. 2019;91:114-122

7. Carlo MI, Hakimi AA, Stewart GD, et al: Familial kidney cancer: Implications of new syndromes and molecular insights. Eur Urol. 2019;76(6):754-764

8. Nguyen KA, Syed JS, Espenschied CR, et al: Advances in the diagnosis of hereditary kidney cancer: Initial results of a multigene panel test. Cancer. 2017;123(22):4363-4371

Method Description

Next-generation sequencing is performed to evaluate the presence of a mutation in all coding regions of the FH gene. See Targeted Genes and Methodology Details for FH Mutation Analysis for details regarding the targeted gene regions identified by this test.(Unpublished Mayo method)

 

A pathology review and macro dissection to enrich for tumor cells is performed prior to slide scraping.

Day(s) Performed

Monday through Friday

Report Available

12 to 20 days

Specimen Retention Time

FFPE tissue block: Unused portions of blocks will be returned within 10 to -14 days after testing is complete; FFPE tissue/cytology slides: Unused slides are stored indefinitely; Digital images are obtained and stored for all slides used in testing.

Performing Laboratory

Mayo Clinic Laboratories in Rochester

Test Classification

This test was developed and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. It has not been cleared or approved by the US Food and Drug Administration.

CPT Code Information

88381-Microdissection, manual

81405

LOINC Code Information

Test ID Test Order Name Order LOINC Value
TFH FH Mutation Analysis, Tumor 49872-5

 

Result ID Test Result Name Result LOINC Value
619659 Result 82939-0
619660 Interpretation 69047-9
619661 Additional Information 48767-8
619662 Specimen 31208-2
619663 Tissue ID 80398-1
619664 Method 85069-3
619665 Disclaimer 62364-5
619666 Released By 18771-6

Forms

If not ordering electronically, complete, print, and send an Oncology Test Request (T729) with the specimen.