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Laboratory Considerations and Logistics

Considerations for identifying NTRK gene fusions in your laboratory vary depending on the tumor type and testing methodology

  • Different approaches to biopsy yield varying sample volumes for diagnostic testing1,2
  • Deviations from required preanalytical steps prior to molecular pathology can affect results2
  • Biopsy sampling and preanalytical variables can affect detection quality of the different methodologies3
  • Average turnaround times vary by testing methodology2
  • Batching logistics vary by testing methodology4

NTRK, neurotrophic tyrosine receptor kinase.

References: 1. Halling KC, Wendel AJ. In situ hybridization: Principles and applications. In: Cagle PT, Allen TC, eds. Basic Concepts of Molecular Biology. New York, NY: Springer Science+Business Media; 2009:109-118. 2. Jennings LJ, Arcila ME, Corless C, et al. Guidelines for validation of next-generation sequencing-based oncology panels: a joint consensus recommendation of the Association for Molecular Pathology and College of American Pathologists. J Mol Diagn. 2017;191(3):343-365. 3. Wilson KD, Schrijver I. Transitioning diagnostic molecular pathology to the genomic era: cancer somatic mutation panel testing. In: Yousef GM, Jothy S, eds. Molecular Testing in Cancer. New York, NY: Springer Science+Business Media; 2014:3-13. 4. How turnaround time can become an issue in laboratory diagnostic testing. December 5, 2017. Diaceutics website. http://www.diaceutics.com/2017/12/05/turnaround-time-can-become-issue-laboratory-diagnostic-testing. Accessed February 23, 2019.

Different approaches to tissue acquisition are used across different tumor types

  • Different volumes of tumor samples are used to support the patient’s diagnostic workup1
  • Degree of biomarker testing associated with different cancer indications can vary significantly, together with volume of tumor sample used2
  • For tumor types which yield small samples, such as lung cancer, on-site evaluation of tissue at biopsy can help ensure the sample is adequate for testing1
Biopsy
Type
Amount
available
Amount used
for diagnosis
Tissue used for routine
biomarker tests*
Tissue remaining
for additional tests
Lung3,4 FNA 200 μm 38 μm 100 μm 62 μm
Colorectal Biopsy >1000 μm 38 μm 136 μm >826 μm
GBM5 Surgical 1600 μm 30 μm 122 μm 1448 μm
Cholangiocarcinoma6 FNA Surgical 200 μm 2600 μm 62 μm 62 μm 64 μm 64 μm 74 μm 2474 μm
Pancreatic FNA/FNB 200 μm 30 μm Not routine 170 μm
Head and Neck7 Surgical/FNA 600 μm ~50 μm 22 μm 528 μm
Melanoma8 Surgical >1000 μm ~50 μm 110 μm >840 μm
Sarcoma9 Biopsy 1500 μm ~50 μm 122 μm 1328 μm
Thyroid10 FNA 3-4 slides 3-4 slides Not routine None
Giloma5 Surgical 1600 μm 30 μm 122 μm 1448 μm
GIST11 Surgical 2600 μm 14 μm 42 μm 2544 μm
Breast/secretory breast12 CNB 1500 μm 42 μm 142 μm 1316 μm

*These volumes are based on best practice recommendations and will likely vary in real-world clinical practice.

Planning for
your practice

Tissue availability for NTRK gene fusion testing can vary significantly depending on tumor type.2 Thus, it is important to work together with your multidisciplinary team to ensure optimal sample availability for testing

Watch Dr Andrew Turk discuss the critical nature of a quality tissue sample for NTRK gene fusion testing

CNB, core needle biopsy; FNA, fine needle aspiration; FNB, fine needle biopsy; GBM, glioblastoma; GIST, gastrointestinal stromal tumor; NTRK, neurotrophic tyrosine receptor kinase.

References: 1. Han Y, Li J. Clin Chem Lab Med. 2017;55(12):1817-1833. 2. Jennings LJ, Arcila ME, Corless C, et al. Guidelines for validation of next-generation sequencing-based oncology panels: a joint consensus recommendation of the Association for Molecular Pathology and College of American Pathologists. J Mol Diagn. 2017;19(3):341-365. 3. Travis WD. Update on small cell carcinoma and its differentiation from squamous cell carcinoma and other non-small cell carcinomas. Mod Pathol. 2012;25:S18-S30. 4. Bergman RA, Afifi AK, Heidger PM. Blood. In: Bergman RA, ed. Anatomy Atlases: Atlas of Microscopic Anatomy. www.anatomyatlases.org/MicroscopicAnatomy/Section04/Section04.shtml. Accessed March 4, 2019. 5. Poca MA, Martínez-Ricarte FR, Gandara DF, et al. Target location after deep cerebral biopsies using low-volume air injection in 75 patients. Results and technical note. Acta Neurochir (Wien). 2017;159(10):1939-1946. 6. Hartman DJ, Slivka A, Giusto DA, Krasinskas AM. Tissue yield and diagnostic efficacy of fluoroscopic and cholangioscopic techniques to assess indeterminate biliary strictures. Clin Gastroenterol Hepatol. 2012;10:1042-1048. 7. Grégoire V, Lefebvre J-L, Licitra L, Felip E. Squamous cell carcinoma of the head and neck: EHNS-ESMO-ESTRO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2010;21 (suppl 5):v184-v186. 8. Dummer R, Hauschild A, Lindenblatt N, Pentheroudakis G, Keilhotz U; ESMO Guidelines Committee. Cutaneous melanoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2015;26 (suppl 5):v126-v132. 9. Casali PG, Bielack S, Abecassis N, et al. Bone sarcomas: ESMO-PaedCan-EUROCAN Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2010;21 (suppl 5): iv79-iv95. 10. Pacini F, Castagna MG, Brilli L, Pentheroudakis G; ESMO Guidelines Working Group. Thyroid cancer: ESMO clinical practice guidelines for diagnosis, treatment, and follow-up. Ann Oncol. 2012;23(suppl 7):vii110-vii119. 11. Rüschoff J, Hanna W, Bilous M, et al. HER2 testing in gastric cancer: a practical approach. Mod Pathol. 2012;25:637-650. 12. Lee AHS, Carder P, Deb R. Guidelines for non-operative diagnostic procedures and reporting in breast cancer screening. In: Lee AHS, ed. Pathology: the Science Behind the Cure. London, UK: The Royal College of Pathologists; June 2016. Document G150.

Preanalytical steps prior to molecular pathology may affect the quality of the sample result1,2

Preanalytical step Areas of requirement Possible consequence of error
Fixation
  • Preserves tissue
  • Prevents autolysis
  • Preserves nucleic acids
  • Artificial mutations may be created during fixation, resulting in false positives when a highly sensitive technique (such as NGS) is used
  • Decalcification of bone lesions for an excessive time can cause loss of nuclear staining and can macerate tissues, thus impacting downstream test quality
  • Failure of test due to inappropriate fixation
Specimen handling
  • Minimum 3-4 identifiers to match both request form and specimen
  • Legible and accurate forms
  • Delay in TAT—missed window for treatment
  • Sample error, patient does not receive correct treatment at the right time
Tissue processing
  • To ensure all endogenous water is removed from tissue, ensuring good tissue morphology and preservation of antigen and DNA
  • DNA quality affected due to fragmentation of DNA with tissue processing
Embedding
  • Specimens must be oriented correctly at embedding to ensure section includes epidermis to subcutis; this ensures pathologist can stage disease
  • Incorrect embedding of biopsy can lead to lesion being lost, inability to stage disease correctly, or inappropriate diagnosis2

FFPE samples are the standard in molecular pathology testing1-3

Preanalytical step FFPE tissue samples Fresh-frozen tissues
Processing
  • Commonly by fixation in cold 10% NBF for the shortest possible times:
    • 6-12 h for small biopsies
    • 8-18 h for large surgical resections
  • Embedded in optimal cutting temperature compound, frozen rapidly at −80°C, or immersed in liquid nitrogen (−190°C)
  • Cut into serial frozen sections at −15°C to −25°C, using a cryostat for histological and molecular diagnoses
Advantages
  • FFPE samples provide commendable cellular morphology and preserve the entire tumor structure for assessment of tumor cellularity
  • Simple long-term storage: Can be kept in archives at room temperature for years
  • Optimal for providing high-quality DNA or RNA
  • More conducive to analysis of long DNA segments (>1000 bp)
Disadvantages
  • DNA cross-linking can impact nucleic acid isolation, making it more challenging
  • DNA fragmentation restricts the analysis of long DNA segments:
    • Sequencing of DNA segments between 300 and 1000 bp succeeds inconsistently
    • Tiling of contiguous regions to accurately identify breakpoints with sufficient coverage can be challenging
  • Cytosine deamination can result in the generation of random sequence artifacts, which can potentially lead to false positives using highly sensitive techniques
  • Additional pre-extraction handling procedures are required to ensure robust DNA/RNA template quality
  • Fast sample processing and staining may lead to ambiguity in cell morphology (tumor vs normal tissue)
  • Highly controlled storage conditions:
    • Fresh surgical samples need to be kept in liquid nitrogen
    • Sections that are mounted on glass slides are required to be kept at −80°C until further use

Planning for
your practice

While fresh-frozen samples provide a higher-quality template, sample handling difficulties limit routine use in a real-world laboratory setting1

bp, base pairs; DNA, deoxyribonucleic acid; FFPE, formalin-fixed paraffin-embedded; NBF, neutral buffered formalin; NGS, next-generation sequencing; RNA, ribonucleic acid; TAT, turnaround time.

References: 1. Han Y, Li J. Clin Chem Lab Med. 2017;55(12):1817-1833. 2. Rolls G. Steps to better processing and embedding. Leica Biosystems. https://www.leicabiosystems.com/pathologyleaders/steps-to-better-processing-and-embedding/. Accessed March 3, 2019. 3. Frampton GM, Fichtenholtz A, Otto GA, et al. Development and validation of a clinical cancer genomic profiling test based on massively parallel DNA sequencing. Nat Biotechnol. 2013;31(11):1023-1031.

Biopsy sampling and preanalytical variables affect detection quality of the different methodologies1

NGS

  • RNA is a very fragile molecule and is easily degraded2
  • Fixation may result in fragmented DNA/RNA template and this may impact the accurate detection of breakpoint with sufficient coverage2
  • Multiple biomarker results can be detected from a single sample3,4

IHC

  • Variability in fixation (ie, time of fixation and fixative type) can significantly impact quality of results:
    • Incomplete fixation can produce heterogeneous staining5
    • Prolonged fixation can result in loss of immunoreactivity5

FISH

  • Volume of material required is linked to number of probes and reactions required
    • Limited to the clinical assessment of a small number of oncogenic markers6

RT-PCR

  • Samples have to be appropriately processed to ensure high-quality testing:
    • PCR inhibitors that may exist within the biological sample can impact test quality7
    • DNA or RNA contamination could result in false positives7
    • Fixation may result in fragmented template5

Planning for
your practice

Samples should be carefully reviewed by pathologists to identify tumor-rich areas and ensure biopsy sample contains high tumor content that can support all methodologies1

DNA, deoxyribonucleic acid; FISH, fluorescence in situ hybridization; IHC, immunohistochemistry; NGS, next-generation sequencing; RNA, ribonucleic acid; RT-PCR, reverse-transcription polymerase chain reaction.

References: 1. Han Y, Li J. Clin Chem Lab Med. 2017;55(12):1817-1833. 2. Meyerson M, Gabriel S, Getz G. Advances in understanding cancer genomes through second-generation sequencing. Nat Rev Genet. 2010;11(10):685-696. 3. Serrati S, De Summa S, Pilato B, et al. Next-generation sequencing: advances and applications in cancer diagnosis. Onco Targets Ther. 2016;9:7355-7365. 4. Jennings LJ, Arcila ME, Corless C, et al. Guidelines for validation of next-generation sequencing-based oncology panels: a joint consensus recommendation of the Association for Molecular Pathology and College of American Pathologists. J Mol Diagn. 2017;19(3):341-365. 5. Fitzgibbons PL, Cooper K. Immunohistochemistry of biomarkers. In Cagle PT, Allen TC, eds. Basic Concepts of Molecular Pathology. New York, NY: Springer Science+Business Media; 2009:133-137. 6. Church AJ, Calicchio ML, Nardi V, et al. Recurrent EML4-NTRK3 fusions in infantile fibrosarcoma and congenital mesoblastic nephroma suggest a revised testing strategy. Mod Pathol. 2018;31(3):463-473. 7. Olsen JL. Polymerase chain reaction. In: Vohr HW, ed. Encyclopedia of Immunotoxicology. Berlin, Germany; Springer Verlag 2016:715-720.

Average turnaround times* vary by methodology used1

Laboratory TATs associated with common gene fusion methodologies

*This may be due to individual batching considerations within the laboratory.

  • When comparing average TAT associated with different methodologies, NGS typically takes the longest time6
    • This is largely due to the complexities associated with the test set up and bioinformatics analysis6
  • IHC, FISH, and RT-PCR have faster turnaround times, reflecting their incorporation within established laboratory workflows supporting time efficiencies6

Recognizing potential issues that can affect testing turnaround time

Sequential reflex testing

  • May be required or necessary to confirm NTRK gene fusion–positive results determined by one method (ie, FISH/IHC) that requires another molecular method (ie, NGS)7

Sample handling and preanalytical errors

  • General sample shipping and storage requirements may impact transit time8
  • Inappropriate handling of primary samples may result in suboptimal DNA quality, thus requiring repeat testing, which will extend TAT6,8

Sample batching

  • Labs may test once or twice a week. This enables a cost-efficient service but results in delays of TAT (eg, waiting until you have enough samples to build a library to optimize cost/run because reagents are usually one-time use only)9

Methodology

  • Testing method used will dictate turnaround time (ie, TAT for IHC is generally shorter than FISH, IHC/FISH shorter than NGS, etc)2,3,5,10

DNA-/RNA-based NGS

  • Factors specific to NGS—such as DNA/RNA extraction, library prep, sequencing time, data analysis/annotation, and reporting—all contribute to relatively longer NGS TATs, which can range from 10-30 days11

Planning for
your practice

Although NGS typically has a longer TAT, its detection capabilities simultaneously analyze multiple targets, allowing for the detection of more genomic alterations12-15

Watch Drs Michelle Shiller and Erin Rudzinski explore key considerations to optimize turnaround time in NTRK gene fusion testing

Test Your Knowledge

True or false.
Testing turnaround time is limited only by the detection method (ie, whether a tumor sample is tested by NGS, IHC, FISH, or RT-PCR)?
True
False

DNA, deoxyribonucleic acid; FISH, fluorescence in situ hybridization; IHC, immunohistochemistry; NGS, next-generation sequencing; NTRK, neurotrophic tyrosine receptor kinase; RNA, ribonucleic acid; RT-PCR, reverse-transcription polymerase chain reaction; TAT, turnaround time.

References: 1. How turnaround time can become an issue in laboratory diagnostic testing. December 5, 2017. Diaceutics website. http://www.diaceutics.com/2017/12/05/turnaround-time-can-become-issue-laboratory-diagnostic-testing. Accessed February 23, 2019. 2. Rudzinski ER, Lockwood CM, Stohr BA, et al. Pan-Trk immunohistochemistry identifies NTRK rearrangements in pediatric mesenchymal tumors. Am J Surg Pathol. 2018;42(7):927-935. 3. Indiana University Department of Medical and Molecular Genetics. Specimen criteria for clients. http://geneticslab.medicine.iu.edu/Files/Specimen%20Criteria%20for%20Clients%20V07182018.pdf. Accessed March 1, 2019. 4. Real time PCR FAQs. Source BioScience website. https://www.sourcebioscience.com/services/genomics/frequently-asked-questions/real-time-pcr-faqs. Accessed March 1, 2019. 5. Devarakonda S. Expert highlights benefits of next-generation sequencing for NSCLC. http://www.targetedonc.com/news/expert-highlights-benefits-of-nextgeneration-sequencing-for-nsclc. Accessed March 1, 2019. 6. Wilson KD, Schrijver I. Transitioning diagnostic molecular pathology to the genomic era: cancer somatic mutation panel testing. In: Yousef GM, Jothy S, eds. Molecular Testing in Cancer. New York, NY: Springer Science+Business Media; 2014:3-13. 7. Farago AF, Taylor MS, Doebele RC, et al. Clinicopathologic features of non–small-cell lung cancer harbouring an NTRK gene fusion. JCO Precis Oncol. 2018. 8. Han Y, Li J. Clin Chem Lab Med. 2017;55(12):1817-1833. 9. Batch vs continuous flow specimen processing. Lab CE website. https://www.labce.com/spg1837894_batch_versus_continuous_flow_specimen_processing.aspx. Accessed February 26, 2019. 10. Goodwin S, McPherson JD, McCombie WR. Coming of age: ten years of next-generation sequencing technologies. Nat Rev Genet. 2016;17(6):333-351. 11. Jennings LJ, Arcila ME, Corless C, et al. Guidelines for validation of next-generation sequencing-based oncology panels: a joint consensus recommendation of the Association for Molecular Pathology and College of American Pathologists. J Mol Diagn. 2017;191(3):341-365. 12. Serrati S, De Summa S, Pilato B, et al. Next-generation sequencing: advances and applications in cancer diagnosis. Onco Targets Ther. 2016;9:7355-7365. 13. Kummar S, Lassen UN. Target Oncol. 2018:13(5):545-556. 14. Jang JS, Wang X, Vedell PT, et al. Custom gene capture and next-generation sequencing to resolve discordant ALK status by FISH and IHC in lung adenocarcinoma. J Thorac Oncol. 2016;11(11):1891-1900. 15. Gounder MM, Ali SM, Robinson V, et al. Impact of next-generation sequencing (NGS) on diagnostic and therapeutic options in soft-tissue and bone sarcoma. J Clin Oncol. 2017;35(suppl). Abstract 11001.

Laboratory considerations associated with common methodologies, which aid the implementation of efficient workflows

  • Key drivers for batching are:
    • Cost efficiency1
    • Provision of high-quality routine clinical services using limited resources1
NGS IHC FISH RT-PCR
Estimated cost per sample $$$2 $3 $$3,4 $5
Complexity Complex2 Simple3 Simple-complex5* Simple6
Estimated sample numbers for cost efficiency Large2 Small3 Varies4* Small5
Common batching practice Runs are commonly performed once sufficient samples have been received2 Runs are commonly performed daily3 Runs are commonly performed on dedicated days or once sufficient samples have been received4 Runs are commonly performed on dedicated days or in an ad-hoc manner5

*Depends on number of reactions and fusions to detect.5

FISH, fluorescence in situ hybridization; IHC, immunohistochemistry; NGS, next-generation sequencing; RT-PCR, reverse-transcription polymerase chain reaction.

References: 1. Next Generation Sequencing Implementation Guide. Silver Spring, MD: Association of Public Health Laboratories; 2016. 2. Serratì S, De Summa S, Pilato B, et al. Next-generation sequencing: advances and applications in cancer diagnosis. Onco Targets Ther. 2016;9:7355-7365. 3. Chen ZE, Lin F. Overview of predictive biomarkers and integration of IHC into molecular pathology. In: Lin F, Prichard J, eds. Handbook of Practical Immunohistochemistry: Frequently Asked Questions. New York, NY: Springer Science+Business Media; 2015:91-104. 4. Hu L, Ru K, Zhang L, et al. Fluorescence in situ hybridization (FISH): an increasingly demanded tool for biomarker research and personalized medicine. Biomark Res. 2014;2(1):1-13. 5. Darawi MN, Ai-Vyrn C, Ramasamy K, et al. Allele-specific polymerase chain reaction for the detection of Alzheimer’s disease-related single nucleotide polymorphisms. BMC Med Genet. 2013;14:27. 6. Peter M, Gilbert E, Delattre O. A multiplex real-time PCR assay for the detection of gene fusions observed in solid tumors. Lab Invest. 2001;81(6):905-912.