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Characterization of Endoscopic Ultrasound Fine-Needle Aspiration Cytology by Targeted Next-Generation Sequencing and Theranostic Potential
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The Molecular Characterization and Theranostic Potential of Endoscopic Ultrasound Fine-Needle Aspiration Malignant Cytology Using Targeted Next-Generation Sequencing Ferga C. Gleeson,* Benjamin R. Kipp,‡ Sarah E. Kerr,‡ Jesse Voss,‡ Konstantinos N. Lazaridis,*,§ David A. Katzka,* and Michael J. Levy* *Division of Gastroenterology and Hepatology, ‡Department of Laboratory Medicine and Pathology, §Center for Individualized Medicine, Mayo Clinic, Rochester, Minnesota
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Determination of tumor genetic architecture based on tissue analysis yields important information on signaling pathways involved in cancer pathogenesis and plays a growing role in choosing the optimal medical management of malignancies. Specifically, the advent of next-generation sequencing has led to a rapidly evolving era of relatively inexpensive, high-throughput DNA sequencing of tumors. One such example is multiplexed tumor genotyping (ie, panel testing) of more than 2800 mutations across 50 commonly mutated cancer-associated genes. This resulting mutational landscape shows medically actionable pathogenic alterations to optimize antitumor therapy. We recently assessed the performance and outcome of targeted next-generation sequencing with archived endoscopic ultrasound fine-needle aspirates across a broad range of primary and metastatic sites with encouraging accuracy. As a result, endoscopic ultrasound has the potential to move from a test for diagnosis or confirmation of malignancy, to one in which it could facilitate the personalization of cancer-directed therapy. Keywords: Endoscopic Ultrasound Fine-Needle Aspiration; Malignant Cytology; Targeted Next-Generation Sequencing; Theranostics; Individualized Medicine.
he treatment decision model in oncology practices is undergoing a paradigm shift with increasing emphasis to customize potential therapy not just by histology alone but also to incorporate tumor molecular characteristics that represent the biological pathways driving tumorigenesis. Consequently, there is an increasing need for routine comprehensive DNA clinical testing for selected tumors to aid therapy triage for an individual patient, a practice often referred to as theranostics or personalized medicine. Formalin-fixed, paraffinembedded specimens are currently the specimen of choice for tumor genetic profiling. However, ancillary testing of endoscopic ultrasound fine-needle aspiration (EUS-FNA) samples is becoming more widely performed in clinical practice. Our objectives were to identify key cytology specimen selection criteria suitable for next-generation sequencing (NGS) and to determine the prevalence and spectrum of
T
pathogenic alterations with theranostic intent. We chose a cohort of patients with primary gastric gastrointestinal tumors (GISTs), malignant lymph nodes from locally advanced primary rectal cancer, and adrenal gland metastasis from treatment-naive lung cancer.1,2
Technological Primer Targeted NGS enables sequencing of specific segments of the genome to detect pathogenic point mutations and small insertions/deletions at reduced cost and turnaround time, with enhanced sensitivity when compared with whole-exome or whole-genome sequencing. Sequencing of cytology samples has long been sought because of the theoretical advantage of containing a more pure tumor cell population in contrast to matrix-rich histologic specimens. Furthermore, cytology specimen processing has superior preservation of nucleic acids given the use of alcohol-based fixation and direct smearing when compared with formalin fixation of histology specimens.3–5
Deep Sequencing of Multiplex Polymerase Chain Reaction Amplicons After cytology slide selection and DNA extraction, multiplex polymerase chain reaction (PCR) was performed by amplifying DNA with the Ion Ampliseq Cancer Hotspot Panel v2 and the Ion Ampliseq Library Kit 2.0 (Life Technologies, Carlsbad, CA). This Ampliseq kit targets 2855 possible mutations within 50 cancerassociated genes, including the following: ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, Abbreviations used in this paper: EGFR, epidermal growth factor receptor; EUS FNA, endoscopic ultrasound fine-needle aspiration; GIST, gastrointestinal stromal tumor; NGS, next-generation sequencing; PCR, polymerase chain reaction. © 2014 by the AGA Institute 1542-3565/$36.00 http://dx.doi.org/10.1016/j.cgh.2014.10.017
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FGFR3, FLT3, GNAQ, GNAS, GNA11, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, and VHL. The PCR product underwent library preparation using the TruSeq Nano DNA Sample Preparation Kit (Illumina, San Diego, CA).
Data Analysis Identification of a cell variant was made by an internal bioinformatics pipeline and alignments were reviewed manually by a molecular geneticist to verify whether variants were pathogenic or not (Figure 1). Each variant was reviewed using multiple public databases (Catalogue of Somatic Mutations in Cancer) and software programs (eg, Alamut). Patients with variants with an allele frequency of more than 5% were flagged for review. Variants with minor allele frequencies greater than 1% or silent changes without evidence of pathogenicity were considered common single-nucleotide polymorphism and were not reported. Remaining variants were flagged as pathogenic if the alteration was listed in the Catalogue of Somatic Mutations in Cancer and/or there were functional data suggesting pathogenicity. Remaining variants were classified as variants of unknown significance.
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Findings Cytology Sample Slide Characteristics for Successful Next-Generation Sequencing The selected criteria for optimal NGS sequencing were obtained from archived adrenal metastasis cytology slides. Slide cellularity was triaged by a molecular pathologist as low (300–1000 cells), moderate (1000–5000 cells), or high (>5000 cells). The degree of necrosis was categorized subjectively as low, moderate, or high (>50% of tumor cells). Slides with low, moderate, and high cellularity underwent successful NGS 22%, 25%, and 91% of the time. Slides yielding less than 5 ng/mL or more than 5 ng/mL of DNA were NGS successful in 4% and = Figure 1. Cytology slide sample workflow. Cytology slides were evaluated and triaged by a cytopathologist for tumor cell percentage and DNA extraction eligibility. Selected slides were imaged for photographic archives within the cytopathology laboratory. After DNA extraction, and assessment for suitable quality and quantity, the NGS process using a multigene cancer panel was used. After extensive analysis, a molecular geneticist manually reviewed the results to identify pathogenic variants. Clinically, the presence or absence of a multigene mutation profile then was determined and documented.
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93%, respectively. These findings suggest that a critical cell mass of approximately 5000 viable cells and a DNA concentration more than 5 ng/mL will provide an excellent chance of successful sequencing. To date, we estimate more than a 95% success rate in formalin-fixed, paraffinembedded samples that have at least 1.2 cm2 of tissue from unstained slides and have at least 5 ng/mL of recoverable DNA.
Cytology Pathogenic Alteration Profile of Endoscopic Ultrasound Fine-Needle Aspiration Luminal and Extraluminal Locations Exon sequencing in advanced lung cancer patients with adrenal metastasis showed pathogenic alterations in 10 of 50 evaluated genes in 89% of evaluated patients (TP53 [57%], KRAS [29%], STK11 [14%], EGFR [7%], RB1 [7%], PIK3CA [4%], BRAF [4%], GNAS [4%], PTEN [4%], and RET [4%]) (Figure 2). EGFR and KRAS pathogenic alterations were mutually exclusive. In primary gastric GIST patients, the mutational analysis and kinase genotype showed KIT and PDGFRA
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mutations in 58% and 26%, respectively. No PIK3CA, BRAF, KRAS, NRAS, or FGFR3 pathogenic alterations were identified in the wild-type subset. Complete sequencing of malignant lymph node samples was achieved in 102 patients whereby 89% had 194 pathogenic alterations (2 [1–3] alterations per patient) identified in 19 of 50 evaluated genes. Genotyping showed mutations in TP53 (35%), APC (22%), KRAS (17%), FBXW7 (5%), PIK3CA (4%), BRAF (3%), and SMAD4 (3%). The following genes were identified in less than 2% of the identified alterations: CDKN2A, CTNNB1, ERBB2, IDH1, HRAS, GNAS, FLT3, STK11, SMARCB1, PTPN11, and PTEN.
Importance of Findings Advanced colorectal cancer represents one of the best examples, highlighting why multigene testing may be necessary to stratify treatment approaches. Current Food and Drug Administration–approved genetic assays for colorectal rectal cancer can detect a maximum of 7 KRAS mutations (codons 12 and 13). Their presence predicts an unfavorable response to anti-EGFR immunotherapy.
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Figure 2. (A) Scanned low-magnification (1) image of Papanicolau-stained touch preparation cytology slide from the fineneedle core with high cellularity for adenocarcinoma. (B) Higher magnification (200). (C) Snapshot of amplicon reads showing an EGFR c.2573T>G, p.Leu858Arg mutation.
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However, recent data suggest that mutations beyond the 7 tested KRAS mutations, most notably KRAS codon 61 mutations, also predict an unfavorable response to anti–epidermal growth factor (EGFR) therapies. In our small series, rare KRAS mutations in codons 19 and 146 were identified. Furthermore, hot spot mutations in BRAF (V600E) and NRAS (codons 12, 13, and 61) also may predict an unfavorable treatment response.
Translation of Findings Into Routine Clinical Diagnosis and Treatment
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In non-small-cell lung cancer, the presence of an EGFR mutation in 11% of patients suggests that those patients are sensitive to anti-EGFR–tyrosine kinase inhibitor therapy. However, molecular profiling of EGFR–tyrosine kinase inhibitor–resistant adenocarcinomas may help to identify patients who would most benefit from alternative single- or dual-pathway inhibition therapy, potentially leading to a revision in current molecular testing guidelines. Similarly, in patients with gastric GIST, up to 32% are anticipated to have primary imatinib resistance as a result of identified mutations. Finally, among patients with rectal cancer, the identification of KRAS, NRAS, or BRAF mutations suggested that 42% were unlikely to respond to anti-EGFR therapy. Among KRAS, NRAS, or BRAF wild-type patients, alterations in 8 genes linked to alternative therapies, rather than cetuximab or panitumumab as the biologic agent of choice, were identified in 44%. We now increasingly can provide more effective individualized therapies for cancer as part of mutationdriven treatment algorithms that target the genomic profile of a patient’s tumor or metastatic site with the aid of EUS FNA. All testing to date has been performed on archival specimens. A formal validation study is needed to satisfy Clinical Laboratory Improvement Amendments and the College of American Pathologists requirements for a clinical test. After completion of this, a clinical assay will be available to patients undergoing EUS FNA to obtain adequate specimens for NGS mutation panel testing. It is estimated that it will cost between $2000 and $3000 per evaluated sample. Recently, studies by other investigators also showed the utility of an Ion Ampliseq Cancer Hotspot Panel, but primarily were performed on formalin-fixed paraffinembedded tissue specimens. These studies showed that the Ampliseq NGS technology also could detect somatic mutations with very high accuracy when compared with previous mutation results. Although the cost of a NGS panel is more than a single-gene mutation assay, NGS is able to provide considerably higher value owing to its ability to multiplex hundreds of hotspot mutation gene regions within a single reaction. An important caveat of this technique is the principle of intertumor heterogeneity, which refers to the disparate genomic profile that frequently exists between the
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primary tumor and sites of metastases.6,7 In other words, a tumor pathway identified in the primary tumor may be different from that identified in a distant site. This genetic alteration may result from administered therapy, immune response, or represent a temporal event.8–10 Such an occurrence may explain, at least in part, the mixed response to targeted therapies toward one of the identified pathways. Whether the most appropriate genomic architecture to target may be that of metastatic sites rather than the primary tumor, or a combined approach to target genetic anomalies for all disease sites, needs to be investigated further.6,12,13
Challenges, Pitfalls, and Future Perspectives Cytologic material obtained through EUS-FNA cytologic material can be analyzed with immunocytochemical assessments, gene expression patterns by PCR, microsatellite loss analysis, and fluorescence in situ hybridization analysis.14–20 We have reported the utility of targeted NGS of luminal and extraluminal sites using a 50-gene cancer panel to expand tumor information. It has to be kept in mind, however, that the pursuit of improved patient management requires a balance between the risks associated with tissue acquisition vs the utility of the resulting genetic testing and impact on patient care and outcome. This new information has not been shown to be critical in all tumors yet, even though we suspect that the ability to increase the breadth of genetic testing beyond a 50-gene cancer panel, and to assess for actionable rearrangements and copy number variants from such diminutive biopsy specimens, greatly could expand the clinical utility. These panels should focus on clinically actionable alterations and all efforts to reduce testing costs should be considered as larger and more expensive genetic tests become more sought after. Performance of these types of tissue analysis also will necessitate laboratory expertise not only for accuracy, but with acceptable turnaround times and an assessment of the clinical relevance of the detected variants.
Conclusions In the postgenomic era, it is possible and critical to accurately define tumor-specific, genotype-based subpopulations that are most likely to benefit from individualized pharmacologic interventions. The analytic sensitivity and parallel multigene approach of NGS for the detection of mutations with routine EUS-FNA cytology specimens, from luminal and extraluminal sites, offers tremendous promise to personalized patient care for specific primary and metastatic tumor-targeted therapies. The development of techniques to optimize cellular density in collected specimens also will be important for optimal analysis.
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Uncited References This section consists of references that are included in the reference list but are not cited in the article text. Please either cite each of these references in the text or, alternatively, delete it from the reference list. If you do not provide further instruction for this reference, we will retain it in its current form and publish it as an “un-cited reference” with your article.11
References
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1. Gleeson FC, Kipp BR, Levy MJ, et al. Lung cancer adrenal gland metastasis: optimal fine-needle aspirate and touch preparation smear cellularity characteristics for successful theranostic nextgeneration sequencing. Cancer Cytopathol 2014 Epub ahead of print. 2. Gleeson FC, Kipp BR, Kerr SE, et al. Kinase genotype analysis of gastric gastrointestinal stromal tumor cytology samples using targeted next-generation sequencing. Clin Gastroenterol Hepatol 2014 Epub ahead of print.
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colorectal adenocarcinomas and their corresponding metastases. Clin Cancer Res 2010;16:790–799. 12. Longo DL. Tumor heterogeneity and personalized medicine. N Engl J Med 2012;366:956–957. 13. Crockford A, Jamal-Hanjani M, Hicks J, et al. Implications of intratumour heterogeneity for treatment stratification. J Pathol 2014;232:264–273. 14. Carrara S, Cangi MG, Arcidiacono PG, et al. Mucin expression pattern in pancreatic diseases: findings from EUS-guided fineneedle aspiration biopsies. Am J Gastroenterol 2011; 106:1359–1363. 15. Nodit L, McGrath KM, Zahid M, et al. Endoscopic ultrasoundguided fine needle aspirate microsatellite loss analysis and pancreatic endocrine tumor outcome. Clin Gastroenterol Hepatol 2006;4:1474–1478. 16. Al-Haddad M, Wallace MB. Molecular diagnostics of non-small cell lung cancer using mediastinal lymph nodes sampled by endoscopic ultrasound-guided needle aspiration. Cytopathology 2006;17:3–9.
3. Bellevicine C, Vita GD, Malapelle U, et al. Applications and limitations of oncogene mutation testing in clinical cytopathology. Semin Diagn Pathol 2013;30:284–297.
17. Levy MJ, Oberg TN, Campion MB, et al. Comparison of methods to detect neoplasia in patients undergoing endoscopic ultrasound-guided fine-needle aspiration. Gastroenterology 2012;142:1112–1121.
4. Bediaga NG, Davies MP, Acha-Sagredo A, et al. A microRNAbased prediction algorithm for diagnosis of non-small lung cell carcinoma in minimal biopsy material. Br J Cancer 2013; 109:2404–2411.
18. Bournet B, Pointreau A, Souque A, et al. Gene expression signature of advanced pancreatic ductal adenocarcinoma using low density array on endoscopic ultrasound-guided fine needle aspiration samples. Pancreatology 2012;12:27–34.
5. Fischer AH, Cibas ES, Howell LP, et al. Role of cytology in the management of non-small-cell lung cancer. J Clin Oncol 2011; 29:3331–3332; author reply 3332–3333.
19. Henkes DN, Patel SN, Rosenkranz LA, et al. The utility of UroVysion fluorescence in situ hybridization in pancreatic fineneedle aspiration samples directed and obtained by endoscopic ultrasonography. Arch Pathol Lab Med 2013;137:64–71.
6. Kosmidou V, Oikonomou E, Vlassi M, et al. Tumour heterogeneity revealed by KRAS, BRAF and PIK3CA pyrosequencing: KRAS and PIK3CA intratumour mutation profile differences and their therapeutic implications. Hum Mutat 2013 Epub ahead of print. 7. Bedard PL, Hansen AR, Ratain MJ, et al. Tumour heterogeneity in the clinic. Nature 2013;501:355–364. 8. Gerlinger M, Rowan AJ, Horswell S, et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med 2012;366:883–892. 9. Marte B. Tumour heterogeneity. Nature 2013;501:327. 10. Meacham CE, Morrison SJ. Tumour heterogeneity and cancer cell plasticity. Nature 2013;501:328–337. 11. Baldus SE, Schaefer KL, Engers R, et al. Prevalence and heterogeneity of KRAS, BRAF, and PIK3CA mutations in primary
20. van Eijk R, Licht J, Schrumpf M, et al. Rapid KRAS, EGFR, BRAF and PIK3CA mutation analysis of fine needle aspirates from nonsmall-cell lung cancer using allele-specific qPCR. PLoS One 2011;6:e17791.
Reprint requests Address requests for reprints to: Ferga C. Gleeson, MD, Division of Gastroenterology and Hepatology, Mayo Clinic, 200 1st Street SW, Rochester, MinQ3 nesota 55905. e-mail: [email protected]; fax: (507) 266-3931. Conflicts of interest The authors disclose no conflicts.
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Funding Supported by the Center for Individualized Medicine, Mayo Clinic, Rochester, Q19 Q5 MN.
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The Molecular Characterization and Theranostic Potential of Endoscopic Ultrasound Fine-Needle Aspiration Malignant Cytology Using Targeted Next-Generation Sequencing Ferga C. Gleeson,* Benjamin R. Kipp,‡ Sarah E. Kerr,‡ Jesse Voss,‡ Konstantinos N. Lazaridis,*,§ David A. Katzka,* and Michael J. Levy* *Division of Gastroenterology and Hepatology, ‡Department of Laboratory Medicine and Pathology, §Center for Individualized Medicine, Mayo Clinic, Rochester, Minnesota
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Determination of tumor genetic architecture based on tissue analysis yields important information on signaling pathways involved in cancer pathogenesis and plays a growing role in choosing the optimal medical management of malignancies. Specifically, the advent of next-generation sequencing has led to a rapidly evolving era of relatively inexpensive, high-throughput DNA sequencing of tumors. One such example is multiplexed tumor genotyping (ie, panel testing) of more than 2800 mutations across 50 commonly mutated cancer-associated genes. This resulting mutational landscape shows medically actionable pathogenic alterations to optimize antitumor therapy. We recently assessed the performance and outcome of targeted next-generation sequencing with archived endoscopic ultrasound fine-needle aspirates across a broad range of primary and metastatic sites with encouraging accuracy. As a result, endoscopic ultrasound has the potential to move from a test for diagnosis or confirmation of malignancy, to one in which it could facilitate the personalization of cancer-directed therapy. Keywords: Endoscopic Ultrasound Fine-Needle Aspiration; Malignant Cytology; Targeted Next-Generation Sequencing; Theranostics; Individualized Medicine.
he treatment decision model in oncology practices is undergoing a paradigm shift with increasing emphasis to customize potential therapy not just by histology alone but also to incorporate tumor molecular characteristics that represent the biological pathways driving tumorigenesis. Consequently, there is an increasing need for routine comprehensive DNA clinical testing for selected tumors to aid therapy triage for an individual patient, a practice often referred to as theranostics or personalized medicine. Formalin-fixed, paraffinembedded specimens are currently the specimen of choice for tumor genetic profiling. However, ancillary testing of endoscopic ultrasound fine-needle aspiration (EUS-FNA) samples is becoming more widely performed in clinical practice. Our objectives were to identify key cytology specimen selection criteria suitable for next-generation sequencing (NGS) and to determine the prevalence and spectrum of
T
pathogenic alterations with theranostic intent. We chose a cohort of patients with primary gastric gastrointestinal tumors (GISTs), malignant lymph nodes from locally advanced primary rectal cancer, and adrenal gland metastasis from treatment-naive lung cancer.1,2
Technological Primer Targeted NGS enables sequencing of specific segments of the genome to detect pathogenic point mutations and small insertions/deletions at reduced cost and turnaround time, with enhanced sensitivity when compared with whole-exome or whole-genome sequencing. Sequencing of cytology samples has long been sought because of the theoretical advantage of containing a more pure tumor cell population in contrast to matrix-rich histologic specimens. Furthermore, cytology specimen processing has superior preservation of nucleic acids given the use of alcohol-based fixation and direct smearing when compared with formalin fixation of histology specimens.3–5
Deep Sequencing of Multiplex Polymerase Chain Reaction Amplicons After cytology slide selection and DNA extraction, multiplex polymerase chain reaction (PCR) was performed by amplifying DNA with the Ion Ampliseq Cancer Hotspot Panel v2 and the Ion Ampliseq Library Kit 2.0 (Life Technologies, Carlsbad, CA). This Ampliseq kit targets 2855 possible mutations within 50 cancerassociated genes, including the following: ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, Abbreviations used in this paper: EGFR, epidermal growth factor receptor; EUS FNA, endoscopic ultrasound fine-needle aspiration; GIST, gastrointestinal stromal tumor; NGS, next-generation sequencing; PCR, polymerase chain reaction. © 2014 by the AGA Institute 1542-3565/$36.00 http://dx.doi.org/10.1016/j.cgh.2014.10.017
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FGFR3, FLT3, GNAQ, GNAS, GNA11, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, and VHL. The PCR product underwent library preparation using the TruSeq Nano DNA Sample Preparation Kit (Illumina, San Diego, CA).
Data Analysis Identification of a cell variant was made by an internal bioinformatics pipeline and alignments were reviewed manually by a molecular geneticist to verify whether variants were pathogenic or not (Figure 1). Each variant was reviewed using multiple public databases (Catalogue of Somatic Mutations in Cancer) and software programs (eg, Alamut). Patients with variants with an allele frequency of more than 5% were flagged for review. Variants with minor allele frequencies greater than 1% or silent changes without evidence of pathogenicity were considered common single-nucleotide polymorphism and were not reported. Remaining variants were flagged as pathogenic if the alteration was listed in the Catalogue of Somatic Mutations in Cancer and/or there were functional data suggesting pathogenicity. Remaining variants were classified as variants of unknown significance.
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Findings Cytology Sample Slide Characteristics for Successful Next-Generation Sequencing The selected criteria for optimal NGS sequencing were obtained from archived adrenal metastasis cytology slides. Slide cellularity was triaged by a molecular pathologist as low (300–1000 cells), moderate (1000–5000 cells), or high (>5000 cells). The degree of necrosis was categorized subjectively as low, moderate, or high (>50% of tumor cells). Slides with low, moderate, and high cellularity underwent successful NGS 22%, 25%, and 91% of the time. Slides yielding less than 5 ng/mL or more than 5 ng/mL of DNA were NGS successful in 4% and = Figure 1. Cytology slide sample workflow. Cytology slides were evaluated and triaged by a cytopathologist for tumor cell percentage and DNA extraction eligibility. Selected slides were imaged for photographic archives within the cytopathology laboratory. After DNA extraction, and assessment for suitable quality and quantity, the NGS process using a multigene cancer panel was used. After extensive analysis, a molecular geneticist manually reviewed the results to identify pathogenic variants. Clinically, the presence or absence of a multigene mutation profile then was determined and documented.
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93%, respectively. These findings suggest that a critical cell mass of approximately 5000 viable cells and a DNA concentration more than 5 ng/mL will provide an excellent chance of successful sequencing. To date, we estimate more than a 95% success rate in formalin-fixed, paraffinembedded samples that have at least 1.2 cm2 of tissue from unstained slides and have at least 5 ng/mL of recoverable DNA.
Cytology Pathogenic Alteration Profile of Endoscopic Ultrasound Fine-Needle Aspiration Luminal and Extraluminal Locations Exon sequencing in advanced lung cancer patients with adrenal metastasis showed pathogenic alterations in 10 of 50 evaluated genes in 89% of evaluated patients (TP53 [57%], KRAS [29%], STK11 [14%], EGFR [7%], RB1 [7%], PIK3CA [4%], BRAF [4%], GNAS [4%], PTEN [4%], and RET [4%]) (Figure 2). EGFR and KRAS pathogenic alterations were mutually exclusive. In primary gastric GIST patients, the mutational analysis and kinase genotype showed KIT and PDGFRA
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mutations in 58% and 26%, respectively. No PIK3CA, BRAF, KRAS, NRAS, or FGFR3 pathogenic alterations were identified in the wild-type subset. Complete sequencing of malignant lymph node samples was achieved in 102 patients whereby 89% had 194 pathogenic alterations (2 [1–3] alterations per patient) identified in 19 of 50 evaluated genes. Genotyping showed mutations in TP53 (35%), APC (22%), KRAS (17%), FBXW7 (5%), PIK3CA (4%), BRAF (3%), and SMAD4 (3%). The following genes were identified in less than 2% of the identified alterations: CDKN2A, CTNNB1, ERBB2, IDH1, HRAS, GNAS, FLT3, STK11, SMARCB1, PTPN11, and PTEN.
Importance of Findings Advanced colorectal cancer represents one of the best examples, highlighting why multigene testing may be necessary to stratify treatment approaches. Current Food and Drug Administration–approved genetic assays for colorectal rectal cancer can detect a maximum of 7 KRAS mutations (codons 12 and 13). Their presence predicts an unfavorable response to anti-EGFR immunotherapy.
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Figure 2. (A) Scanned low-magnification (1) image of Papanicolau-stained touch preparation cytology slide from the fineneedle core with high cellularity for adenocarcinoma. (B) Higher magnification (200). (C) Snapshot of amplicon reads showing an EGFR c.2573T>G, p.Leu858Arg mutation.
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However, recent data suggest that mutations beyond the 7 tested KRAS mutations, most notably KRAS codon 61 mutations, also predict an unfavorable response to anti–epidermal growth factor (EGFR) therapies. In our small series, rare KRAS mutations in codons 19 and 146 were identified. Furthermore, hot spot mutations in BRAF (V600E) and NRAS (codons 12, 13, and 61) also may predict an unfavorable treatment response.
Translation of Findings Into Routine Clinical Diagnosis and Treatment
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In non-small-cell lung cancer, the presence of an EGFR mutation in 11% of patients suggests that those patients are sensitive to anti-EGFR–tyrosine kinase inhibitor therapy. However, molecular profiling of EGFR–tyrosine kinase inhibitor–resistant adenocarcinomas may help to identify patients who would most benefit from alternative single- or dual-pathway inhibition therapy, potentially leading to a revision in current molecular testing guidelines. Similarly, in patients with gastric GIST, up to 32% are anticipated to have primary imatinib resistance as a result of identified mutations. Finally, among patients with rectal cancer, the identification of KRAS, NRAS, or BRAF mutations suggested that 42% were unlikely to respond to anti-EGFR therapy. Among KRAS, NRAS, or BRAF wild-type patients, alterations in 8 genes linked to alternative therapies, rather than cetuximab or panitumumab as the biologic agent of choice, were identified in 44%. We now increasingly can provide more effective individualized therapies for cancer as part of mutationdriven treatment algorithms that target the genomic profile of a patient’s tumor or metastatic site with the aid of EUS FNA. All testing to date has been performed on archival specimens. A formal validation study is needed to satisfy Clinical Laboratory Improvement Amendments and the College of American Pathologists requirements for a clinical test. After completion of this, a clinical assay will be available to patients undergoing EUS FNA to obtain adequate specimens for NGS mutation panel testing. It is estimated that it will cost between $2000 and $3000 per evaluated sample. Recently, studies by other investigators also showed the utility of an Ion Ampliseq Cancer Hotspot Panel, but primarily were performed on formalin-fixed paraffinembedded tissue specimens. These studies showed that the Ampliseq NGS technology also could detect somatic mutations with very high accuracy when compared with previous mutation results. Although the cost of a NGS panel is more than a single-gene mutation assay, NGS is able to provide considerably higher value owing to its ability to multiplex hundreds of hotspot mutation gene regions within a single reaction. An important caveat of this technique is the principle of intertumor heterogeneity, which refers to the disparate genomic profile that frequently exists between the
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primary tumor and sites of metastases.6,7 In other words, a tumor pathway identified in the primary tumor may be different from that identified in a distant site. This genetic alteration may result from administered therapy, immune response, or represent a temporal event.8–10 Such an occurrence may explain, at least in part, the mixed response to targeted therapies toward one of the identified pathways. Whether the most appropriate genomic architecture to target may be that of metastatic sites rather than the primary tumor, or a combined approach to target genetic anomalies for all disease sites, needs to be investigated further.6,12,13
Challenges, Pitfalls, and Future Perspectives Cytologic material obtained through EUS-FNA cytologic material can be analyzed with immunocytochemical assessments, gene expression patterns by PCR, microsatellite loss analysis, and fluorescence in situ hybridization analysis.14–20 We have reported the utility of targeted NGS of luminal and extraluminal sites using a 50-gene cancer panel to expand tumor information. It has to be kept in mind, however, that the pursuit of improved patient management requires a balance between the risks associated with tissue acquisition vs the utility of the resulting genetic testing and impact on patient care and outcome. This new information has not been shown to be critical in all tumors yet, even though we suspect that the ability to increase the breadth of genetic testing beyond a 50-gene cancer panel, and to assess for actionable rearrangements and copy number variants from such diminutive biopsy specimens, greatly could expand the clinical utility. These panels should focus on clinically actionable alterations and all efforts to reduce testing costs should be considered as larger and more expensive genetic tests become more sought after. Performance of these types of tissue analysis also will necessitate laboratory expertise not only for accuracy, but with acceptable turnaround times and an assessment of the clinical relevance of the detected variants.
Conclusions In the postgenomic era, it is possible and critical to accurately define tumor-specific, genotype-based subpopulations that are most likely to benefit from individualized pharmacologic interventions. The analytic sensitivity and parallel multigene approach of NGS for the detection of mutations with routine EUS-FNA cytology specimens, from luminal and extraluminal sites, offers tremendous promise to personalized patient care for specific primary and metastatic tumor-targeted therapies. The development of techniques to optimize cellular density in collected specimens also will be important for optimal analysis.
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Uncited References This section consists of references that are included in the reference list but are not cited in the article text. Please either cite each of these references in the text or, alternatively, delete it from the reference list. If you do not provide further instruction for this reference, we will retain it in its current form and publish it as an “un-cited reference” with your article.11
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Reprint requests Address requests for reprints to: Ferga C. Gleeson, MD, Division of Gastroenterology and Hepatology, Mayo Clinic, 200 1st Street SW, Rochester, MinQ3 nesota 55905. e-mail: [email protected]; fax: (507) 266-3931. Conflicts of interest The authors disclose no conflicts.
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Funding Supported by the Center for Individualized Medicine, Mayo Clinic, Rochester, Q19 Q5 MN.
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