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Weill Cornell Medicine to Expand Tumor Exome Test (EXaCT-1 identifies alterations within tumors) to Thousands of Advanced Cancer Patients

Posted in CANCER BIOLOGY & Innovations in Cancer Therapy, Genome Biology, Genomic Testing: Methodology for Diagnosis, tagged EXaCT-1 assay, exome, panel sequencing, where DNA is transcribed into RNA, whole exome sequencing on July 20, 2016| Leave a Comment »

Weill Cornell Medicine to Expand Tumor Exome Test (EXaCT-1 identifies alterations within tumors) to Thousands of Advanced Cancer Patients

Reporter: Aviva Lev-Ari, PhD, RN

PRECISION MEDICINE TEAM AT WEILL CORNELL MEDICINE AND NEWYORK-PRESBYTERIAN WILL INTRODUCE WHOLE EXOME SEQUENCING TEST, EXACT-1, TO CLINICAL SETTING

	Tumor cells from a biopsy of a patient enrolled in the EXaCT-1 pilot program that investigators are culturing in the lab for further biologic studies and drug testing. Image credit: Dr. Loredana Puca

NEW YORK (November 13, 2015) — A powerful new test that can reveal untapped therapies for patients with advanced cancers by scanning thousands of their genes will soon be available for patients at Weill Cornell Medicine and NewYork-Presbyterian/Weill Cornell Medical Center. The test, EXaCT-1, identifies alterations within tumors — some of which drive cancerous growth — on a magnitude up to hundreds of times greater than similar technologies designed to pinpoint the most precise ways of treating the disease.

Weill Cornell Medicine recently received approval for EXaCT-1 by the New York State Department of Health. The test was developed by the institutions’ precision medicine team. In May, the team published findings on its first 97 patients who underwent the test and found that scanning a patient’s tumor to look for any genomic mutations — rather than limiting the screen to mutations commonly associated with a given patient’s tumor type — worked. In 92 percent of cases in the pilot program, the precision medicine team was able to recommend new treatment options based on the test’s findings. Now that the state has approved the test, precision medicine leaders will begin the process to implement it for large-scale clinical use for oncology patients treated at NewYork-Presbyterian/Weill Cornell. Until that time, patients with advanced cancers will be able to access EXaCT-1 through the Caryl and Israel Englander Institute for Precision Medicine at Weill Cornell Medicine, the research enterprise of the two institutions’ joint precision medicine efforts.

Unlike these focused tests, typically called panel sequencing, the EXaCT-1 assay takes an unbiased, exploratory look at more than 21,000 genes in cells both healthy and malignant, allowing researchers to find alterations in the cancer-development process in unexpected regions of the exome, where DNA is transcribed into RNA. This type of test, known as whole exome sequencing, can be effective in advanced-stage patients for whom other treatments have failed because it uncovers mutations that the less comprehensive tests miss. In practice, this means, for example, that a patient with bladder cancer whom EXaCT-1 shows to share a mutation associated with breast cancer might benefit from a drug typically prescribed to fight the latter type of tumor.

	Dr. Olivier Elemento Photo credit: Roger Tully

“That’s the nice thing about sequencing the entire tumor genome – we cover genes that other tests will miss,” said Dr. Olivier Elemento, head of the Englander Institute for Precision Medicine’s computational biology group, an associate professor of physiology and biophysics and head of the laboratory of cancer systems biology in the HRH Prince Alwaleed Bin Talal Bin Abdulaziz Al-Saud Institute for Computational Biomedicine at Weill Cornell. “This test is ideal for patients with advanced cancer because it allows us to identify mutations that may be related to the resistance of their disease, and helps us to pinpoint the best way to treat them.”

The screen requires a blood sample and a sample of the patient’s tumor. Computational biologists at the Englander Institute analyze the data and generate patient-and physician-friendly reports that summarize the key clinical and genetic findings. Once the precision medicine team has reviewed the results, it consults with the patient’s oncologist at NewYork-Presbyterian/Weill Cornell to help decide which treatment options and clinical trials may best target the patient’s disease.

Read more @SOURCE

SOURCE

http://weill.cornell.edu/news/pr/2015/11/new-treatment-options-for-advanced-stage-cancer-patients-revealed-through-next-generation-sequencing.html

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Multiple Lung Cancer Genomic Projects Suggest New Targets, Research Directions for Non-Small Cell Lung Cancer

Posted in Biological Networks, Biomarkers & Medical Diagnostics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cancer Prevention: Research & Programs, Computational Biology/Systems and Bioinformatics, Gene Regulation and Evolution, Genome Biology, Health Economics and Outcomes Research, Personalized and Precision Medicine & Genomic Research, tagged antitumor therapy, Bioinformatics, Cancer - General, Cell Biology, Computational Biology/Systems and Bioinformatics, DNA, DNA Sequencing, driver mutations, gene expression, genetics, genome, genomics, informatics, Lung cancer, mutation, mutational analysis, National Institutes of Health, Non-small cell lung cancer, Personalized medicine, Proceedings of the National Academy of Sciences of the United States of America, Prostate cancer, research, smoking, The Cancer Genome Atlas (TCGA), The Clinical Lung Cancer Genome Project (CLCGP), whole exome sequencing on September 5, 2014| 2 Comments »

Multiple Lung Cancer Genomic Projects Suggest New Targets, Research Directions for Non-Small Cell Lung Cancer

Curator, Writer: Stephen J. Williams, Ph.D.

UPDATED 10/10/2021

(photo credit: cancer.gov)

A report Lung Cancer Genome Surveys Find Many Potential Drug Targets, in the NCI Bulletin,

http://www.cancer.gov/ncicancerbulletin/091812/page2

summarizes the clinical importance of five new lung cancer genome sequencing projects. These studies have identified genetic and epigenetic alterations in hundreds of lung tumors, of which some alterations could be taken advantage of using currently approved medications.

The reports, all published this month, included genomic information on more than 400 lung tumors. In addition to confirming genetic alterations previously tied to lung cancer, the studies identified other changes that may play a role in the disease.

Collectively, the studies covered the main forms of the disease—lung adenocarcinomas, squamous cell cancers of the lung, and small cell lung cancers.

“All of these studies say that lung cancers are genomically complex and genomically diverse,” said Dr. Matthew Meyerson of Harvard Medical School and the Dana-Farber Cancer Institute, who co-led several of the studies, including a large-scale analysis of squamous cell lung cancer by The Cancer Genome Atlas (TCGA) Research Network.

Some genes, Dr. Meyerson noted, were inactivated through different mechanisms in different tumors. He cautioned that little is known about alterations in DNA sequences that do not encode genes, which is most of the human genome.

Four of the papers are summarized below, with the first described in detail, as the Nature paper used a multi-‘omics strategy to evaluate expression, mutation, and signaling pathway activation in a large cohort of lung tumors. A literature informatics analysis is given for one of the papers.  Please note that links on GENE names usually refer to the GeneCard entry.

Paper 1. Comprehensive genomic characterization of squamous cell lung cancers[1]

The Cancer Genome Atlas Research Network Project just reported, in the journal Nature, the results of their comprehensive profiling of 230 resected lung adenocarcinomas. The multi-center teams employed analyses of

• microRNA
• Whole Exome Sequencing including
  • Exome mutation analysis
  • Gene copy number
  • Splicing alteration
• Methylation
• Proteomic analysis

Summary:

Some very interesting overall findings came out of this analysis including:

• High rates of somatic mutations including activating mutations in common oncogenes
• Newly described loss of function MGA mutations
• Sex differences in EGFR and RBM10 mutations
• driver roles for NF1, MET, ERBB2 and RITI identified in certain tumors
• differential mutational pattern based on smoking history
• splicing alterations driven by somatic genomic changes
• MAPK and PI3K pathway activation identified by proteomics not explained by mutational analysis = UNEXPLAINED MECHANISM of PATHWAY ACTIVATION

however, given the plethora of data, and in light of a similar study results recently released, there appears to be a great need for additional mining of this CGAP dataset. Therefore I attempted to curate some of the findings along with some other recent news relevant to the surprising findings with relation to biomarker analysis.

Makeup of tumor samples

230 lung adenocarcinomas specimens were categorized by:

Subtype

33% acinar

25% solid

14% micro-papillary

9% papillary

8% unclassified

5% lepidic

4% invasive mucinous
Gender

Smoking status

81% of patients reported past of present smoking

The authors note that TCGA samples were combined with previous data for analysis purpose.

A detailed description of Methodology and the location of deposited data are given at the following addresses:

Publication TCGA Web Page: https://tcga-data.nci.nih.gov/docs/publications/luad_2014/

Sequence files: https://cghub.ucsc.edu

Results:

Gender and Smoking Habits Show different mutational patterns

 

WES mutational analysis

1. a) smoking status

– there was a strong correlations of cytosine to adenine nucleotide transversions with past or present smoking. In fact smoking history separated into transversion high (past and previous smokers) and transversion low (never smokers) groups, corroborating previous results.

→ mutations in groups              Transversion High                   Transversion Low

TP53, KRAS, STK11,                 EGFR, RB1, PI3CA

     KEAP1, SMARCA4 RBM10

 

1. b) Gender

Although gender differences in mutational profiles have been reported, the study found minimal number of significantly mutated genes correlated with gender. Notably:

• EGFR mutations enriched in female cohort
• RBM10 loss of function mutations enriched in male cohort

Although the study did not analyze the gender differences with smoking patterns, it was noted that RBM10 mutations among males were more prevalent in the transversion high group.

Whole exome Sequencing and copy number analysis reveal Unique, Candidate Driver Genes

Whole exome sequencing revealed that 62% of tumors contained mutations (either point or indel) in known cancer driver genes such as:

KRAS, EGFR, BRMF, ERBB2

However, authors looked at the WES data from the oncogene-negative tumors and found unique mutations not seen in the tumors containing canonical oncogenic mutations.

Unique potential driver mutations were found in

TP53, KEAP1, NF1, and RIT1

The genomics and expression data were backed up by a proteomics analysis of three pathways:

1. MAPK pathway
2. mTOR
3. PI3K pathway

…. showing significant activation of all three pathways HOWEVER the analysis suggested that activation of signaling pathways COULD NOT be deduced from DNA sequencing alone. Phospho-proteomic analysis was required to determine the full extent of pathway modification.

For example, many tumors lacked an obvious mutation which could explain mTOR or MAPK activation.

 

Altered cell signaling pathways included:

• Increased MAPK signaling due to activating KRAS
• Higher mTOR due to inactivating STK11 leading to increased proliferation, translation

Pathway analysis of mutations revealed alterations in multiple cellular pathways including:

• Reduced oxidative stress response
• Nucleosome remodeling
• RNA splicing
• Cell cycle progression
• Histone methylation

Summary:

Authors noted some interesting conclusions including:

1. MET and ERBB2 amplification and mutations in NF1 and RIT1 may be unique driver events in lung adenocarcinoma
2. Possible new drug development could be targeted to the RTK/RAS/RAF pathway
3. MYC pathway as another important target
4. Cluster analysis using multimodal omics approach identifies tumors based on single-gene driver events while other tumor have multiple driver mutational events (TUMOR HETEROGENEITY)

Paper 2. A Genomics-Based Classification of Human Lung Tumors[2]

The paper can be found at

http://stm.sciencemag.org/content/5/209/209ra153

by The Clinical Lung Cancer Genome Project (CLCGP) and Network Genomic Medicine (NGM),*,†

Paper Summary

This sequencing project revealed discrepancies between histologic and genomic classification of lung tumors.

Methodology

– mutational analysis by whole exome sequencing of 1255 lung tumors of histologically

defined subtypes

– immunohistochemistry performed to verify reclassification of subtypes based on sequencing data

Results

• 55% of all cases had at least one oncogenic alteration amenable to current personalized treatment approaches
• Marked differences existed between cluster analysis within and between preclassified histo-subtypes
• Reassignment based on genomic data eliminated large cell carcinomas
• Prospective classification of 5145 lung cancers allowed for genomic classification in 75% of patients
• Identification of EGFR and ALK mutations led to improved outcomes

Conclusions:

It is feasible to successfully classify and diagnose lung tumors based on whole exome sequencing data.

Paper 3. Genomic Landscape of Non-Small Cell Lung Cancer in Smokers and Never-Smokers[3]

A link to the paper can be found here with Graphic Summary: http://www.cell.com/cell/abstract/S0092-8674%2812%2901022-7?cc=y?cc=y

Methodology

• Whole genome sequencing and transcriptome sequencing of cancerous and adjacent normal tissues from 17 patients with NSCLC
• Integrated RNASeq with WES for analysis of
  • Variant analysis
  • Clonality by variant allele frequency anlaysis
  • Fusion genes
• Bioinformatic analysis
  • PathScan, KEGG for pathway analysis
  • COSMIC for reported mutations
  • ChimeraScan, defuse, BreakFusion for fusion protein analysis

Results

• 3,726 point mutations and more than 90 indels in the coding sequence
• Smokers with lung cancer show 10× the number of point mutations than never-smokers
• Novel lung cancer genes, including DACH1, CFTR, RELN, ABCB5, and HGF were identified

• Tumor samples from males showed high frequency of MYCBP2 MYCBP2 involved in transcriptional regulation of MYC.

• Variant allele frequency analysis revealed 10/17 tumors were at least biclonal while 7/17 tumors were monoclonal revealing majority of tumors displayed tumor heterogeneity
• Novel pathway alterations in lung cancer include cell-cycle and JAK-STAT pathways
• 14 fusion proteins found, including ROS1-ALK fusion. ROS1-ALK fusions have been frequently found in lung cancer and is indicative of poor prognosis[4].
• Novel metabolic enzyme fusions
• Alterations were identified in 54 genes for which targeted drugs are available.           Drug-gable mutant targets include: AURKC, BRAF, HGF, EGFR, ERBB4, FGFR1, MET, JAK2, JAK3, HDAC2, HDAC6, HDAC9, BIRC6, ITGB1, ITGB3, MMP2, PRKCB, PIK3CG, TERT, KRAS, MMP14

Table. Validated Gene-Fusions Obtained from Ref-Seq Data

Note: Gene columns contain links for GeneCard while Gene function links are to the    gene’s GO (Gene Ontology) function.

GeneA (5′)	GeneB (3′)	GeneA function (link to Gene Ontology)	GeneB function (link to Gene Ontology)		known function (refs)
GRIP1	TNIP1	glutamate receptor IP	transcriptional repressor
SGMS1	STK10	sphingolipid synthesis	ser/thr kinase
RASSF3	TTYH2	GTP-binding protein	chloride anion channel
KDELR2	ROS1, GOPC	ER retention seq. binding	proto-oncogenic tyr kinase
ACSL4	DCAF6	fatty acid synthesis	?
MARCH8	PRKG1	ubiquitin ligase	cGMP dependent protein kinase
APAF1	UNC13B, TLN1	caspase activation	cytoskeletal
EML4	ALK	microtubule protein	tyrosine kinase		♦
EDR3,PHC3	LOC441601	polycomb pr/DNA binding	?
DKFZp761L1918,RHPN2	ANKRD27	Rhophilin (GTP binding pr	ankyrin like
VANGL1	HAO2	tetraspanin family	oxidase
CACNA2D3	FLNB	VOC Ca++ channel	filamin (actin binding)

† Author’s Note:

There has been a recent literature on the importance of the EML4-ALK fusion protein in lung cancer. EML4-ALK positive lung tumors were found to be les chemo sensitive to cytotoxic therapy[5] and these tumor cells may exhibit an epitope rendering these tumors amenable to immunotherapy[6]. In addition, inhibition of the PI3K pathway has sensitized EMl4-ALK fusion positive tumors to ALK-targeted therapy[7]. EML4-ALK fusion positive tumors show dependence on the HSP90 chaperone, suggesting this cohort of patients might benefit from the new HSP90 inhibitors recently being developed[8].

Table. Significantly mutated genes (point mutations, insertions/deletions) with associated function.

Gene	Function
TP53	tumor suppressor
KRAS	oncogene
ZFHX4	zinc finger DNA binding
DACH1	transcription factor
EGFR	epidermal growth factor receptor
EPHA3	receptor tyrosine kinase
ENSG00000205044
RELN	cell matrix protein
ABCB5	ABC Drug Transporter

Table. Literature Analysis of pathways containing significantly altered genes in NSCLC reveal putative targets and risk factors, linkage between other tumor types, and research areas for further investigation.

Note: Significantly mutated genes, obtained from WES, were subjected to pathway analysis (KEGG Pathway Analysis) in order to see which pathways contained signicantly altered gene networks. This pathway term was then used for PubMed literature search together with terms “lung cancer”, “gene”, and “NOT review” to determine frequency of literature coverage for each pathway in lung cancer. Links are to the PubMEd search results.

KEGG pathway Name	# of PUBMed entries containing Pathway Name, Gene ANDLung Cancer
Cell cycle	1237
Cell adhesion molecules (CAMs)	372
Glioma	294
Melanoma	219
Colorectal cancer	207
Calcium signaling pathway	175
Prostate cancer	166
MAPK signaling pathway	162
Pancreatic cancer	88
Bladder cancer	74
Renal cell carcinoma	68
Focal adhesion	63
Regulation of actin cytoskeleton	34
Thyroid cancer	32
Salivary secretion	19
Jak-STAT signaling pathway	16
Natural killer cell mediated cytotoxicity	11
Gap junction	11
Endometrial cancer	11
Long-term depression	9
Axon guidance	8
Cytokine-cytokine receptor interaction	8
Chronic myeloid leukemia	7
ErbB signaling pathway	7
Arginine and proline metabolism	6
Maturity onset diabetes of the young	6
Neuroactive ligand-receptor interaction	4
Aldosterone-regulated sodium reabsorption	2
Systemic lupus erythematosus	2
Olfactory transduction	1
Huntington’s disease	1
Chemokine signaling pathway	1
Cardiac muscle contraction	1
Amyotrophic lateral sclerosis (ALS)	1

A few interesting genetic risk factors and possible additional targets for NSCLC were deduced from analysis of the above table of literature including HIF1-α, mIR-31, UBQLN1, ACE, mIR-193a, SRSF1. In addition, glioma, melanoma, colorectal, and prostate and lung cancer share many validated mutations, and possibly similar tumor driver mutations.

 please click on graph for larger view

Paper 4. Mapping the Hallmarks of Lung Adenocarcinoma with Massively Parallel Sequencing[9]

For full paper and graphical summary please follow the link: http://www.cell.com/cell/abstract/S0092-8674%2812%2901061-6

Highlights

• Exome and genome characterization of somatic alterations in 183 lung adenocarcinomas
• 12 somatic mutations/megabase
• U2AF1, RBM10, and ARID1A are among newly identified recurrently mutated genes
• Structural variants include activating in-frame fusion of EGFR
• Epigenetic and RNA deregulation proposed as a potential lung adenocarcinoma hallmark

Summary

Lung adenocarcinoma, the most common subtype of non-small cell lung cancer, is responsible for more than 500,000 deaths per year worldwide. Here, we report exome and genome sequences of 183 lung adenocarcinoma tumor/normal DNA pairs. These analyses revealed a mean exonic somatic mutation rate of 12.0 events/megabase and identified the majority of genes previously reported as significantly mutated in lung adenocarcinoma. In addition, we identified statistically recurrent somatic mutations in the splicing factor gene U2AF1 and truncating mutations affecting RBM10 and ARID1A. Analysis of nucleotide context-specific mutation signatures grouped the sample set into distinct clusters that correlated with smoking history and alterations of reported lung adenocarcinoma genes. Whole-genome sequence analysis revealed frequent structural rearrangements, including in-frame exonic alterations within EGFR and SIK2 kinases. The candidate genes identified in this study are attractive targets for biological characterization and therapeutic targeting of lung adenocarcinoma.

Paper 5. Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer[10]

Highlights

• Whole exome and transcriptome (RNASeq) sequencing 29 small-cell lung carcinomas
• High mutation rate 7.4 protein-changing mutations/million base pairs
• Inactivating mutations in TP53 and RB1
• Functional mutations in CREBBP, EP300, MLL, PTEN, SLIT2, EPHA7, FGFR1 (determined by literature and database mining)
• The mutational spectrum seen in human data also present in a Tp53-/- Rb1-/- mouse lung tumor model

 

Curator Graphical Summary of Interesting Findings From the Above Studies

The above figure (please click on figure) represents themes and findings resulting from the aforementioned studies including

questions which will be addressed in Future Posts on this site.

UPDATED 10/10/2021

The following article uses RNASeq to screen lung adenocarcinomas for fusion proteins in patients with either low or high tumor mutational burden. Findings included presence of MET fusion proteins in addition to other fusion proteins irrespective if tumors were driver negative by DNASeq screening.

High Yield of RNA Sequencing for Targetable Kinase Fusions in Lung Adenocarcinomas with No Mitogenic Driver Alteration Detected by DNA Sequencing and Low Tumor Mutation Burden

Source:

High Yield of RNA Sequencing for Targetable Kinase Fusions in Lung Adenocarcinomas with No Mitogenic Driver Alteration Detected by DNA Sequencing and Low Tumor Mutation Burden

Ryma Benayed, Michael Offin, Kerry Mullaney, Purvil Sukhadia, Kelly Rios, Patrice Desmeules, Ryan Ptashkin, Helen Won, Jason Chang, Darragh Halpenny, Alison M. Schram, Charles M. Rudin, David M. Hyman, Maria E. Arcila, Michael F. Berger, Ahmet Zehir, Mark G. Kris, Alexander Drilon and Marc Ladanyi

Clin Cancer Res August 1 2019 (25) (15) 4712-4722; DOI: 10.1158/1078-0432.CCR-19-0225

Abstract

Purpose: Targeted next-generation sequencing of DNA has become more widely used in the management of patients with lung adenocarcinoma; however, no clear mitogenic driver alteration is found in some cases. We evaluated the incremental benefit of targeted RNA sequencing (RNAseq) in the identification of gene fusions and MET exon 14 (METex14) alterations in DNA sequencing (DNAseq) driver–negative lung cancers.

Experimental Design: Lung cancers driver negative by MSK-IMPACT underwent further analysis using a custom RNAseq panel (MSK-Fusion). Tumor mutation burden (TMB) was assessed as a potential prioritization criterion for targeted RNAseq.

Results: As part of prospective clinical genomic testing, we profiled 2,522 lung adenocarcinomas using MSK-IMPACT, which identified 195 (7.7%) fusions and 119 (4.7%) METex14 alterations. Among 275 driver-negative cases with available tissue, 254 (92%) had sufficient material for RNAseq. A previously undetected alteration was identified in 14% (36/254) of cases, 33 of which were actionable (27 in-frame fusions, 6 METex14). Of these 33 patients, 10 then received matched targeted therapy, which achieved clinical benefit in 8 (80%). In the 32% (81/254) of DNAseq driver–negative cases with low TMB [0–5 mutations/Megabase (mut/Mb)], 25 (31%) were positive for previously undetected gene fusions on RNAseq, whereas, in 151 cases with TMB >5 mut/Mb, only 7% were positive for fusions (P < 0.0001).

Conclusions: Targeted RNAseq assays should be used in all cases that appear driver negative by DNAseq assays to ensure comprehensive detection of actionable gene rearrangements. Furthermore, we observed a significant enrichment for fusions in DNAseq driver–negative samples with low TMB, supporting the prioritization of such cases for additional RNAseq.

Translational Relevance

Inhibitors targeting kinase fusions have shown dramatic and durable responses in lung cancer patients, making their comprehensive detection critical. Here, we evaluated the incremental benefit of targeted RNA sequencing (RNAseq) in the identification of gene fusions in patients where no clear mitogenic driver alteration is found by DNA sequencing (DNAseq)–based panel testing. We found actionable alterations (kinase fusions or MET exon 14 skipping) in 13% of cases apparently driver negative by previous DNAseq testing. Among the driver-negative samples tested by RNAseq, those with low tumor mutation burden (TMB) were significantly enriched for gene fusions when compared with the ones with higher TMB. In a clinical setting, such patients should be prioritized for RNAseq. Thus, a rational, algorithmic approach to the use of targeted RNA-based next-generation sequencing (NGS) to complement large panel DNA-based NGS testing can be highly effective in comprehensively uncovering targetable gene fusions or oncogenic isoforms not just in lung cancer but also more generally across different tumor types.

A Commentary is in the same issue at https://clincancerres.aacrjournals.org/content/25/15/4586?iss=15

Wake Up and Smell the Fusions: Single-Modality Molecular Testing Misses Drivers

by Kurtis D. Davies and Dara L. Aisner

Abstract

Multitarget assays have become common in clinical molecular diagnostic laboratories. However, all assays, no matter how well designed, have inherent gaps due to technical and biological limitations. In some clinical cases, testing by multiple methodologies is needed to address these gaps and ensure the most accurate molecular diagnoses.

See related article by Benayed et al., p. 4712

In this issue of Clinical Cancer Research, Benayed and colleagues illustrate the growing need to consider multiple molecular testing methodologies for certain clinical specimens (1). The rapidly expanding list of actionable molecular alterations across cancer types has resulted in the wide adoption of multitarget testing approaches, particularly those based on next-generation sequencing (NGS). NGS-based assays are commonly viewed as “one-stop shops” to detect a vast array of molecular variants. However, as Benayed and colleagues discuss, even well-designed and highly vetted NGS assays have inherent gaps that, under certain circumstances, are ideally addressed by analyzing the sample using an alternative approach.

In the article, the authors examined a cohort of lung adenocarcinoma patient samples that had been deemed “driver- negative” via MSK-IMPACT, an FDA-cleared test that is widely considered by experts in the field to be one of the best examples of a DNA-based large gene panel NGS assay (2). Of 589 driver-negative cases, 254 had additional material amenable for a different approach: RNA-based NGS designed specifically for gene fusion and oncogenic gene isoform detection. After accounting for quality control failures, 232 samples were successfully sequenced, and, among these, 36 samples (representing an astonishing 15.5% of tested cases) were found to be positive for a driver gene fusion or oncogenic isoform that had not been detected by DNA-based NGS. The real-world value derived from this orthogonal testing schema was more than theoretical, with 8 of 10 (80%) patients demonstrating clinical benefit when treated according to the alteration identified via the RNA-based approach.

To detect gene rearrangements that lead to oncogenic gene fusions (and to detect mutations and insertions/deletions that lead to MET exon 14 skipping), MSK-IMPACT employs hybrid capture-based enrichment of selected intronic regions from genomic DNA. While this approach has proven to be successful in a variety of settings, there are associated limitations that were determined in this study to underlie the discrepancies between MSK-IMPACT and the RNA-based assay. First, some introns that are involved in clinically actionable rearrangement events are very large, thus requiring substantial sequencing capital that can represent a disproportionate fraction of the assay. Despite the ability via NGS to perform sequencing at a large scale, this sequencing capacity is still finite, and thus decisions must be made to sacrifice coverage of certain large genomic regions to ensure sufficient sequencing depth for other desired genomic targets. In the case of MSK-IMPACT (and most other DNA-based NGS assays), certain important introns in NTRK3 and NRG1 are not included in covered content, simply because they are too large (>90 Kb each). The second primary problem with DNA-based analysis of introns is that they often contain highly repetitive elements that are extremely difficult to assess via NGS due to their recurring presence across the genome. Attempts to sequence these regions are largely unfruitful because any sequencing data obtained cannot be specifically aligned/mapped to the desired targeted region of the genome (3). This is particularly true for intron 31 of ROS1, because it contains two repetitive long interspersed nuclear elements, and many DNA-based assays, including MSK-IMPACT, poorly cover this intron (4). In this study by Benayed and colleagues, the most common discrepant alteration was fusion involving ROS1, which accounted for 10 of 36 (28%) cases. At least six of these, those that demonstrated fusion to ROS1 exon 32, were likely directly explained by incomplete intron 31 sequencing. RNA-based analysis is able to overcome the above described limitations owing to the simple fact that sequencing is focused on exons post-splicing and the need to sequence introns is entirely avoided (Fig. 1).

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Figure 1.

Schematic representation of underlying genomic complexities that can lead to false-negative gene fusion results in DNA-based NGS analysis. In some cases, RNA-based approaches may overcome the limitations of DNA-based testing.

Lack of sufficient intronic coverage could not account for all of the discrepancies between DNA-based and RNA-based analysis however. Six samples in the cohort were found to be positive for MET exon 14 skipping based on RNA. In five of these, genomic alterations in MET introns 13 or 14 were observed, however they did not conform to canonical splice site alterations and thus were not initially called (although this was addressed by bioinformatics updates). In RNA-based testing, however, determination of exon skipping is simplified such that, regardless of the specific genomic alteration that interferes with splicing, absence of the exon in the transcript is directly observed (5). In another two of the discrepant cases, tumor purity was observed to be low in the sample, meaning that the expected variant allele frequency (VAF) for a genomic event would also likely be low, potentially below detectable levels. However, overexpression of the fusions at the transcript level was theorized to compensate for low VAF (Fig. 1). Additional explanations for discordant findings between the assays included sample-specific poor sequencing in selected introns and complex rearrangements that hindered proper capture (Fig. 1).

The take home message from Benayed and colleagues is simply this: there is no perfect assay that will detect 100% of the potential actionable alterations in patient samples. Even an extremely well designed, thoroughly vetted, and FDA-cleared assay such as MSK-IMPACT will have inherent and unavoidable “holes” due to intrinsic limitations. The solution to this dilemma, as adeptly described by Benayed and colleagues, is additional testing using a different approach. While in an ideal world every clinical tumor sample would be tested by multiple modalities to ensure the most comprehensive clinical assessment, the reality is that these samples are often scant and testing is fiscally burdensome (and often not reimbursed). Therefore, algorithms to determine which samples should be reflexed to secondary assays after testing with a primary assay are critical for maximizing benefit. In this study, the first algorithmic step was lack of an identified driver (because activated oncogenic drivers tend to exist exclusively of each other), which amounted to 23% of samples tested with the primary assay. In addition, the authors found a significantly higher rate of actionable gene fusions in samples with a low (<5 mut/Mb) tumor mutational burden, meaning that this metric, which was derived from the primary assay, could also be used to help inform decision making regarding additional testing. While this scenario is somewhat specific to lung cancer, similar approaches could be prescribed on a cancer type–specific basis.

These findings should be considered a “wake-up call” for oncologists in regard to the ordering and interpretation of molecular testing. It is clear from these and other published findings that advanced molecular analysis has limitations that require nuanced technical understanding. As this arena evolves, it is critical for oncologists (and trainees) to gain an increased comprehension of how to identify when the “gaps” in a test might be most clinically relevant. This requires a level of technical cognizance that has been previously unexpected of clinical practitioners, yet is underscored by the reality that opportunities for effective targeted therapy can and will be missed if the treating oncologist is unaware of how to best identify patients for whom additional testing is warranted. This study also highlights the mantra of “no test is perfect” regardless of prestige of the testing institution, number of past tests performed, or regulatory status. NGS, despite its benefits, does not mean all-encompassing. It is only through the adaptability of laboratories to utilize knowledge such as is provided by Benayed and colleagues that advances in laboratory medicine can be quickly deployed to maximize benefits for oncology patients.

References:

1. Comprehensive genomic characterization of squamous cell lung cancers. Nature 2012, 489(7417):519-525.
2. A genomics-based classification of human lung tumors. Science translational medicine 2013, 5(209):209ra153.
3. Govindan R, Ding L, Griffith M, Subramanian J, Dees ND, Kanchi KL, Maher CA, Fulton R, Fulton L, Wallis J et al: Genomic landscape of non-small cell lung cancer in smokers and never-smokers. Cell 2012, 150(6):1121-1134.
4. Takeuchi K, Soda M, Togashi Y, Suzuki R, Sakata S, Hatano S, Asaka R, Hamanaka W, Ninomiya H, Uehara H et al: RET, ROS1 and ALK fusions in lung cancer. Nature medicine 2012, 18(3):378-381.
5. Morodomi Y, Takenoyama M, Inamasu E, Toyozawa R, Kojo M, Toyokawa G, Shiraishi Y, Takenaka T, Hirai F, Yamaguchi M et al: Non-small cell lung cancer patients with EML4-ALK fusion gene are insensitive to cytotoxic chemotherapy. Anticancer research 2014, 34(7):3825-3830.
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10. Peifer M, Fernandez-Cuesta L, Sos ML, George J, Seidel D, Kasper LH, Plenker D, Leenders F, Sun R, Zander T et al: Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer. Nature genetics 2012, 44(10):1104-1110.

Other posts on this site which refer to Lung Cancer and Cancer Genome Sequencing include:

Multi-drug, Multi-arm, Biomarker-driven Clinical Trial for patients with Squamous Cell Carcinoma called the Lung Cancer Master Protocol, or Lung-MAP launched by NCI, Foundation Medicine, and Five Pharma Firms

US Personalized Cancer Genome Sequencing Market Outlook 2018 –

Comprehensive Genomic Characterization of Squamous Cell Lung Cancers

International Cancer Genome Consortium Website has 71 Committed Cancer Genome Projects Ongoing

Non-small Cell Lung Cancer drugs – where does the Future lie?

Lung cancer breathalyzer trialed in the UK

Diagnosing Lung Cancer in Exhaled Breath using Gold Nanoparticles

Multi-drug, Multi-arm, Biomarker-driven Clinical Trial for patients with Squamous Cell Carcinoma called the Lung Cancer Master Protocol, or Lung-MAP launched by NCI, Foundation Medicine, and Five Pharma Firms

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