Molecular Profiling Technology​

Caris Life Sciences offers unique precision medicine services that are designed to maximize the chances of success for clinical trials and address many patient accrual challenges facing biopharma partners in the world of precision medicine.​

DNA Sequencing

Technical Specs

Technical Information Next-Generation Sequencing
Sample Requirements FFPE block or 10 unstained slides with a minimum of 20% malignant origin for DNA.
Needle biopsy is also acceptable (4-6 cores).
Tumor Enrichment (when necessary) Microdissection to isolate and increase the number of cancer cells to improve test performance
and increase the chance for successful testing from small tumor samples
Number of Genes ~22,000 genes
Average Depth of Coverage 1,000x for 720+ clinical and research genes; 400-500x for all other genes
Positive Percent Agreement (PPA) > 95% for base substitutions at ≥ 5% mutant allele frequency;
> 95% for indels at ≥ 5% mutant allele frequency;
>90% for copy number alterations (amplifications ≥ 6 copies)
Negative Percent Agreement (NPA) >99%
Genomic and Transcriptomic Signatures and Panels Microsatellite Instability (MSI),
Tumor Mutational Burden (TMB)
Caris FOLFIRSTai™ – AI predictor of FOLFOX response in metastatic colorectal adenocarcinoma
Caris GPSai™ Genomic Prevalence Score – CUP, atypical presentation or clinical ambiguity cases

Mutations

Point Mutations and Indels (DNA)

Copy Number Alterations​

Point Mutations, Indels and Copy Number Alterations (DNA)

Microsatellite Instability (MSI)

Caris Molecular Intelligence® tumor profiling includes microsatellite instability (MSI) testing via next-generation sequencing (NGS). MSI is caused by failure of the DNA mismatch repair (MMR) system. High levels of MSI correlate to an increased neoantigen burden, which may make the tumor more sensitive to immunotherapy. MSI status is reported on pages one and two of the MI Profile Report, as well as in the NGS section in the Appendix.

MSI-High Status Across Caris Molecular Intelligence Cases​

Earlier studies have associated MSI-High status with benefit to immunotherapy in metastatic colorectal cancer. Recent data, however, show that MSI is a useful indicator for predicting response to pembrolizumab in any solid tumor type.1

Traditionally, MSI is detected through polymerase chain reaction (PCR) by fragment analysis (FA) of five conserved satellite regions and comparing cancer tissue to normal tissue to identify differences in tandem repeats. To validate MSI testing via NGS, Caris evaluated more than 7,000 target microsatellite loci and compared the results from PCR for 2,189 cases across 26 different tumor types. This data was published in Cancer Medicine and demonstrated that MSI testing with Caris’ NGS platform is highly concordant with the traditional standard method of PCR-FA and is a more efficient and cost-effective approach to identifying patient candidates for immunotherapy.2

Traditional Approach: normal and cancer tissue required.

Caris Approach: no normal tissue required; saving resources, costs and time.

Attributions:

  1. D. T. Le, et al. Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade. Science. Published Online 8 June 2017. DOI: 10.1126/science.aan6733.
  2. Vanderwalde, A., Spetzler, D., Xiao, N., Gatalica, Z. and Marshall, J. (2018), Microsatellite instability status determined by next-generation sequencing and compared with PD-L1 and tumor mutational burden in 11,348 patients. Cancer Med. doi:10.1002/cam4.1372

Tumor Mutational Burden (TMB)​

TMB is a Pan-Tumor Biomarker for IO Response

TMB by Whole Exome Sequencing measures the total number of non-synonymous, somatic mutations identified per megabase
(Mb) of the genome coding area of DNA (a megabase is 1,000,000 DNA basepairs).

  • Non-synonymous mutations are changes in DNA that result in amino acid changes in the protein.1,2
  • The new protein changes result in new shapes (neo-antigens) that are considered to be foreign to the immune system.1,3
  • Immune checkpoint inhibitors are able to stimulate and allow the immune system to detect these neo-antigens and destroy the tumor.2
  • Germline (inherited) mutations are not included in TMB because the immune system has a higher likelihood of recognizing these alterations as normal.4

TMB has emerged as an important biomarker when considering immunotherapy in solid tumors. This is highlighted by the recent U.S. FDA accelerated approval of pembrolizumab (KEYTRUDA®) for the treatment of adult and pediatric patients with unresectable or metastatic tumor mutational burden-high (TMB-H) [≥10 mutations/megabase (mut/Mb)] solid tumors that have progressed following prior treatment and who have no satisfactory alternative treatment options. This approval is based on the results of the KEYNOTE-158 trial, which achieved an overall response rate of 29% (95% CI: 21, 39), with a 4% complete response rate and 25% partial response rate.5

TMB is included with all Caris Molecular Intelligence orders (MI Profile™ and MI Tumor Seek™) and is performed using Whole Exome Sequencing

Genomic profiling with Caris Molecular Intelligence can help you make more informed therapy decisions when
considering immune checkpoint inhibitors.

In addition, Caris has been working in collaboration with the Friends of Cancer Research TMB Harmonization Project to systematically characterize and standardize TMB testing and reporting to a common industry standard6. Based on this collective work and exciting KEYNOTE-158 result and drug approval, Caris has updated the TMB high/low threshold to reflect greater than or equal to 10 mutations per megabase across all solid tumors, aligning the testing results to pembrolizumab for TMB-H cases.5

Attributions:

1. Snyder A. N Engl J Med. 2014; 371:2189-2199. doi:10.1056/ NEJMoa1406498
2. Le DT. N Engl J Med. 2015;372:2509-2520. doi:10.1056/NEJMoa1500596
3. Rosenberg JE. The Lancet. 2016; 387(10031):1909-1920. doi:10.1016/S0140-6736(16)00561-4.
4. Stewart TJ. Oncogene. 2008;27:5894-5903. doi:10.1038/onc.2008.268
5. U.S. Food and Drug Administration. (2020, June 16). FDA approves pembrolizumab for adults and children with TMB-H solid tumors [Press release].
6. Stenzinger, A, Allen, JD, Maas, J, et al. Tumor mutational burden standardization initiatives: Recommendations for consistent tumor mutational burden assessment in clinical samples to guide immunotherapy treatment decisions. Genes Chromosomes Cancer. 2019; 58: 578– 588. https://doi.org/10.1002/gcc.22733

Loss of Heterozygosity (LOH)​

Caris Life Sciences® utilizes MI Exome™ (whole exome sequencing) to analyze 250,000 evenly-spaced single nucleotide polymorphisms (SNP) to measure genomic instability in the tumor. Genomic Loss of Heterozygosity (LOH) or genomic instability is often related to defective homologous recombination repair mechanisms. High levels of genomic instability may be indicative of PARPinhibitor and platinum therapy response.

Genomic LOH testing is provided at no additional cost and no increase in specimen requirements or turnaround time when MI Profile™ or MI Tumor Seek™ are ordered. The results can be found in the Genomic Signatures section of the Caris Molecular Intelligence® report, alongside Microsatellite Instability (MSI) and Tumor Mutational Burden (TMB) results.

What is Loss of Heterozygosity (LOH)?

  • Normally, there are 2 copies of every gene in a person’s DNA (excluding sex chromosomes in males)
  • When the copies are not identical, the person is considered heterozygous at that gene location
  • In cancer, DNA damage events can occur in the cell that causes the loss of one copy
    • In the second example shown here, the cell has lost copy B and, therefore, is no longer heterozygous at this location.
  • LOH can occur at the single-gene level or genome wide – which is called Genomic LOH

    • Single-gene level: In a person heterozygous for a tumor suppressor gene (one functional copy and one disabled copy), the loss of the functional copy can lead to cancer, as the person no longer has a working version of the tumor suppressor.
    • Genome-wide: When a person has lost a critical gene involved in DNA repair, chromosome deletions can appear throughout the genome, resulting in LOH at thousands of locations. Even if the DNA repair gene alteration is missed in testing, the detection of genomic LOH can identify a tumor that may be susceptible to drugs that impact the DNA-damage/repair pathway (PARP inhibitors or platinum agents).
    • The Caris assay measures genomic LOH in order to identify cases of potential homologous recombination deficiency that are not identified with standard NGS.
LOH_Graphic

RNA Sequencing​

Technical Specs​

Technical Information Whole Transcriptome Sequencing
Sample Requirements FFPE block or 2-5 unstained slides with a minimum of 20%
malignant origin. Needle biopsy is also acceptable (4-6 cores).
Tumor Enrichment (when necessary) Microdissection to isolate and increase the number of cancer cells to improve test performance
and increase the chance for successful testing from small tumor samples
Number of Genes ~22,000 genes
Average Read Count 60 million
Positive Percent Agreement (PPA) >97%
Negative Percent Agreement (NPA) >99%
Genomic and Transcriptomic Signatures and Panels

Caris GPSai™ Genomic Prevalence Score – CUP, atypical presentation or clinical ambiguity cases
Human Leukocyte Antigen (HLA) Genotype

Fusions

Variant Transcripts​

AR-V7
EGFR vIII
MET Exon 14 Skipping

Human Leukocyte Antigen (HLA) Genotype​

The HLA System Regulates the Immune System

The HLA system or complex is a group of related proteins that are encoded by the major histocompatibility complex (MHC) gene complex in humans.  These cell-surface proteins are responsible for the regulation of the immune system. The MHCs have two classes Class I and Class II that differ by function and dimerization (binding) patterns.   

The proteins encoded by HLAs are those on the outer part of body cells that are unique to that person. The immune system uses the HLAs to differentiate self-cells and non-self-cells. Any cell displaying that person’s HLA type belongs to that person and, therefore, is not an invader. 1

HLAs have multiple roles in disease defense:

  • They may protect against or fail to protect (if down-regulated by an infection) against cancers.2 Cancer researchers are looking to see if there are any relationships between HLA variants and certain cancers.
  • HLA genotypes may have an impact on drug response and prognosis.  There is ongoing research exploring this relationship with cancer immunotherapy.3
  • They tell the body what cells are self and what cells are invaders.4
  • Mutations in HLA may be linked to autoimmune disease (examples: type I diabetes, coeliac disease).5 Medical researchers are examining the relationships between HLA variants and a variety of autoimmune diseases.

HLA Genotyping* is included with all Caris Molecular orders (MI Profile™ and MI Tumor Seek™) and is performed using Whole Transcriptome Sequencing.

HLA Genotyping adds value to the Caris Molecular Report.

Understanding a patient’s complete profile through Caris Molecular Intelligence not only provides the most comprehensive genomic profile that can help identify the best course of today’s treatment, but it can continue to enhance a provider’s clinical approach as evidence evolves.

There is ongoing research that observed a correlation between patients’ HLA genotype with a response to checkpoint blockade immunotherapy. A patient’s genotype may also direct them to clinical trials recruiting for specific genotypes.  Understanding patient HLA genotypes can be utilized in the development of both personalized cancer vaccine development as well as in the immunotherapy biomarker discovery.

*Not available in New York State.

References:

  1. Reference, Genetics Home. “Histocompatibility complex”. Genetics Home Reference. Retrieved 1 May2020.
  2. Nobuoka D, Yoshikawa T, Fujiwara T, Nakatsura T. Peptide intra-tumor injection for cancer immunotherapy: enhancement of tumor cell antigenicity is a novel and attractive strategy. Human vaccines & immunotherapeutics. 2013;9:1234–1236. doi: 10.4161/hv.23990. 
  3. Wang, C., Xiong, C., Hsu, Y., Wang, X., & Chen, L. (2020). Human leukocyte antigen (HLA) and cancer immunotherapy: HLA-dependent and -independent adoptive immunotherapies. Annals Of Blood, 5. doi:10.21037/aob-20-27
  4. Duquesnoy RJ. Histocompatibility testing in organ transplantation. University of Pittsburgh Medical Center Web site [http://tpis.upmc.edu/tpis/immuno/compre.htm];1996.
  5. J. Simmonds and S. C.L. Gough, “ The HLA Region and Autoimmune Disease: Associations and Mechanisms of Action”, Current Genomics (2007) 8: 453. https://doi.org/10.2174/138920207783591690

Artificial Intelligence​

Caris FOLFIRSTai

Caris FOLFIRSTai™ is an Artificial Intelligence-powered predictor of FOLFOX response that utilizes Caris Molecular Intelligence® tumor profiling results. It is intended to be used as an aid in gauging a patient’s likelihood to benefit from FOLFOX chemotherapy (in combination with bevacizumab) as the first-line chemotherapy regimen in metastatic colorectal adenocarcinoma. FOLFIRSTai* is included for all metastatic colorectal adenocarcinoma cases. The FOLFIRSTai results appear on the front page of the Caris report as INCREASED BENEFIT or DECREASED BENEFIT – with additional detail provided about the results on page two of the report. This information provides additional insight for patient response to FOLFOX as a first-line therapeutic option. FOLFIRSTai was validated using two independent data sets:
  • 296 manually curated cases with real-world evidence (data acquired from insurance claims records, electronic medical records and death registries)
    • Median Overall Survival difference between the increased benefit arm and the decreased benefit arm: 11.2 months
  • 149 cases analyzed retrospectively from the randomized, prospective Phase III TRIBE2 study
    • Median Overall Survival difference between the increased benefit arm and the decreased benefit arm: 6.0 months
Patients predicted to have increased benefit to FOLFOX may achieve optimal results by receiving a FOLFOX regimen first in their chemotherapy sequencing plan. Patients predicted to have decreased benefit to FOLFOX may achieve results by receiving an alternate regimen, such as FOLFOXIRI or FOLFIRI, prior to the administration of a FOLFOX regimen.

*Not available in New York State.

Decisions on patient care and treatment must be based on the independent medical judgment of the treating physician, taking into consideration all available information concerning the patient’s condition. 

MI GPSai™​ | Genomic Prevalence Score

Caris has one of the largest and most comprehensive databases of combined molecular and clinical outcomes data in the world, and we are actively employing advanced machine learning capabilities with the database to identify unique molecular signatures. These molecular signatures can be used to better identify cancer subtypes and predict patient response to certain therapies. We are pleased to introduce a tool to help manage cancer of unknown primary (CUP) or cases identified by the ordering physician with atypical clinical presentation or clinical ambiguity.

MI GPSai™* provides a cancer type similarity assessment that compares the genomic (DNA) and transcriptomic (RNA) characteristics of the patient’s tumor against other tumors in the Caris database (e.g. lung cancer tumor submitted for testing has a similar molecular signature as the lung cancers found in the Caris Database, or conversely the molecular signature is not similar to lung cancer, but similar to another tumor type’s molecular signature).

MI GPS ai™ can be added to any solid tumor order by selecting the appropriate box on the tumor profiling requisition. The result is presented as a prevalence score in a convenient tabular format and is populated onto the final Caris report. These results will provide additional insight by assessing how closely tumors match the genomic and transcriptomic signatures of tissue types to help you make more informed treatment decisions.

Caris Molecular Artificial Intelligence (MAI™) uses the power of DEAN (Deliberation Analytics) and machine learning technology to provide oncologists with the most thorough genomic and transcriptomic classifications to inform decision making. Caris MAI™ analyzes historical clinical and outcome data and learns from the past to provide for a better future via molecular subtyping.

*Not available in New York State.

 

 

Other

IHC | Immunohistochemistry

Immunohistochemistry (IHC) determines the level of protein expression in a tumor, which can be used in conjunction with CISH or FISH to validate date or provide complementary information that provides greater insights into various cancer types.

CISH | Chromogenic in Situ Hybridization

Chromogenic in situ hybridization (CISH) is an assay that uses chromogenic probes to visualize specific regions of DNA in a tissue specimen using a bright-field microscope, similar to standard immunohistochemistry. CISH allows for the enumeration of a variety of chromosomal abnormalities including gene amplifications, deletions, and translocations. This assay is often utilized at Caris Life Sciences in the reflex setting; for instance, when further clarity is needed to substantiate a result or when tissue is limited. CISH can be performed as a tissue-sparing alternative to analyze a specific gene of interest.

FISH | Fluorescence in Situ Hybridization

Fluorescence in situ hybridization (FISH) is an assay that uses fluorescent probes to visualize specific nucleic acid regions (DNA or RNA) in a tissue specimen using a fluorescent microscope. FISH allows for the enumeration of a variety of chromosomal abnormalities including gene amplifications, deletions, and translocations. This assay is often employed at Caris Life Sciences in the reflex setting; for instance, when further clarity is needed to substantiate a result or when tissue is limited. FISH can be performed as a tissue-sparing alternative to analyze a specific gene of interest.
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