PATIENTS AT THE CENTER OF EVERYTHING WE DO Main Menu Patients Physicians Test Menu Research POA Careers About MI PORTAL ORDER A TEST MOLECULAR PROFILING TECHNOLOGY 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 RNA AI OTHER 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) ABI1 BRD4 CRLF2 FOXO4 HOXC11 KLF4 MUC1 PAK3 RHOH TAL2 ABL1 BTG1 DDB2 FSTL3 HOXC13 KLK2 MUTYH PATZ1 RNF213 TBL1XR1 ACKR3 BTK DDIT3 GATA1 HOXD11 LASP1 MYCL (MYCL1) PAX8 RPL10 TCEA1 AKT1 C15orf65 DNM2 GATA2 HOXD13 LMO1 NBN PDE4DIP SEPT5 TCL1A AMER1 (FAM123B) CBLC DNMT3A GNA11 HRAS LMO2 NDRG1 PHF6 SEPT6 TERT AR CD79B EIF4A2 GPC3 IKBKE MAFB NKX2-1 PHOX2B SFPQ TFE3 ARAF CDH1 ELF4 HEY1 INHBA MAX NONO PIK3CG SLC45A3 TFPT ATP2B3 CDK12 ELN HIST1H3B IRS2 MECOM NOTCH1 PLAG1 SMARCA4 THRAP3 ATRX CDKN2B ERCC1 HIST1H4I JUN MED12 NRAS PMS1 SOCS1 TLX3 BCL11B CDKN2C ETV4 HLF KAT6A (MYST3) MKL1 NUMA1 POU5F1 SOX2 TMPRSS2 BCL2 CEBPA FAM46C HMGN2P46 KAT6B MLLT11 NUTM2B PPP2R1A SPOP UBR5 BCL2L2 CHCHD7 FANCF HNF1A KCNJ5 MN1 OLIG2 PRF1 SRC VHL BCOR CNOT3 FEV HOXA11 KDM5C MPL OMD PRKDC SSX1 WAS BCORL1 COL1A1 FOXL2 HOXA13 KDM6A MSN P2RY8 RAD21 STAG2 ZBTB16 BRD3 COX6C FOXO3 HOXA9 KDSR MTCP1 PAFAH1B2 RECQL4 TAL1 ZRSR2 Copy Number Alterations Point Mutations, Indels and Copy Number Alterations (DNA) ABL2 BRCA1 CREB3L1 ETV5 GID4 (C17orf39) KNL1 NACA PIM1 SDHC TNFRSF17 ACSL3 BRCA2 CREB3L2 ETV6 GMPS KRAS NCKIPSD PML SDHD TOP1 ACSL6 BRIP1 CREBBP EWSR1 GNA13 KTN1 NCOA1 PMS2 SEPT9 TP53 ADGRA2 BUB1B CRKL EXT1 GNAQ LCK NCOA2 POLE SET TPM3 AFDN CACNA1D CRTC1 EXT2 GNAS LCP1 NCOA4 POT1 SETBP1 TPM4 AFF1 CALR CRTC3 EZH2 GOLGA5 LGR5 NF1 POU2AF1 SETD2 TPR AFF3 CAMTA1 CSF1R EZR GOPC LHFPL6 NF2 PPARG SF3B1 TRAF7 AFF4 CANT1 CSF3R FANCA GPHN LIFR NFE2L2 PRCC SH2B3 TRIM26 AKAP9 CARD11 CTCF FANCC GRIN2A LPP NFIB PRDM1 SH3GL1 TRIM27 AKT2 CARS CTLA4 FANCD2 GSK3B LRIG3 NFKB2 PRDM16 SLC34A2 TRIM33 AKT3 CASP8 CTNNA1 FANCE H3F3A LRP1B NFKBIA PRKAR1A SMAD2 TRIP11 ALDH2 CBFA2T3 CTNNB1 FANCG H3F3B LYL1 NIN PRRX1 SMAD4 TRRAP ALK CBFB CYLD FANCL HERPUD1 MAF NOTCH2 PSIP1 SMARCB1 TSC1 APC CBL CYP2D6 FAS HGF MALT1 NPM1 PTCH1 SMARCE1 TSC2 ARFRP1 CBLB DAXX FBXO11 HIP1 MAML2 NR4A3 PTEN SMO TSHR ARHGAP26 CCDC6 DDR2 FBXW7 HMGA1 MAP2K1 (MEK1) NSD1 PTPN11 SNX29 TTL ARHGEF12 CCNB1IP1 DDX10 FCRL4 HMGA2 MAP2K2 (MEK2) NSD2 PTPRC SOX10 U2AF1 ARID1A CCND1 DDX5 FGF10 HNRNPA2B1 MAP2K4 NSD3 RABEP1 SPECC1 USP6 ARID2 CCND2 DDX6 FGF14 HOOK3 MAP3K1 NT5C2 RAC1 SPEN VEGFA ARNT CCND3 DEK FGF19 HSP90AA1 MCL1 NTRK1 RAD50 SRGAP3 VEGFB ASPSCR1 CCNE1 DICER1 FGF23 HSP90AB1 MDM2 NTRK2 RAD51 SRSF2 VTI1A ASXL1 CD274 (PDL1) DOT1L FGF3 IDH1 MDM4 NTRK3 RAD51B SRSF3 WDCP ATF1 CD74 EBF1 FGF4 IDH2 MDS2 NUP214 RAF1 SS18 WIF1 ATIC CD79A ECT2L FGF6 IGF1R MEF2B NUP93 RALGDS SS18L1 WISP3 ATM CDC73 EGFR FGFR1 IKZF1 MEN1 NUP98 RANBP17 STAT3 WRN ATP1A1 CDH11 ELK4 FGFR1OP IL2 MET NUTM1 RAP1GDS1 STAT4 WT1 ATR CDK4 ELL FGFR2 IL21R MITF PALB2 RARA STAT5B WWTR1 AURKA CDK6 EML4 FGFR3 IL6ST MLF1 PAX3 RB1 STIL XPA AURKB CDK8 EMSY FGFR4 IL7R MLH1 PAX5 RBM15 STK11 XPC AXIN1 CDKN1B EP300 FH IRF4 MLLT1 PAX7 REL SUFU XPO1 AXL CDKN2A EPHA3 FHIT ITK MLLT10 PBRM1 RET SUZ12 YWHAE BAP1 CDX2 EPHA5 FIP1L1 JAK1 MLLT3 PBX1 RICTOR SYK ZMYM2 BARD1 CHEK1 EPHB1 FLCN JAK2 MLLT6 PCM1 RMI2 TAF15 ZNF217 BCL10 CHEK2 EPS15 FLI1 JAK3 MNX1 PCSK7 RNF43 TCF12 ZNF331 BCL11A CHIC2 ERBB2 (HER2/NEU) FLT1 JAZF1 MRE11 PDCD1 (PD1) ROS1 TCF3 ZNF384 BCL2L11 CHN1 ERBB3 (HER3) FLT3 KDM5A MSH2 PDCD1LG2 (PDL2) RPL22 TCF7L2 ZNF521 BCL3 CIC ERBB4 (HER4) FLT4 KDR (VEGFR2) MSH6 PDGFB RPL5 TET1 ZNF703 BCL6 CIITA ERC1 FNBP1 KEAP1 MSI2 PDGFRA RPN1 TET2 BCL7A CLP1 ERCC2 FOXA1 KIAA1549 MTOR PDGFRB RPTOR TFEB BCL9 CLTC ERCC3 FOXO1 KIF5B MYB PDK1 RUNX1 TFG BCR CLTCL1 ERCC4 FOXP1 KIT MYC PER1 RUNX1T1 TFRC BIRC3 CNBP ERCC5 FUBP1 KLHL6 MYCN PICALM SBDS TGFBR2 BLM CNTRL ERG FUS KMT2A (MLL) MYD88 PIK3CA SDC4 TLX1 BMPR1A COPB1 ESR1 GAS7 KMT2C (MLL3) MYH11 PIK3R1 SDHAF2 TNFAIP3 BRAF CREB1 ETV1 GATA3 KMT2D (MLL2) MYH9 PIK3R2 SDHB TNFRSF14 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: 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. 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. 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 ABL BRD3 ETV5 INSR MYB NUMBL PRKCA RSPO3 AKT3 BRD4 ETV6 MAML2 NOTCH1 NUTM1 PRKCB TERT ALK EGFR EWSR1 MAST1 NOTCH2 PDGFRA RAF1 TFE3 ARHGAP26 ERG FGFR1 MAST2 NRG1 PDGFRB RELA TFEB AXL ESR1 FGFR2 MET NTRK1 PIK3CA RET THADA BCR ETV1 FGFR3 MSMB NTRK2 PKN1 ROS1 TMPRSS2 BRAF ETV4 FGR MUSK NTRK3 PPARG RSPO2 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: Reference, Genetics Home. “Histocompatibility complex”. Genetics Home Reference. Retrieved 1 May2020. 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. 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 Duquesnoy RJ. Histocompatibility testing in organ transplantation. University of Pittsburgh Medical Center Web site [http://tpis.upmc.edu/tpis/immuno/compre.htm];1996. 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.