Supplementary Note 1. A sample alteration report. Each alteration nominated by PHIAL is curated to answer specific fields that are intended to guide physician interpretation. Gene Alteration Patient ID Tumor Type KRAS p.a146v DFCI.11-104.03 Lung Adenocarcinoma Sequencing Issues Gene Description (Brief Introduction to this gene) Activating mutations in KRAS are among the most common genetic alterations in human tumors. KRAS mutations play a central role in tumor progression in multiple cancer types, and have been implicated in poor prognosis and resistance to therapy. Gene Frequency (Incidence of alterations in the gene in cancer overall and in this cancer in particular) KRAS alterations are common across numerous malignancies. KRAS mutations are the most common molecular change found in NSCLC. Activating KRAS mutations are found in 20 30% of all patients with lung adenocarcinoma. In TCGA, 25% of lung adenocarcinomas were found to have KRAS mutations (cbio, April 2013). Alteration Function (function of this alteration (activating, likely activating, inactivating, likely inactivating, unknown/uncharacterized) and rationale) This alteration is a known activating mutation, though may be less potent than the more common codon 12 and 13 mutations (PMID: 20570890). Alteration Frequency in this tumor type This specific alteration has not been previously reported in lung adenocarcinoma. A single patient with lung adenocarcinoma patient was found to have a KRAS A146P mutation (COSMIC, April 2013). A146 mutations in KRAS were not found In 2 studies comprised 449 cases of NSCLC in which KRAS was sequenced in its entirety (PMID: 18948947, 18632602). More than 90% of KRAS mutations in lung cancer are codon 12 mutations. Alteration Frequency in other tumor types (if uncommon in this tumor type) This alteration has rarely been found in other cancer types. This alteration has only been reported in 15 colorectal cancer cases in the COSMIC database. An additional 68 cases of A146T have been reported in colorectal cancer in the COSMIC database. However, one systematic study of exon 4 mutations in colorectal cancer demonstrated the presence of A146 mutations in 5% of colon cancers (PMID: 20570890). Prognostic implications (prognostic implications of this alteration and/or of this functional change (activation, loss, etc) Activating mutations in KRAS havehas been shown to be associated with a poor prognosis in NSCLC (Reviewed in PMID: 23401440), though these studies have generally only included codon 12 and 13 mutations. Diagnostic implications (diagnostic implications of this alteration and/or of this functional change (activation, loss, etc) Therapeutic implications (therapeutic implications (sensitivity and resistance) of this alteration and/or of this functional change (activation, loss, etc) Activating KRAS mutations may predict resistance to anti- EGFR therapies, which are FDA- approved for use in lung adenocarcinoma (Reviewed in PMID: 23401440).
Many clinical trials of novel therapeutic agents have been designed specifically for use in KRAS- mutant lung adenocarcinoma (Reviewed in PMID: 23401440). Because activating mutations in KRAS may predict sensitivity to inhibitors of the RAS/RAF/MEK/ERK pathway, many of these trials focus on specific targeting of the MAP- kinase pathway (especially MEK). Preclinical studies have shown that MEK inhibitors, in particular, may be effective for KRAS mutant tumors, and these agents are in clinical trials for patients with KRAS mutant cancers. A recent randomized phase II study of selumetinib (AZD6244) in combination with docetaxel versus docetaxel alone demonstrated an improved PFS in patients with KRAS- mutant, locally advanced, or metastatic NSCLC (PMID: 23200175). However, the parallel mouse lung cancer co- clinical trial suggests that concomitant loss of STK11 with activating mutations in KRAS may confer primary resistance to combination therapy with docetaxel and the MEK- inhibitor selumetinib (AZD6244), possibly through activation of parallel signalling pathways such as AKT and SRC (PMID: 22425996). In addition to selumetinib, several other MEK inhibitors are also in clinical development including trametinib (GSK1120212), GDC- 0973, MEK162, and pimasertib (AS703026 or MSC1936369B). Based on preclinical data, therapies targeting the PI3K/AKT/mTOR pathway are also being investigated in KRAS- mutant lung cancers (Reviewed in PMID: 23401440). Data from preclinical KRAS- mutant, genetically engineered mouse models of NSCLC suggest that dual inhibition of the PI3K and MEK pathways are required, and clinical trials of this combination are underway. Other agents that are being studied in KRAS mutant lung cancers include HSP90 inhibitors (PMID: 23012248). There is preclinical evidence data implicating a synthetic lethal relationship between activated KRAS and CDK440 (PMID: 20609353). Finally, recent clinical data suggests a synthetical lethal interaction of the combination of MEK inhibitors and the BCL- XL inhibitor ABT- 263 (navitoclax) in KRAS mutant cancers (PMID: 23245996). Examples of ongoing clinical trials for KRAS- mutant lung cancers include trials of MEK inhibitors (NCT00890825, NCT01362296). MEK inhibitors + PI3K inhibitors (NCT01449058), MEK inhibitors + RAF inhibitors (NCT01352273), MEK inhibitors + EGFR inhibitors (NCT01229150), MEK inhibitors + IGF1- R inhibitors (NCT01562899), Bortezomib (NCT01833143), HSP90 inhibitors (NCT01427946, NCT01348126) and others (clinicaltrials.gov, April 2013). (Therapies in Clinical Trials, LEVEL A). CATEGORIZATION AND LEVELS OF EVIDENCE CATEGORY: LEVEL A LEVEL B LEVEL C LEVEL D LEVEL E FDAapproved therapies Resistance to erlotinib Therapies in clinical trials MEK inhibitors PI3K inhibitors RAF inhibitors IGF1- R inhibitors Bortezomib HSP90 inhibitors Combinations CDK inhibitors BCL- XL + MEK inhibitors Prognostic Negative Prognostic Marker Diagnostic
Gene Alteration Patient ID Tumor Type STK11 p.g279fs DFCI.11-104.03 Lung Adenocarcinoma Sequencing Issues Gene Description (Brief Introduction to this gene) STK11 is a well- known tumor suppressor (also known as LKB1) that is commonly inactivated in several cancers. Germline mutations in STK11 cause Puetz- Jeghers Syndrome (PJS). Gene Frequency (Incidence of alterations in the gene in cancer overall and in this cancer in particular) STK11 is mutated in 15-20% of patients with lung adenocarcinoma. It is commonly seen in conjunction with KRAS mutations. In TCGA, 15% of lung adenocarcinomas were found to have STK11 mutations or deletions (cbio, April 2013). Alteration Function (function of this alteration (activating, likely activating, inactivating, likely inactivating, unknown/uncharacterized) and rationale) This alteration is likely inactivating, as it is a frameshift mutation that occurs at codon 279 out of 434. Alteration Frequency in this tumor type This specific alteration has not been reported in the COSMIC database for NSCLC, though inactivating mutations in STK11 are common in this tumor type, occurring at a rate of 5-20% of NSCLC. They commonly co- occur with KRAS mutations. Alteration Frequency in other tumor types (if uncommon in this tumor type) N/A Prognostic implications (prognostic implications of this alteration and/or of this functional change (activation, loss, etc) Unknown. Diagnostic implications (diagnostic implications of this alteration and/or of this functional change (activation, loss, etc) Therapeutic implications (therapeutic implications (sensitivity and resistance) of this alteration and/or of this functional change (activation, loss, etc) Loss of STK11 activates the MTOR pathway and therefore may predict sensitivity to inhibitors of this pathway. Preclinical evidence suggests that MTOR inhibitors are effective in cancers with STK11 loss, and there is at least 1 case report of a pancreatic cancer with STK11 loss with a response to an MTOR inhibitor (PMID 21189378, Level C). A clinical trial of everolimus in PJS patients with advanced cancer is now underway. In lung cancer, preclinical evidence suggests that PI3K/MTOR inhibition may be effective specifically in STK11/KRAS mutant lung cancers (PMID: 21998291). A recent mouse lung cancer co- clinical trial suggests that concomitant loss of STK11 with activating mutations in KRAS may confer primary resistance to combination therapy with docetaxel and the MEK- inhibitor selumetinib (AZD6244), possibly through activation of parallel signalling pathways such as AKT and SRC (PMID: 22425996). However, a recent study presented at the AACR 2013 annual meeting demonstrated conflicting results, showing that cancer cell lines with STK11 loss were sensitive to MEK inhibition. ("LKB1 loss leads to activation of the CREB transcription factor and sensitivity to MEK inhibition in human lung cancer", Kaufman and Carbone, AACR 2013 Annual Meeting). Another preclinical study showed that SRC and FAK kinases are activated in STK11- deficient primary and metastatic lung tumors, and these cells are sensitive to the SRC- inhibitor dasatinib and/or the FAK inhibitor PF573228 (PMID: 20541700). Moreover, the addition
of dasatinib restored sensitivity of mouse tumors to the combination of PI3K + MEK1/2 inhibitors, which are effective in KRAS mutant mouse tumors but not in KRAS/STK11 mutant mouse tumors. A recent preclinical study identified phenformin, a mitochondrial inhibitor and analog of the diabetes therapeutic metformin, as selectively inducing apoptosis in LKB1- deficient NSCLC cells. Therapeutic trials in mouse models of NSCLC revealed that tumors with Kras and Lkb1 mutations, but not those with Kras and p53 mutations, showed selective response to phenformin as a single agent, resulting in prolonged survival (PMID: 23352126). A clinical trials of metformin in lung cancer is currently underway (NCT01578551). CATEGORIZATION AND LEVELS OF EVIDENCE CATEGORY: LEVEL A LEVEL B LEVEL C LEVEL D LEVEL E FDAapproved therapies Everolimus Temsirolimus Therapies in clinical trials MEK inhibitors PI3K inhibitors MTOR Inhibitors SRC inhibitors FAK Inhibitors Metformin Combinations Prognostic Diagnostic
Gene Alteration Patient ID Tumor Type ATM p.k208fs DFCI.11-104.03 Lung Adenocarcinoma Sequencing Issues Gene Description (Brief Introduction to this gene) ATM is a known tumor suppressor and has been implicated in multiple cancer types. Germline mutations in ATM cause Ataxia Telangiectasia. Gene Frequency (Incidence of alterations in the gene in cancer overall and in this cancer in particular) ATM inactivation is common and has been implicated in multiple cancer types. Alteration Function (function of this alteration (activating, likely activating, inactivating, likely inactivating, unknown/uncharacterized) and rationale) This alteration is likely inactivating, as it is a frameshift mutation that occurs at codon 208 out of 3057. Alteration Frequency in this tumor type This specific alteration has not been reported in the COSMIC database for NSCLC, though inactivating mutations in ATM have been reported in this tumor type. Alteration Frequency in other tumor types (if uncommon in this tumor type) This specific alteration has not been reported in the COSMIC database for other cancers, though inactivating mutations in ATM are common in several tumors, especially lymphoid tumors such as mantle cell lymphoma and CLL. Prognostic implications (prognostic implications of this alteration and/or of this functional change (activation, loss, etc) N/A Diagnostic implications (diagnostic implications of this alteration and/or of this functional change (activation, loss, etc) Therapeutic implications (therapeutic implications (sensitivity and resistance) of this alteration and/or of this functional change (activation, loss, etc) Inactivation of ATM may predict sensitivity to PARP inhibitors. Preclinical evidence from cell culture and mouse xenograft models demonstrated that ATM- deficient lymphoid tumor cells were sensitive to PARP inhibition (PMID: 22416035). A clinical trial of the PARP inhibitor olaparib that enrolled patients with ATM- deficient gastric cancer was recently completed.
CATEGORIZATION AND LEVELS OF EVIDENCE CATEGORY: LEVEL A LEVEL B LEVEL C LEVEL D LEVEL E FDAapproved therapies Therapies in clinical trials PARP Inhibitors Prognostic Diagnostic