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Whole-exome sequencing and clinical interpretation of formalin-fixed, paraffin-embedded tumor samples to guide precision cancer medicine.

Van Allen EM, Wagle N, Stojanov P, Perrin DL, Cibulskis K, Marlow S, Jane-Valbuena J, Friedrich DC, Kryukov G, Carter SL, McKenna A, Sivachenko A, Rosenberg M, Kiezun A, Voet D, Lawrence M, Lichtenstein LT, Gentry JG, Huang FW, Fostel J, Farlow D, Barbie D, Gandhi L, Lander ES, Gray SW, Joffe S, Janne P, Garber J, MacConaill L, Lindeman N, Rollins B, Kantoff P, Fisher SA, Gabriel S, Getz G, Garraway LA - Nat. Med. (2014)

Bottom Line: The platform employs computational methods for effective clinical analysis and interpretation of WES data.When applied retrospectively to 511 exomes, the interpretative framework revealed a 'long tail' of somatic alterations in clinically important genes.Prospective application of this approach identified clinically relevant alterations in 15 out of 16 patients.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA. [2] Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.

ABSTRACT
Translating whole-exome sequencing (WES) for prospective clinical use may have an impact on the care of patients with cancer; however, multiple innovations are necessary for clinical implementation. These include rapid and robust WES of DNA derived from formalin-fixed, paraffin-embedded tumor tissue, analytical output similar to data from frozen samples and clinical interpretation of WES data for prospective use. Here, we describe a prospective clinical WES platform for archival formalin-fixed, paraffin-embedded tumor samples. The platform employs computational methods for effective clinical analysis and interpretation of WES data. When applied retrospectively to 511 exomes, the interpretative framework revealed a 'long tail' of somatic alterations in clinically important genes. Prospective application of this approach identified clinically relevant alterations in 15 out of 16 patients. In one patient, previously undetected findings guided clinical trial enrollment, leading to an objective clinical response. Overall, this methodology may inform the widespread implementation of precision cancer medicine.

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Clinical sequencing informs clinical trial enrollment and experimental discoveryThe PHIAL output and treatment course for a patient with metastatic lung adenocarcinoma is shown, with the integration of clinical WES occurring during the patient’s first-line therapy allowing subsequent clinical trial enrollment (A). The patient’s time to relapse data for the three treatment regimens received demonstrate that the best and only clinical response occurred with the CDK4 inhibitor (B). Radiographic imaging demonstrates a small reduction in a representative metastatic focus for the patient on the CDK4 inhibitor trial after two cycles of therapy consistent with stable disease (cm: centimeter; measurement is 1.7 × 1.5 cm for baseline mass and 1.3 × 1.3 cm for two month interval scan of the same mass). Per RECIST criteria, overall tumor reduction was 7.9% (C). For another patient, PHIAL nominated a JAK3 missense mutation (D), and given its location in the kinase domain near alterations previously defined as activating, was considered to have inferential evidence (Level E) for being clinically actionable. The crystal structure of JAK3 demonstrates that the arginine at residue 870 directly coordinates the phosphate group of the primary activating tyrosine phosphorylation site (E). To better characterize this alteration, experimental follow-up of this alteration was performed in a Ba/F3 system. Overexpression of the patient’s JAK3 mutation did not suggest an activating phenotype or further consideration of JAK3 inhibitor clinical trial enrollment (F).
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Figure 5: Clinical sequencing informs clinical trial enrollment and experimental discoveryThe PHIAL output and treatment course for a patient with metastatic lung adenocarcinoma is shown, with the integration of clinical WES occurring during the patient’s first-line therapy allowing subsequent clinical trial enrollment (A). The patient’s time to relapse data for the three treatment regimens received demonstrate that the best and only clinical response occurred with the CDK4 inhibitor (B). Radiographic imaging demonstrates a small reduction in a representative metastatic focus for the patient on the CDK4 inhibitor trial after two cycles of therapy consistent with stable disease (cm: centimeter; measurement is 1.7 × 1.5 cm for baseline mass and 1.3 × 1.3 cm for two month interval scan of the same mass). Per RECIST criteria, overall tumor reduction was 7.9% (C). For another patient, PHIAL nominated a JAK3 missense mutation (D), and given its location in the kinase domain near alterations previously defined as activating, was considered to have inferential evidence (Level E) for being clinically actionable. The crystal structure of JAK3 demonstrates that the arginine at residue 870 directly coordinates the phosphate group of the primary activating tyrosine phosphorylation site (E). To better characterize this alteration, experimental follow-up of this alteration was performed in a Ba/F3 system. Overexpression of the patient’s JAK3 mutation did not suggest an activating phenotype or further consideration of JAK3 inhibitor clinical trial enrollment (F).

Mentions: The prospective WES framework was used for clinical decision-making in one demonstrative case. A patient with metastatic lung adenocarcinoma underwent standard clinical genetic testing that revealed wild-type EGFR, KRAS (codon 12 and 13), and ALK status. Mass spectrometry testing of 471 alterations in 41 genes5 revealed an STK11 frameshift deletion. The patient was started on carboplatin, paclitaxel, and bevacizumab (Fig. 5A). In parallel, we applied the clinical WES platform on the FFPE metastatic tumor sample and germline peripheral blood. PHIAL nominated a KRASA146V mutation, along with alterations in STK11 (identical to other testing) and ATM (Fig. 5A, Supplementary Table 8). KRASA146V is a known activating mutation, though possibly less potent than the codon 12 and 13 mutations28. Although activating KRAS mutations are found in 15–30% of all patients with non-small cell lung cancer (NSCLC) and commonly in conjunction with STK11 loss29, this specific KRAS alteration has not been reported in NSCLC20,30–32. KRAS146V was confirmed using the same FFPE tumor sample in a clinical lab that met Clinical Laboratory Improvement Amendments (CLIA) standards (Knight Diagnostic Laboratories, Oregon), and then returned to the patient’s oncologist. After rapidly progressing on combination chemotherapy (Fig. 5B), the patient was enrolled in a phase I CDK4 inhibitor (LY2835219) clinical trial based on preclinical data (Level D) implicating a synthetic lethal relationship between activated KRAS and CDK433. The patient achieved stable disease (per RECIST 1.1 criteria; 7.9% reduction in tumor volume compared to baseline) and was on therapy for 16 weeks (Fig. 5B–C). Of note, this represented the patient’s best and only clinical response to any cancer-directed therapy.


Whole-exome sequencing and clinical interpretation of formalin-fixed, paraffin-embedded tumor samples to guide precision cancer medicine.

Van Allen EM, Wagle N, Stojanov P, Perrin DL, Cibulskis K, Marlow S, Jane-Valbuena J, Friedrich DC, Kryukov G, Carter SL, McKenna A, Sivachenko A, Rosenberg M, Kiezun A, Voet D, Lawrence M, Lichtenstein LT, Gentry JG, Huang FW, Fostel J, Farlow D, Barbie D, Gandhi L, Lander ES, Gray SW, Joffe S, Janne P, Garber J, MacConaill L, Lindeman N, Rollins B, Kantoff P, Fisher SA, Gabriel S, Getz G, Garraway LA - Nat. Med. (2014)

Clinical sequencing informs clinical trial enrollment and experimental discoveryThe PHIAL output and treatment course for a patient with metastatic lung adenocarcinoma is shown, with the integration of clinical WES occurring during the patient’s first-line therapy allowing subsequent clinical trial enrollment (A). The patient’s time to relapse data for the three treatment regimens received demonstrate that the best and only clinical response occurred with the CDK4 inhibitor (B). Radiographic imaging demonstrates a small reduction in a representative metastatic focus for the patient on the CDK4 inhibitor trial after two cycles of therapy consistent with stable disease (cm: centimeter; measurement is 1.7 × 1.5 cm for baseline mass and 1.3 × 1.3 cm for two month interval scan of the same mass). Per RECIST criteria, overall tumor reduction was 7.9% (C). For another patient, PHIAL nominated a JAK3 missense mutation (D), and given its location in the kinase domain near alterations previously defined as activating, was considered to have inferential evidence (Level E) for being clinically actionable. The crystal structure of JAK3 demonstrates that the arginine at residue 870 directly coordinates the phosphate group of the primary activating tyrosine phosphorylation site (E). To better characterize this alteration, experimental follow-up of this alteration was performed in a Ba/F3 system. Overexpression of the patient’s JAK3 mutation did not suggest an activating phenotype or further consideration of JAK3 inhibitor clinical trial enrollment (F).
© Copyright Policy
Related In: Results  -  Collection

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Figure 5: Clinical sequencing informs clinical trial enrollment and experimental discoveryThe PHIAL output and treatment course for a patient with metastatic lung adenocarcinoma is shown, with the integration of clinical WES occurring during the patient’s first-line therapy allowing subsequent clinical trial enrollment (A). The patient’s time to relapse data for the three treatment regimens received demonstrate that the best and only clinical response occurred with the CDK4 inhibitor (B). Radiographic imaging demonstrates a small reduction in a representative metastatic focus for the patient on the CDK4 inhibitor trial after two cycles of therapy consistent with stable disease (cm: centimeter; measurement is 1.7 × 1.5 cm for baseline mass and 1.3 × 1.3 cm for two month interval scan of the same mass). Per RECIST criteria, overall tumor reduction was 7.9% (C). For another patient, PHIAL nominated a JAK3 missense mutation (D), and given its location in the kinase domain near alterations previously defined as activating, was considered to have inferential evidence (Level E) for being clinically actionable. The crystal structure of JAK3 demonstrates that the arginine at residue 870 directly coordinates the phosphate group of the primary activating tyrosine phosphorylation site (E). To better characterize this alteration, experimental follow-up of this alteration was performed in a Ba/F3 system. Overexpression of the patient’s JAK3 mutation did not suggest an activating phenotype or further consideration of JAK3 inhibitor clinical trial enrollment (F).
Mentions: The prospective WES framework was used for clinical decision-making in one demonstrative case. A patient with metastatic lung adenocarcinoma underwent standard clinical genetic testing that revealed wild-type EGFR, KRAS (codon 12 and 13), and ALK status. Mass spectrometry testing of 471 alterations in 41 genes5 revealed an STK11 frameshift deletion. The patient was started on carboplatin, paclitaxel, and bevacizumab (Fig. 5A). In parallel, we applied the clinical WES platform on the FFPE metastatic tumor sample and germline peripheral blood. PHIAL nominated a KRASA146V mutation, along with alterations in STK11 (identical to other testing) and ATM (Fig. 5A, Supplementary Table 8). KRASA146V is a known activating mutation, though possibly less potent than the codon 12 and 13 mutations28. Although activating KRAS mutations are found in 15–30% of all patients with non-small cell lung cancer (NSCLC) and commonly in conjunction with STK11 loss29, this specific KRAS alteration has not been reported in NSCLC20,30–32. KRAS146V was confirmed using the same FFPE tumor sample in a clinical lab that met Clinical Laboratory Improvement Amendments (CLIA) standards (Knight Diagnostic Laboratories, Oregon), and then returned to the patient’s oncologist. After rapidly progressing on combination chemotherapy (Fig. 5B), the patient was enrolled in a phase I CDK4 inhibitor (LY2835219) clinical trial based on preclinical data (Level D) implicating a synthetic lethal relationship between activated KRAS and CDK433. The patient achieved stable disease (per RECIST 1.1 criteria; 7.9% reduction in tumor volume compared to baseline) and was on therapy for 16 weeks (Fig. 5B–C). Of note, this represented the patient’s best and only clinical response to any cancer-directed therapy.

Bottom Line: The platform employs computational methods for effective clinical analysis and interpretation of WES data.When applied retrospectively to 511 exomes, the interpretative framework revealed a 'long tail' of somatic alterations in clinically important genes.Prospective application of this approach identified clinically relevant alterations in 15 out of 16 patients.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA. [2] Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.

ABSTRACT
Translating whole-exome sequencing (WES) for prospective clinical use may have an impact on the care of patients with cancer; however, multiple innovations are necessary for clinical implementation. These include rapid and robust WES of DNA derived from formalin-fixed, paraffin-embedded tumor tissue, analytical output similar to data from frozen samples and clinical interpretation of WES data for prospective use. Here, we describe a prospective clinical WES platform for archival formalin-fixed, paraffin-embedded tumor samples. The platform employs computational methods for effective clinical analysis and interpretation of WES data. When applied retrospectively to 511 exomes, the interpretative framework revealed a 'long tail' of somatic alterations in clinically important genes. Prospective application of this approach identified clinically relevant alterations in 15 out of 16 patients. In one patient, previously undetected findings guided clinical trial enrollment, leading to an objective clinical response. Overall, this methodology may inform the widespread implementation of precision cancer medicine.

Show MeSH
Related in: MedlinePlus