Duplex-Repair enables highly accurate sequencing, despite DNA damage

Accurate DNA sequencing is crucial in biomedicine. Underlying the most accurate methods is the assumption that a mutation is true if altered bases are present on both strands of the DNA duplex. We now show that this assumption can be wrong. We establish that current methods to prepare DNA for sequencing, via ‘End Repair/dA-Tailing,’ may substantially resynthesize strands, leading amplifiable lesions or alterations on one strand to become indiscernible from true mutations on both strands. Indeed, we discovered that 7–17% and 32–57% of interior ‘duplex base pairs’ from cell-free DNA and formalin-fixed tumor biopsies, respectively, could be resynthesized in vitro and potentially introduce false mutations. To address this, we present Duplex-Repair, and show that it limits interior duplex base pair resynthesis by 8- to 464-fold, rescues the impact of induced DNA damage, and affords up to 8.9-fold more accurate duplex sequencing. Our study uncovers a major Achilles’ heel in sequencing and offers a solution to restore high accuracy.

Duplex-Repair enables highly accurate sequencing, despite DNA damage

Accurate DNA sequencing is crucial in biomedicine. Underlying the most accurate methods is the assumption that a mutation is true if altered bases are present on both strands of the DNA duplex. We now show that this assumption can be wrong. We establish that current methods to prepare DNA for sequencing, via End Repair/dA-Tailing, may substantially resynthesize strands, leading amplifiable lesions or alterations on one strand to become indiscernible from true mutations on both strands. Indeed, we discovered that 7-17% and 32-57% of interior duplex base pairs from cell-free DNA and formalin-fixed tumor biopsies, respectively, could be resynthesized in vitro and potentially introduce false mutations. To address this, we present Duplex-Repair, and show that it limits interior duplex base pair resynthesis by 8- to 464-fold, rescues the impact of induced DNA damage, and affords up to 8.9-fold more accurate duplex sequencing. Our study uncovers a major Achilles heel in sequencing and offers a solution to restore high accuracy.

IMT-05 PHASE III RANDOMIZED CLINICAL TRIAL OF AFTV FOR NEWLY DIAGNOSED GLIOBLASTOMA

BACKGROUND The highly fatal glioblastoma has an extremely poor prognosis and development of a new treatment is desired. Local treatment is limited due to its high invasiveness, and immunotherapy utilizing self-immune mechanism is theoretically expected. An autologous formalin-fixed tumor vaccine (AFTV) is a vaccine that is prepared using formalin-fixed tumor tissue and recognizes tumor antigen peptides to induce cytotoxic T cells. We have previously conducted three clinical trials using AFTV for patients with newly diagnosed glioblastoma since 2004. The third trial was a double-blind multicenter phase IIb/III trial with 63 case registries, which did not make a significant difference in OS (study group 25 months, placebo group 31 months), the total removal group showed excellent clinical results (3-year survival rate; 65%, median survival; not reached). Since the study was designed to go to Phase III if the test group was not inferior to the placebo group, so it went on to go to Phase III. METHODS Target patients will be 80 patients with newly diagnosed glioblastoma undergoing pathologic diagnosis, who have undergone total removal of contrast-enhanced lesions and receive standard chemoradiation therapy. STUDY DESIGN Double-blind, 3-year enrollment period, 18-month observation period. Stratification factor: Photodynamic therapy (PDT), facility, age, KPS. Administration method: After standard chemoradiotherapy, in parallel with maintenance chemotherapy, a total of 9 times intradermal administration of vaccine. Primary endpoint: PFS of FAS patients, Secondary endpoints: 18 months PFS of the FAS patient, OS, PFS of the ITT analysis target case. Based on the results of the IIb trial, we limited the registered patients with total tumor removal, and in view of the fact that the prognosis of patients with combined PDT and AFTV were excellent, PDT was added to the stratification factor. We outline our efforts and problems aimed at clinical approval of AFTV for glioblastoma.

Rate of Clinical Complete Response for 1 Year or More in Bone-Metastatic Breast Cancer after Comprehensive Treatments including Autologous Formalin-Fixed Tumor Vaccine

Introduction. No effective treatment has been developed for bone-metastatic breast cancer. We found 3 cases with clinical complete response (cCR) of the bone metastasis and longer overall survival of the retrospectively examined cohort treated comprehensively including autologous formalin-fixed tumor vaccine (AFTV). Patients and Methods. AFTV was prepared individually for each patient from their own formalin-fixed and paraffin-embedded breast cancer tissues. Results. Three patients maintained cCR status of the bone metastasis for 17 months or more. Rate of cCR for 1 year or more appeared to be 15% (3/20) after comprehensive treatments including AFTV. The median overall survival time (60.0 months) and the 3- to 8-year survival rates after diagnosis of bone metastasis were greater than those of historical control cohorts in Japan (1988–2002) and in the nationwide population-based cohort study of Denmark (1999–2007). Conclusion. Bone-metastatic breast cancer may be curable after comprehensive treatments including AFTV, although larger scale clinical trial is required.