Genomic Profiling of the Residual Disease of Advanced-stage Ovarian Cancer After Neoadjuvant Chemotherapy
Condition: Advanced-stage Ovarian Cancer
MeSH terms: Ovarian Neoplasms; Carcinoma, Ovarian Epithelial
Study type: Observational

Summary: 
      Tumor response to NAC predicts survival and can be considered a surrogate prognostic marker.
      Three tiered chemotherapy response score (CRS) of omental tissue sections showed a
      significant association with survival. In patients with CRS 1 or 2, NAC selects a
      subpopulation of chemotherapy resistant tumor cells. This study will examine comprehensive
      molecular analyses on the residual disease of 104 clinically defined high-grade serous
      carcinoma after NAC, including next-generation sequencing on 14 matched pretreatment
      biopsies. This information together with immune marker expression and BRCA expression, will
      provide a unique opportunity to guide biomarker-driven adjuvant studies targeting these
      chemotherapy-resistant tumor cells.
    

Detailed description: 
      DNA extracted from FFPE block will be used. Capture sequencing and immunohistochemistry will
      be performed.

      Immunohistochemistry The formalin-fixed, paraffin-embedded tissue blocks were sectioned at 4
      µm thickness onto Superfrost Plus glass slides (Thermo Fisher Scientific, Waltham, MA, USA).
      These sections were deparaffinized in xylene and rehydrated through graded alcohols.
      Immunohistochemical staining was performed using an automatic immunostaining instrument
      (Ventana Benchmark XT; Ventana Medical Systems), according to the manufacturer's
      recommendations. Antigen retrieval was performed using a Cell Conditioning Solution (CC1;
      Ventana Medical Systems). The sections were incubated with antibodies against PD-L1
      (prediluted, clone SP263, Ventana Medical Systems), CD8 (prediluted, clone C8/144B, Dako,
      Glostrup, Denmark), PD-1 (1:50, clone NAT105, Cell Marque, Rocklin, CA, USA), Foxp3 (1:50,
      clone 236A/E7, Abcam, Cambridge, UK), ICOS/CD278 (1:50, clone SP98, Thermo Fisher
      Scientific), and LAG-3 (1:100, clone EPR4392(2), Abcam). After chromogenic visualization
      using an ultraView Universal DAB Detection Kit (Ventana Medical Systems), the slices were
      counterstained with hematoxylin, dehydrated in graded alcohols and xylene, and then embedded
      in mounting solution. Appropriate positive and negative controls were concurrently stained to
      validate the staining method.

      Targeted Capture Sequencing on NextSeq550 using SureSelect XT Sample Preparation DNA was
      extracted from microdissected tumor rich areas from formalin fixed paraffin embedded tumor
      sections using DNAeasy kit (QIAGEN, Venlo, Netherlands). Two hundred fifty nanogram of
      genomic DNA in 50μl TE buffer was fragmented to a median size of 150bp using the Covaris-E220
      AFA instrument (Covaris, Woburn, MA) with the following settings: peak incident power 175
      watts, duty factor 10.0%, cycles per burst 200 cycles, run time 500 sec at 4oC. DNA fragments
      were evaluated using capillary electrophoresis on High Sensitivity D1000 ScreenTape and
      Reagents (TapeSation4200, Cat.No.5067-5584, 5585; Agilent Technologies, Santa Clara, CA,
      USA). End repair, 3'-end adenylation and sequencing adapter ligation of DNA fragments were
      performed following the manufacture's protocol (SureSelect XT Reagent kit, HSQ Cat.No.G9611A,
      G9611B; Agilent Technologies, Santa Clara, CA, USA). After each steps, fragments were
      purified using AMPure XP beads (Cat. No. A63382; Beckman Coulter, High Wycombe, UK). The
      resulting DNA was amplified by 14 cycles PCR amplification (SureSelect Herculase Ⅱ Fusion
      Enzyme with dNTP Combo 200 RXN kit, Cat. No. 600677; Agilent Technologies, Santa Clara, CA,
      USA). The quality of the PCR product was assessed by capillary electrophoresis with DNA1000
      TapeScreen and Reagents (TapeSation4200, Cat.No.5067-5582, 5583; Agilent Technologies, Santa
      Clara, CA, USA) and the concentration was measured by Qubit 2.0 Fluorometer (Qubit dsDNA HS
      Assay Kit, Cat. No. Q32854; Thermofisher Scientific, Inc, Waltham, MA).

      Capture and Enrichment Target enrichment was performed according to the manufacturer's
      instructions (SureSelect XT Custom 0.5Mb-2.9Mb library, Cat. No. 5190-4816, 4817; Agilent
      Technologies, Santa Clara, CA, USA). The resulting captured libraries with indexing primers
      were amplified by 12 cycles of PCR (SureSelect Herculase Ⅱ Fusion Enzyme with dNTP Combo 200
      RXN kit, Cat. No. 600677; Agilent Technologies, Santa Clara, CA, USA) and purified again.
      Quantity and quality of indexed library DNA were verified by capillary electrophoresis with
      High Sensitivity D1000 ScreenTape and Reagents (TapeSation4200, Cat.No.5067-5584, 5585;
      Agilent Technologies, Santa Clara, CA, USA) and Qubit 2.0 Fluorometer (Qubit dsDNA HS Assay
      Kit, Cat. No. Q32854; Thermofisher Scientific, Inc, Waltham, MA).

      Sequencing We diluted resulting libraries to 4nmol/l and pooled by combining 5μl of each
      diluted library for normalization. Subsequently, pooled library was denatured into single
      strands by using fresh 0.2N NaOH and 200mM Tris-HCl, pH7. A PhiX control was denatured in the
      same way. The concentration of final loading library was 1.8pmol/l and Phix was spiked in at
      1%. A standard flow cell was loaded on the Illumina NextSeq550 and sequencing was conducted
      with 2X100bp paired-end reads using NextSeq550 reagent v2 kit with high-output 300 cycles.
      (NextSeq550 System user guide part #15069765-V02 Mar 2016, NextSeq550 Reagent V2 Kit
      High-output 300 cycles Cat. No. FC-404-2004; Illumina, CA).
    

Intervention name(s): chemotherapy
Intervention type(s): Drug
Intervention description(s): All of the patients in this study underwent NAC regimens consisting of taxane and platinum combination chemotherapy. After NAC, all of the patients underwent interval debulking surgery. Subsequently, additional cycles of chemotherapy were administered after IDS to complete a total of 6 cycles at the discretion of the treating physician.
Observational model: Cohort
Time perspective: Retrospective
Sampling method: Non-Probability Sample
Study population: 
        tirtiary medical center
      
Biospecimen description: 
      Formalin-fixed paraffin embedded
    
Keywords: advanced-stage ovarian cancer; neoadjuvant chemotherapy; residual disease; genomic profiling; immunohistochemistry