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A. Rice
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P2.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 234)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P2.04-085 - A Comparative Analysis of Cancer Hotspot Mutation Profiles in Circulating Tumour Cells, Circulating Tumour DNA and Matched Primary Lung Tumour (ID 2451)
09:30 - 09:30 | Author(s): A. Rice
- Abstract
Background:
Blood based mutation profile analyses are becoming an increasingly important non-invasive form of mutation screening in cancer. Many have reported on single mutation comparisons between blood based and primary tumour tissue, but limited information is available on multiplex comparisons between the DNA extracted from circulating tumour cells (CTC), circulating free tumour DNA in the plasma (ctDNA) against the current standard of FFPE analysis of primary tumour.
Methods:
Pre-operative whole blood samples were collected from 30 patients who underwent thoracic surgery. CTCs were isolated using ScreenCell MB devices from 6ml of whole blood, and 1ml aliquots of plasma were removed from 9ml of EDTA samples. Matching FFPE samples were retrieved from post-resection primary tumour tissue in three 10µm PCR rolls. DNA was extracted from the CTCs, ctDNA and matched FFPE tissues using Qiagen kits (QIAamp DNA Micro kit, QIAamp DNA blood mini kit and QIAamp FFPE tissue kit, respectively). The 90 (30 matched triplicates) DNA samples were sequenced by Illumina HiSeq using Z3 cancer panel (Illumina, San Diego). Agreements of variant calls were compared between the three DNA substrates and a kappa statistic was reported using Stata 13.
Results:
Between 2011 and 2013, samples from 30 consenting patients were obtained. In total, 10 had primary lung cancer, 19 had secondary lung cancer, and 1 (intentionally included) had no evidence of cancer. From the 90 samples, a total of 18,821 variant calls were identified after the removal of known 1,048 germline variants. Within the hotspot panel alone, the mean (SD) number of variant calls per patient was 151 (44) on FFPE samples, 136 (49) on CTC samples and 463(108) on ctDNA samples. There was good agreement between CTCs and FFPE of 79.8% with a Kappa statistic of 0.42 (P<0.001). Agreement between ctDNA and FFPE was much poorer at 12.7% with a Kappa statistic of -0.40 (P=1.000). The results also suggested poor agreement between CTC and ctDNA of 16.1% with a Kappa statistic of -0.32 (P=1.000). Focusing on single gene comparisons on the multiplex platform, agreement was considerably better for KRAS and EGFR for CTCs compared to ctDNA at 44% versus 11% for KRAS and 92% versus 9% for EGFR respectively. Discordances were largely due to an increased number of variants that were identified in ctDNA and not in CTC or FFPE tissue.
Conclusion:
Our results suggest on a next generation sequencing platform that the global genetic variant profile between DNA extracted from CTC had good agreement with FFPE primary tumour tissue, and the agreement for ctDNA and FFPE was much poorer. This was observed to be an increase in the number of variants detected on single gene analysis and may be due to processing, sample or analytic difficulties with ctDNA.
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P3.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 235)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/09/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P3.04-011 - A Validation Study for the Use of ROS-1 Immunohistochemistry in Screening for ROS-1 Translocations in Lung Cancer (ID 2826)
09:30 - 09:30 | Author(s): A. Rice
- Abstract
Background:
ROS-1 translocations are a rare genetic abnormality in lung cancers that, when identified, are a target for personalised therapy. The current test of choice is FISH, although with a rate of no more than 1-2%, screening using FISH is an expensive proposition. A further possibility is using immunohistochemistry (IHC) as a screening tool and commercial antibodies are now available that identify the ROS-1 protein in tumour cells. We present our data in undertaking a validation study for potential diagnostic usage.
Methods:
Given the relative rarity of the translocation and the fact the most driver mutations occur in isolation, a test cohort of cases was selected from patients recruited to phase 1 of the Cancer Research UK-Stratified Medicine Project (CRUK-SMP), who were identified as negative for EGFR, KRAS and/or BRAF mutations, as well as ALK translocations. Negative cases were then screened with an antibody for ROS-1 (D4D6, Cell Signalling, 1 in 300 dilution) and scored as negative, weakly positive or moderately positive, along with the percentage of positive cells. Cases were then sent for FISH analysis for the ROS-1 translocation, with a cut-off of > or = to15%, and the sensitivity and specificity of positive staining for ROS-1 was generated.
Results:
From 170 patients recruited from our institution into CRUK-SMP phase 1, a total of 103 patients were wild type for the above mutations (90 for all 4 genetic abnormalities. 9 further cases had failed tests for one and 4 for two mutations (6 carcinoids, 38 squamous cell carcinomas, 5 small cell carcinoma, 2 adenosquamous carcinoma, 1 pleomorphic carcinoma, 3 large cell carcinoma, 2 large cell neuroendocrine cell carcinoma, 7 non-small cell carcinoma (on biopsy) and 39 adenocarcinomas). 39 cases were tested (adenocarcinoma = 37, adenosquamous carcinoma = 2) with FISH, and one case was positive (78% positive cells). FISH testing was negative in 35 cases with scores of 1-8%, and three cases failed. The one positive case was positive on IHC (>90% of cells, moderate staining). In the 35 cases negative for FISH, four cases showed variable positivity on IHC (20, 40,50, 90%, moderate staining) and five cases showed weak focal staining (<5, <5, 10, 20, 30%, weak staining). The remainder were negative on IHC. All non-adenocarcinomas were negative on IHC. Several cases show positive staining of entrapped background pneumocytes and alveolar macrophages, making scoring problematic in some adenocarcinomas.
Conclusion:
Moderate staining for ROS-1 using IHC, independent of percentage positive cells, showed high sensitivity (100%) for tumours that contained a high level of translocated cells. However, specificity was at best 50%, even if a cut-off of 50% positive cells was applied. Pathologists also need to be aware of background staining so cases are not interpreted as false positives.
