Virtual Library

 x 
My Library Virtual Library FAQ

Start Your Search

S. Popat



Author of

  • +

    P1.03 - Poster Session 1 - Technology and Novel Development (ID 150)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
    • +

      P1.03-006 - The efficiency of detection of <em>KRAS</em>, <em>EGFR </em>and <em>BRAF </em>mutations in primary lung cancer via peripheral blood circulating tumour cells (ID 2762)

      09:30 - 09:30  |  Author(s): S. Popat

      • Abstract

      Background
      Circulating tumour cells (CTCs) are present in the blood of a proportion of patients with lung cancer. However, it is currently unclear how suitable CTCs are for use in the detection of predictive genetic mutations. We sought to determine the utility of DNA extracted from CTCs to screen for the underlying primary tumour mutation.

      Methods
      Using ScreenCell™ MB devices, from 20/01/12 to 25/01/2013, CTCs were captured in peripheral blood of 100 patients who underwent surgery for lung cancer at The Royal Brompton Hospital. DNA was extracted using QIAamp DNA Micro kit (QIAGEN) followed by whole-genome amplification using GenomePlex® SingleCell WGA kit (Sigma). DNA from matched primary tumours was used as reference. Mutation detection in EGFR and KRAS genes was undertaken using cobas®4800 (Roche) and single-strand conformation analysis for BRAF gene. Sensitivity and specificity analyses were undertaken to measure predictive performance of mutation testing in CTCs.

      Results
      The DNA extracted from CTCs, were of sufficient quality to allow mutation analyses to be successfully performed in 100%, 99%, and 98% of samples for EGFR, KRAS, and BRAF genes, respectively. In CTC DNA, the KRAS mutation rate (codons 12/13 and 61) was 9.1% and concordance with the primary tumour was 78.8%. Six mutations were detected in CTCs, but not in primary tumours, and 13 mutations in primary tumours were not detected in corresponding CTC samples. Three mutations were detected in matched CTC and primary tumour specimens. One mutation in EGFR was detected in CTC DNA and 3 mutations were detected in primary tumours. In all cases, the mutations were detected in discordant specimens. The concordance between mutations detection in CTCs and primary tumours was 95.8%. BRAF V600E mutation was not detected in any sample. In general, the results suggested low sensitivity but high specificity (Table). Due to low number of EGFR mutations detected, test performance results require further validation.

      The performance of mutation testing in circulating tumour cells
      Statistic KRAS EGFR
      Sensitivity (95% CI), % 18.8 (4.05-45.6) 0.0 (0.0-70.8)
      Specificity (95% CI), % 91.8 (83.0-96.9) 98.9 (94.1-100)
      Positive predictive value (95% CI), % 33.3 (7.49-70.1) 0.0 (0.0-97.5)
      Negative predictive value (95% CI), % 83.8 (73.8-91.1) 96.8 (91.0-99.3)

      Conclusion
      The result of our study indicates that the DNA extracted from CTCs can be used to screen for primary tumour mutations with reasonable concordance. Differences in the mutation results from the CTC and primary tumours needs to be explored in more detail and may be due to issues related to processing and / or tumour versus CTC heterogeneity.

  • +

    P3.06 - Poster Session 3 - Prognostic and Predictive Biomarkers (ID 178)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
    • +

      P3.06-027 - A comparison of FISH and immunohistochemistry in the detection of ALK rearrangement in lung adenocarcinoma (ID 2392)

      09:30 - 09:30  |  Author(s): S. Popat

      • Abstract

      Background
      Personalised treatments for lung cancer are being increasingly employed. Around 4% of pulmonary adenocarcinomas have rearrangements of the ALK gene resulting in oncogenic fusion proteins. Such cases are sensitive to the small molecule tyrosine kinase inhibitor Crizotinib, and detection of the ALK rearrangement is crucial to the treatment of these patients. Currently the only approved test for the detection of ALK-positive NSCLC is FISH. Various immunohistochemical assays have been proposed as alternatives or screening tools, which are cheaper, quicker and easier to perform and interpret than FISH. Validation of these immunohistochemical tests is therefore important.

      Methods
      15 FISH positive cases were obtained from local archives at the Royal Marsden and Royal Brompton Hospitals, with accompanying data on crizotinib therapy and clinical response. A further 14 FISH negative and FISH uncertain cases were also retrieved. 3 antibodies were optimised for immunohistochemical detection of ALK: D5F3, marketed by Ventana and using their proprietary automated system, ALK1 (Dako), and 5A4 (Abcam). All three antibodies were applied to sections from the archival cases. Antibodies were semiquantitatively scored on their intensity (0-3) and proportion of malignant cells stained (0-100%). Cutoffs for positivity/negativity were set by ROC analysis to optimise correct classification.

      Results
      Positivity according to the Vysis FISH assay (i.e. appropriately abnormal FISH signal in at least 15% of cells) was taken as the gold standard. The Ventana assay (D5F3) was markedly more intense but showed higher background than the other two antibodies. ROC analysis of staining intensity showed that the Ventana and Dako antibodies should be considered positive when staining is of 'moderate' intensity or above. The Abcam assay was discriminatory when any positivity was seen. Scoring of proportion did not improve specificity or sensitivity with any of the antibody tests. Subsequent analyses were of intensity scoring. Taking all cases together, all 3 antibodies were highly specific (100%) and sensitive (Ventana 86%, Abcam 79% and Dako 71%). In excision specimens, all 3 assays showed sensitivity of 83%. In cytology and small biopsy specimens, Ventana performed the best (specificity of 88% compared to 75% for Dako and 63% for Abcam) although the numbers are small. No ‘false positive’ (-ve FISH, +ve IHC) cases were seen; the only disparity between FISH and IHC were 'false negative' cases. Two cases were FISH-positive but universally IHC-negative (with all 3 assays). Of these, one was treated with crizotinib, and failed to respond.

      Conclusion
      IHC is a highly specific (100%) and sensitive (71%-86%) test for ALK rearrangement in archival FFPE tumour tissue. In this study of 15 ALK-rearranged tumours, the Ventana D5F3 assay performed the best, despite having relatively high background staining. Occasional cases are FISH-positive but IHC-negative. One such case was not responsive to Crizotinib, further supporting IHC as a test predictive of drug response. IHC has the potential to replace FISH for the detection of candidates for Crizotinib therapy. However, further work is required to direct the treatment of tumours with discordant FISH and immunohistochemical tests.

  • +

    P3.10 - Poster Session 3 - Chemotherapy (ID 210)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Medical Oncology
    • Presentations: 1
    • +

      P3.10-026 - Outcomes of patients undergoing adjuvant platinum-vinorelbine chemotherapy for resected non-small cell lung cancer (NSCLC) (ID 1566)

      09:30 - 09:30  |  Author(s): S. Popat

      • Abstract

      Background
      Cisplatin-vinorelbine adjuvant chemotherapy significantly improves survival in resected NSCLC. We evaluated outcomes of patients receiving adjuvant chemotherapy in our institution between 2006-2011.

      Methods
      Outcomes of stage IB -IIIA NSCLC patients who received platinum-vinorelbine following radical lung surgery were collected and analysed to assess overall survival (OS), progression-free survival (PFS), and treatment intensity.

      Results
      53 patients were identified (23:30, M:F), mean age 62, and 35% were adenocarcinoma. Resected stage was 1B-3A, with one-third stage 3A. When tested, EGFR mutation prevalence was 33% (39% never smokers; 61% ever smokers). There was one death from chemotherapy toxicity. Median chemotherapy cycles given was 4. There was no difference in PFS or OS in patients having carboplatin compared with cisplatin. A significantly improved OS in patients that received ≥3 cycles of chemotherapy was observed (HR=0.25, 0.07-0.97, p=0.04). 60.4% patients had relapsed at last follow-up. Radically treatable disease on relapse was detected by surveillance imaging.

      Conclusion
      Our small dataset indicates that four cycles of adjuvant platinum-vinorelbine chemotherapy is deliverable in the real world setting, and that less than 3 chemotherapy cycles is associated with poorer outcomes.

  • +

    P3.12 - Poster Session 3 - NSCLC Early Stage (ID 206)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Medical Oncology
    • Presentations: 1
    • +

      P3.12-016 - Frequency in EGFR, K-RAS, BRAF and ALK mutations in a cohort of cancers: ALK translocations are more frequently seen in advanced disease (ID 2696)

      09:30 - 09:30  |  Author(s): S. Popat

      • Abstract

      Background
      Routine mutation testing for potential targeted therapy is a challenge in the management of lung cancer. As part of a feasibility study on the implementation of molecular testing in the United Kingdom, Cancer Research UK (CRUK) set up a stratified medicine program testing EGFR, K-RAS, BRAF mutations and ALK translocation on lung cancer samples. We present the result from the first ten months from two hospitals feeding into one diagnostic molecular laboratory.

      Methods
      Mutations detection in EGFR and KRAS genes was undertaken using cobas®4800 (Roche) and single-strand conformation analysis for BRAF gene. ALK translocations were screened using Vysis ALK break apart rearrangement probe.

      Results
      A total of 94 resections all (but one) in stages I - IIIA were analysed, with mutations found entirely within adenocarcinomas (n=64), with a frequency of mutations/translocation identification of 30% (K-RAS) , 12.5% (EGFR), 1.5% (ALK) and 1.5% (BRAF) (Figure 1). Over the same period, 360 biopsies from patients with non-resectable/advanced disease, were analysed, the majority being stage 4, with a frequency of 24% (K-RAS), 10%.6 (EGFR), 4.3% and 1.9% (Figure 2). Figure 1 Figure 2

      Conclusion
      The data suggest that frequency of K-RAS, EGFR and BRAF mutations are similar in early and advanced non-small cell carcinoma, however ALK translocations were observed to be more frequent in patients with advanced disease. This may have implications when considering which patients should undergo FISH testing for ALK translocation and entry into clinical trials. This study was funded by Cancer Research UK, Astra Zenica and Pfizer.

  • +

    P3.20 - Poster Session 3 - Early Detection and Screening (ID 174)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Imaging, Staging & Screening
    • Presentations: 1
    • +

      P3.20-009 - Diagnostic performance of a filter-based antibody-independent peripheral blood circulating tumour cell capture paired with cytomorphologic criteria for the diagnosis of lung cancer (ID 2782)

      09:30 - 09:30  |  Author(s): S. Popat

      • Abstract

      Background
      The ability to capture and characterise peripheral blood circulating tumour cells (CTCs) has the potential for the development of a blood test for cancer. A number of technological platforms are available to obtain CTCs including filtration-based devices utilising advances of antibody independent capture of cells. This technique shows promising results in experimental conditions; however, its performance has not yet been well evaluated in a clinical setting. We have evaluated diagnostic performance of filtration-based technology using cytomorphologic criteria in patients undergoing surgery for lung cancer.

      Methods
      From 06/03/2012 to 24/01/2013 we obtained and processed blood from 74 patients undergoing surgery for known or suspected lung cancer using ScreenCell[TM] Cyto devices. Captured cells were stained using H&E and independently assessed by two pathologists (AGN, AR) for the presence of atypical cells suspicious for cancer. Results were reported as confirmed cancer, suspicious or no evidence for cancer. Diagnostic performance was evaluated against the reference of cancer identified within surgically obtained specimens reported by a principal pathologist. Sensitivity and specificity analyses were undertaken. Inter-observer agreement was established by kappa-statistics.

      Results
      According to histopathology assessment, 42 patients (56.7%) had primary lung cancer, 18 patients (24.3%) had metastatic cancer (predominantly of colorectal origin), and 14 patients (18.9%) had benign lung diseases. The proportion of patients in which cells suspicious for cancer were identified was 39 (52.7%) and 42 (56.7%) as reported by two pathologists. Among those cases, 6 (15.4%) and 14 (33.3%) were reported as confirmatory. The agreement between the pathologists was 77% corresponding to a kappa-statistics of 53.7% indicating moderate agreement. In metastatic cancer patients, suspicious cells were discovered in 10 (55.6%) and 9 (50%) cases by two pathologists. In non-cancer patients, suspicious cell were found in 6 (42.8%) and 5 (35.7%) cases by two pathologists, respectively. The test performance for the diagnosis of cancer using cytomorphological criteria yielded poor-to-moderate sensitivity and specificity values, high positive predictive values and low negative predictive values (Table).

      The performance of the diagnosis of cancer using filter-based antibody-independent technique of CTCs trapping
      Statistic Pathologist 1 Pathologist 2
      Sensitivity (95% CI), % 55.0 (41.6-67.9) 61.7 (48.2-73.9)
      Specificity (95% CI), % 57.1 (28.9-82.3) 64.3 (35.1-87.2)
      Positive Predictive Value (95% CI), % 84.6 (69.5-94.1) 88.1 (74.4-96.0)
      Negative Predictive Value (95% CI), %
      		22.9 (10.4-40.1) 28.1 (13.7-46.7)

      Conclusion
      The results of our study highlight the potential of filter-based antibody-independent technology to develop an accurate blood test for the diagnosis of cancer in the peripheral blood. However, conventional cytomorphological criteria used for the diagnosis provide inadequate sensitivity and specificity. Improved performance with immunocytochemistry is still required prior to further clinical validation.