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A.G. Nicholson



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

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      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 - 16:30  |  Author(s): A.G. Nicholson

      • 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.

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    P1.19 - Poster Session 1 - Imaging (ID 179)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Imaging, Staging & Screening
    • Presentations: 1
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      P1.19-003 - Test performance of PET-CT for mediastinal lymph node staging of pulmonary carcinoid tumours (ID 854)

      09:30 - 16:30  |  Author(s): A.G. Nicholson

      • Abstract

      Background
      PET-CT is a standard investigation to stage the mediastinum in non-small cell lung cancer when radical management is planned. The absence or presence of mediastinal lymph node involvement on PET-CT informs surgical selection (with or without further nodal sampling). The clinical utility of PET-CT in carcinoid tumours is uncertain as its test performance at identifying mediastinal lymph node disease in these tumours is as yet undefined with such tumours being rare and FDG avidity often considered to be variable or low. As such, it is argued whether PET-CT serves the same purpose in selecting patients for radical management in carcinoid tumours as it does with other non-small cell lung cancers. The aim of this study was to determine the test performance of PET-CT for mediastinal lymph node staging of pulmonary carcinoid tumours by collating a multicentre database.

      Methods
      We collated retrospective data from 7 institutions by performing a search on pathological databases for a consecutive series of patients who underwent thoracic surgery for a carcinoid tumour from Nov 1999 - Jan 2013. Preoperative PET-CT staging reports (prior to surgery with lymph node dissection) were obtained from patients’ records and compared against the reference standard of pathologic results obtained from lymph node dissection, and test performance reported using sensitivity and specificity.

      Results
      From Nov 1999 - Jan 2013, a total of 247 patients from 7 institutions underwent surgery for a carcinoid tumour with a corresponding preoperative PET-CT scan. The mean age of the patients was 61 (SD 15) and 84 were male (34%). The pathologic sub-type was typical carcinoid in 217 patients (88%) and atypical carcinoid in 30 patients (12%). The mean SUV uptake in the primary tumour was 4.8 (SD 4). Results from lymph node dissection were obtained in 213 patients. PET-CT reported uptake at mediastinal lymph nodes in 19 patients, of which only 3 were positive on subsequent pathology. Pathological results, from lymph node dissection carried out in 213 patients at the time of surgery, found 8 patients with mediastinal lymph node positive disease, of which only 3 had been picked up in preoperative PET-CT staging. The calculated sensitivity and specificity of PET-CT to identify mediastinal lymph node disease was 38% (95% CI 8-76%) and 93% (88-96%) respectively.

      Conclusion
      In non-small cell lung cancer, preoperative PET-CT is used for nodal and distant staging to assist in the selection of patients for radical treatment. British Thoracic Society guidelines for the radical management of patients with lung cancer recommend “radical treatment without further mediastinal lymph node sampling if there is no significant uptake in normal sized mediastinal lymph nodes on PET-CT scanning”. In carcinoid tumours, our results of the largest cohort to date suggest that PET-CT has a poor sensitivity but good specificity for the presence of mediastinal lymph node metastases in the staging of pulmonary carcinoid tumours. Therefore lymph node metastases cannot accurately be ruled out in carcinoid tumours with a negative PET-CT. If treatment decisions are based on the N2 status, invasive mediastinal staging should be undertaken in carcinoid tumours.

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

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Medical Oncology
    • Presentations: 1
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      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 - 16:30  |  Author(s): A.G. Nicholson

      • 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.

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

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Imaging, Staging & Screening
    • Presentations: 1
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      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 - 16:30  |  Author(s): A.G. Nicholson

      • 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.