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A. Montero-Fernandez



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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. Montero-Fernandez

      • 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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    P1.09 - Poster Session 1 - Combined Modality (ID 212)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Combined Modality
    • Presentations: 1
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      P1.09-003 - Prognostic impact of secondary pathologic findings on the outcome of patients with resected locally advanced non-small cell lung cancer previously treated with inductive chemotherapy or chemoradiation (ID 296)

      09:30 - 16:30  |  Author(s): A. Montero-Fernandez

      • Abstract

      Background
      Half of non-small cell lung cancers are diagnosed in locally-advanced stages (LA-NSCLC), and warrant multidisciplinary treatments. Inductive and adjuvant therapies obtain the same survival benefit after surgery, but the first ones also provide prognostic information. Mediastinal downstaging and pathologic complete response preclude a better outcome. However, the value of minor pathologic features has been scarcely analyzed. We report the impact of lymphovascular invasion (LVI) and tumor necrosis (TN) on the prognosis of resected LA-NSCLC after inductive chemotherapy (iCT) or chemoradiation (iCRT).

      Methods
      We retrospectively reviewed 50 resected LA-NSCLC treated with iCT or iCRT in our center from October-2004 to June-2012. Three patients died due to early surgery complications and were not analyzed. The impact of ILV, TN, pneumonitis, extracapsular (EI) and pleural invasion (PI) on disease free (DFS) and overall survival (OS) was analyzed in the remaining patients

      Results
      Our series included 42 men and 5 women aged between 47-82 years. Non-specified NSCLC was diagnosed in nine, adenocarcinoma in two, and squamous carcinoma in 36. Thirty-seven received cisplatin-based and 10 carboplatin-based chemotherapy. Concurrent radiation was administered in 21. Pneumonectomy was performed in 15, lobectomy in 26, and segmentectomy in 6. The presence of pneumonitis, EI, or PI in the resected specimens did not impact on the outcome of the patients. However, DFS and OS clearly worsened when LVI (34.1 vs. 14.1 months, p= 0.01; and 43 vs. 29, p= 0.005; respectively), and absence of TN (31.6 vs. 24.3, p= 0.045; and 42 vs. 33, p= 0.041, respectively) were found. Radiation, cis- or carboplatin administration, and treatment length did not modify LVI and TN incidence

      Conclusion
      Minor pathologic features as LVI and TN significantly impact on the prognosis of resected LA-NSCLC after iCT or iCRT. Implications of that should be further analyzed. Figure 1. DFS and OS Kaplan-Meier curves according to the presence of LVI. Figure 1

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    P2.09 - Poster Session 2 - Combined Modality (ID 213)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Combined Modality
    • Presentations: 1
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      P2.09-002 - Prognostic impact of conventional and modified measurement of mediastinal lymph node involvement in the outcome of patients with resected locally advanced non-small cell lung cancers previously treated with inductive chemo or chemoradiation (ID 299)

      09:30 - 16:30  |  Author(s): A. Montero-Fernandez

      • Abstract

      Background
      Mediastinal lymph node involvement (MLNI) is the strongest prognostic factor of resected locally advanced non-small cell lung cancers (LA-NSCLC). The 7[th] edition of the TNM is based in purely topographic criteria: N2 is applied for ipsilateral MLNI and N3 for contralateral and unresectable MLNI. Different authors have proposed many other prognostic classifications of MLNI

      Methods
      We retrospectively reviewed 45 resected LA-NSCLC previously treated with inductive chemo or chemoradiation in our center between October-2004 to June-2012. Three patients died due to early surgical complication were not analyzed. Systematic mediastinal dissection was performed in all of them. The prognostic impact of pN2 was statistically compared to number of resected nodes; number of affected areas; number of metastatic nodes; and ratio of metastatic/resected nodes

      Results
      Our series included 37 men and 5 women with a mean age of 64.64 years-old. There were diagnosed eight non-specified NSCLC, two adenocarcinomas, and 32 squamous carcinomas. Cis- and carboplatin-based chemotherapy was administered in 35 and seven patients, respectively. Concurrent radiation was administered in 21. pN2 MLNI clearly worsened both disease free (DSF) and overall survival (OS) of the patients (p= 0.006; and p= 0.018, respectively). Proportion of patients with pN2 MLNI was lower when radiation was applied (p= 0.033) and did not significantly vary according to chemotherapeutic regimen or histology. The number of resected nodes did not impact on survival (DFS p= 0.261, and OS p= 0.277), but OS was better in patients with a lower rate of pathologically detected nodes (p= 0.046). The number of metastatic nodes according to the Tokio score did not impact on survival. The Regina Elena and the Mount Sinai ratios of metastatic nodes according to the number of resected nodes neither impacted

      Conclusion
      The presence of pN2 MLNI according to the 7[th] edition of the TNM classification is the strongest mediastinal prognostic factor in our series of resected LA-NSCLC previously treated with inductive chemo or chemoradiation. No other tested classification of MLNI could predict significant differences in survival. The prognostic significance of the lower detection rate of resected nodes after inductive treatment should be explored. Figure 1. DFS and OS Kaplan-Meier curves according to MLNI. Figure 1

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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. Montero-Fernandez

      • 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.