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P.J.F. Snijders



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    P1.06 - Poster Session 1 - Prognostic and Predictive Biomarkers (ID 161)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.06-021 - Validation of DNA Hypermethylation Analysis in Sputum for the Diagnosis of Lung Cancer (ID 1774)

      09:30 - 09:30  |  Author(s): P.J.F. Snijders

      • Abstract

      Background
      Lung cancer has the highest mortality of all cancers worldwide with a 5 year survival rate of <15%. The prognosis improves dramatically when the disease is detected at an early stage, and when curative treatment is possible. Current (low dose CT) screening and diagnostic procedures are suboptimal with low specificity. Thus, novel detection methods for lung cancer as stand alone or in combination with other methods are needed. DNA hypermethylation of biomarkers in sputum have shown to distinguish lung cancer cases from cancer-free controls. The aim of the present study was to validate the usage of DNA hypermethylation of biomarkers in sputum samples of lung cancer patients and controls for lung cancer diagnosis, in comparison with sputum cytology.

      Methods
      We prospectively collected sputum of lung cancer patients and controls during 3-9 days in the Amsterdam and Nieuwegein area, The Netherlands. From this sputum bank, a learning set (n=80 lung cancer patients, n=91 controls) and validation set (n=173 lung cancer patients, n=164 controls) were randomly composed. DNA promoter hypermethylation of the following biomarkers was assessed by means of quantitative methylation specific PCR: RASSF1A, APC, cytoglobin, 3OST2, PRDM14, FAM19A4 and PHACTR3. Cut-off values for positive hypermethylation were calculated using Youden’s index. Sputum cytology analysis was performed for all sputum samples. McNemar’s test was used to compare the difference between sensitivity of hypermethylation and sputum cytology for lung cancer diagnosis. A two-sided p-value <0.05 was considered significant.

      Results
      RASSF1A was best able to distinguish cases from controls, with sensitivity of 37-41% and specificity of 91-97% in both learning and validation sets. In multivariate analysis, a panel of RASSF1A, 3OST2 and PRDM14 showed highest sensitivity of 82% [95% confidence interval (CI): 76 – 88%] with a specificity of 68% [95% CI: 61 – 74%] in the learning set, with consistent results in the validation set. Molecular analysis was superior (P<0.001) over sputum cytology (sensitivity of 15%). The sensitivity of the biomarker panel did not improve when it was combined with sputum cytology. There was no association observed between DNA hypermethylation and clinical parameters such as age, smoking status, tumor stage, and histology.

      Conclusion
      This study validates hypermethylation analysis in sputum for the diagnosis of lung cancer. RASSF1A hypermethylation showed high specificity and thereby can have an important role in lung cancer diagnosis in symptomatic patients. A panel of biomarkers RASSF1A, 3OST2 and PRDM14 showed high sensitivity, but relatively low specificity.

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    P1.17 - Poster Session 1 - Bronchoscopy, Endoscopy (ID 182)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Pulmonology + Endoscopy/Pulmonary
    • Presentations: 1
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      P1.17-008 - Results of a close surveillance strategy for subjects with pre-invasive endobronchial squamous lesions (ID 2678)

      09:30 - 09:30  |  Author(s): P.J.F. Snijders

      • Abstract

      Background
      The dismal overall 5-year survival of non-small cell lung cancer (NSCLC) patients is mainly due to advanced stage of disease at time of initial diagnosis in most and the inability to cure metastatic disease in all patients. In contrast, the prognoses of in situ mucosal and small parenchymal lesions are excellent. Early detection strategies might result in the identification of early-stage, (pre-)invasive lesions that are still eligible for curative treatment. The present study was set out to characterize the risk of lung cancer development in a cohort of high-risk subjects harboring pre-invasive endobronchial lesions and to assess the results of surveillance using autofluorescence bronchoscopy (AFB) and computed tomography (CT) scan.

      Methods
      Between November 1995 and December 2012, one hundred and sixty-four at risk individuals with pre-invasive endobronchial lesions were monitored by repeated AFB and CT. During the course of surveillance, progression of lesions to cancer (in situ), recurrences and second primary cancers were treated with different modalities (e.g. endobronchial techniques, surgery, radiotherapy), depending on tumor stage and location. Log-rank tests were performed to examine the relation between baseline characteristics and progression-free and overall survival (PFS and OS, respectively). Cox regression was used for multivariate survival analysis.

      Results
      Demographical and clinical variables of the cohort are shown (Table). At inclusion, 80 individuals were identified with one or more high-grade pre-invasive lesions (severe dysplasia or CIS; HGD), whereas 84 subjects were identified solely with lower grade pre-invasive lesions (LGD). During close surveillance (median follow-up (FU) of 30 months, range 4-152), sixty-one lung cancers were detected (26 CT-detected, 35 AFB-detected cancers) in 55 individuals within a median time to event of 16.5 months. Mean PFS was similar between individuals with radiographically occult lesions vs. FU after surgery for early-stage NSCLC/ENT ca (122.3 vs. 126.9 months, p=0.237) and COPD vs. non-COPD (118.8 vs. 136.8 months, p=0.162). There was a relatively large difference in PFS between LGD and HGD groups (142.6 vs. 93.7 months, p=0.057). Independent risk determinants for OS were indication for surveillance (FU after surgery for early-stage NSCLC/ENT ca vs. radiographically occult lesions, p=0.008) and COPD-status (COPD vs. non-COPD, p<0.001).

      Referral for radiographically occult lesion Follow-up after surgery for early-stage NSCLC / ENT ca
      total
      individuals, n 164 92 72
      Gender
      male 134 72 62
      female 30 20 10
      Age at baseline
      years, mean (range) 64.2 (42-83) 64.8 (42-81) 64.0 (43-82)
      Smoking status
      current smoker 75 44 31
      former smoker 74 36 38
      unknown 15 12 3
      Smoking history
      Pack-years, mean (range) 45 (4-137) 45 (4-120) 40 (15-137)
      COPD-status
      COPD 100 56 44
      non-COPD 45 22 23
      unknown 19 14 5
      AF Bronchoscopies
      Number, mean (range) 7 (1-27) 5 (2-27) 6 (1-18)
      CT-scans
      Number, mean (range) 3 (0-20) 2 (0-20) 3 (0-18)
      No. of detected lung cancers
      During surveillance period 61 29 32
      Parenchymal cancer 21 12 9
      Site-specific lesion progression 24 13 11
      Interval cancer 10 4 6
      Recurrences previous primaries 6 0 6
      Patient outcome
      alive 80 56 24
      died of lung cancer 33 13 20
      died of other/unknown cause 51 23 28

      Conclusion
      Our findings demonstrate that individuals with pre-invasive endobronchial lesions are at high risk of developing (second primary) lung cancers. Combined surveillance using AFB in addition to CT screening facilitated early detection and early (endobronchial) intervention in most patients. Future clinical trials are warranted to determine whether the current approach improves patient outcome.

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    P2.18 - Poster Session 2 - Pathology (ID 176)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Pathology
    • Presentations: 1
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      P2.18-022 - <strong>Do <i>EGFR</i>- and <i>KRAS</i>-mutations occur in squamous cell lung carcinomas?</strong> (ID 3398)

      09:30 - 09:30  |  Author(s): P.J.F. Snijders

      • Abstract

      Background
      Adenocarcinoma (ADC) of the lungs may harbor EGFR- and KRAS-mutations, which are relevant for treatment decisions. Approximately 35% of non-small cell lung cancer (NSCLC) biopsies are diagnosed as not-otherwise-specified (NOS).To improve segregation between ADC and squamous cell carcinoma (SqCC), the classification of lung cancer was updated in 2011, adding immunohistochemistry (IHC) for p63 and TTF-1 to the diagnostic algorithm. The aim of our study was to investigate the hypothesis, that additional IHC reliably delineates lung cancer harboring EGFR- and KRAS-mutations.

      Methods
      From an institutional lung cancer database of specimens routinely analyzed for the presence of EGFR- or KRAS-mutations (n=816), cases harboring a mutation were selected (n=343) and corresponding original histological diagnoses and IHC for TTF-1, p63 and PAS-D were collected. Cases with a pattern compatible with SqCC were histologically reassessed.

      Results
      From the 343 cases 25% were resection specimen, 70% biopsy and 5% cytology specimens. 69% of cases had a KRAS-mutation and 31% an EGFR-mutation. IHC-data were conclusive in 89%. The combination of positive TTF-1 and/or mucin stain and a negative p63 stain, favoring ADC, was found in 264 cases (77%). Six (1.7%) specimens were positive for p63 only, favoring SqCC.

      Conclusion
      The current 2011 classification of lung tumors, based on histology and immunohistochemistry for TTF-1, p63 and mucin, segregates specimens of ADC and SqCC sufficiently well. Our study results support the use of IHC in the diagnosis of lung cancer.

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    P2.20 - Poster Session 2 - Early Detection and Screening (ID 173)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Imaging, Staging & Screening
    • Presentations: 1
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      P2.20-004 - DNA copy number aberrations in endobronchial lesions: a validated predictor for cancer (ID 1166)

      09:30 - 09:30  |  Author(s): P.J.F. Snijders

      • Abstract

      Background
      Individuals who present with squamous metaplastic and dysplastic lesions are considered at high risk of lung cancer. However, these lesions behave erratically and only a minority progresses towards lung cancer. Therefore, biomarkers need to be discovered that can aid in assessing an individual’s risk for subsequent cancer. We recently identified a DNA copy number aberration (CNA)-classifier, including changes at 3p26.3-p11.1, 3q26.2-29, and 6p25.3-24.3, as a risk predictor for cancer in individuals presenting with endobronchial squamous metaplasia (van Boerdonk et al, AJRCCM, 2011). The current study was set out to validate this classifier in an independent series of endobronchial squamous metaplastic and dysplastic lesions.

      Methods
      DNA copy number profiles (i.e., chromosomal gains and losses) were determined in a set of endobronchial lesions (8 squamous metaplasia (SqM), and 28 dysplasias (Dys) of various grades), identified and biopsied during autofluorescence bronchoscopy, of 36 high-risk subjects using a nested case-control design. Of the 36 patients, 12 cases had a carcinoma in situ or invasive carcinoma at the same site at follow-up (median 11 months, range 4-24), while 24 controls remained cancer-free (median 78 months, range 21-142). DNA copy number profiles were related to lesion outcome. The prediction accuracy of the predefined CNA-based classifier to predict endobronchial carcinoma (in situ) in this series was determined.

      Results
      All SqM and Dys lesions of controls showed no or a relatively low number of CNAs (i.e., quiescent profile with on average 0.2% altered probe features, range 0.0 – 2.4%), while the majority of lesions of cases showed multiple CNAs (i.e. highly aberrant profile with on average 38.8% altered probe features, range 0.0 – 76.7%). The previously defined CNA-classifier demonstrated 92% accuracy for cancer (in situ) prediction in the current series. All nine subjects with CNA-classifier-positive endobronchial lesions at baseline had cancer as final outcome (i.e., a positive predictive value of 100%). The negative predictive value of the classifier was 89%, i.e., all 24 controls and 3 cases were classified as being low-risk.

      Conclusion
      CNAs are a highly accurate biomarker for assessing the progression risk of endobronchial squamous metaplastic and dysplastic lesions. This classifier could assist in selecting subjects with endobronchial lesions who might benefit from more aggressive therapeutic interventions.

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    P3.21 - Poster Session 3 - Diagnosis and Staging (ID 171)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Prevention & Epidemiology
    • Presentations: 1
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      P3.21-007 - <em>EGFR</em> mutation analysis in sputum of lung cancer patients: a multicenter multitechnique study (ID 1782)

      09:30 - 09:30  |  Author(s): P.J.F. Snijders

      • Abstract

      Background
      Mutations in the epidermal growth factor receptor (EGFR) gene have been identified in lung adenocarcinomas and are associated with a high response to EGFR tyrosine kinase inhibitors. EGFR mutations can be detected in tumour tissue, cytology specimens and blood from lung cancer patients. Thus far, EGFR mutation analysis has not been systematically demonstrated for sputum samples. The aim of the present study was to determine whether EGFR mutation analysis is feasible on sputum samples, employing different assays in a multicenter study.

      Methods
      Sputum samples were collected from 10 lung cancer patients with confirmed EGFR mutation in their tumour tissue, 10 lung cancer patients without evidence of an EGFR mutation, and 10 patients with chronic obstructive pulmonary disease (COPD). DNA was isolated from the sputum and used for mutation analysis by Cycleave PCR, COLD-PCR, PangaeaBiotech SL technology (PST), and High Resolution Melting, respectively. Targeted resequencing (TruSeq Amplicon Cancer Panel) and droplet digital PCR were additionally performed on the 10 samples with EGFR mutation.

      Results
      Dependent on the assay, EGFR mutations could be detected in 30-50% of the sputum samples of patients with EGFR mutations (Table). The different techniques revealed consistent results, with slightly higher sensitivity for PST. Neither the lung cancer patients without EGFR mutation nor the COPD controls tested positive for EGFR mutations in their sputum samples, indicating high clinical specificity of all assays.

      Subject Gender Age (years) Tumour stage EGFR mutation status of tumour tissue[1] EGFR mutation analysis on sputum specimens[2]
      Cycleave PCR COLD-PCR PST[3] HRM-sequencing Cytology[4]
      A F 72 IV Del E746-A750 0 0 0 0 0
      B M 66 I Del E746-A750 0 2 0 0 0
      C[6] F 78 IV Del E746-A750 1 1 1 1 2
      D F 46 III Del E746-A750 0 0 1 0 0
      E[6] M 54 IV Del E746-A750 1 1 1 1 0
      F F 49 III Del E746-A750 & c.2369C>T [p.T790M] 0 0 0 0 0
      G F 54 IV Del E746-A750 & c.2369C>T [p.T790M] 0 0 1[5] 0 1
      H F 73 IV c.2753T>G [p.L858R] 0 0 0 0 0
      I F 61 IV c.2753T>G [p.L858R] 0 0 0 0 0
      J[6] M 60 IV Del E746-A750 1 1 1 1 2
      [1 ]del E746-A750= deletion exon 19 [2] mutation identified: 0=no, 1=yes, 2=dubious [3] exclusively del19 and L858R were assessed [4] tumour cells: 0=no, 1=yes, 2=in related sample of same patient [5 ]only del19 detected [6 ]TSACP and ddPCR both tested EGFR mutation (del19) positive.

      Conclusion
      EGFR mutations can be detected in sputum samples from patients with EGFR-mutated non-small cell lung cancer, which may replace biopsy procedure for some patients.