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Prolonged sampling of spontaneous sputum improves sensitivity of hypermethylation analysis for lung cancer
  1. A Jasmijn Hubers1,
  2. Daniëlle A M Heideman1,
  3. Gerarda J M Herder2,
  4. Sjaak A Burgers3,
  5. Peter J Sterk4,
  6. Peter W Kunst4,
  7. Henk J Smit5,
  8. Pieter E Postmus6,
  9. Birgit I Witte7,
  10. Sylvia Duin1,
  11. Peter J F Snijders1,
  12. Egbert F Smit6,
  13. Erik Thunnissen1
  1. 1Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands
  2. 2Department of Pulmonary Diseases, Sint Antonius Hospital, Nieuwegein, The Netherlands
  3. 3Department of Thoracic Oncology, NKI - Antoni van Leeuwenhoek Hospital, Amsterdam, The Netherlands
  4. 4Department of Pulmonary Diseases, Academic Medical Center, Amsterdam, The Netherlands
  5. 5Department of Pulmonary Diseases, Sint Lucas Andreas Hospital, Amsterdam, The Netherlands
  6. 6Department of Pulmonary Diseases, VU University Medical Center, Amsterdam, The Netherlands
  7. 7Department of Epidemiology and Biostatistics, VU University Medical Center, Amsterdam, The Netherlands
  1. Correspondence to Dr Erik Thunnissen, Department of Pathology, VU University Medical Center, De Boelelaan 1117, Amsterdam 1081 HV, The Netherlands; e.thunnissen{at}


Aims The adequacy of lung cancer diagnosis with sputum cytology depends on duration of sputum sampling. The aim of this methodological study was to determine whether the hypermethylation detection rate of RASSF1A, adenomatous polyposis coli (APC) and cytoglobin (CYGB) is influenced by the duration of sputum collection.

Methods Prospective sputum samples were collected from 53 lung cancer patients and 47 chronic obstructive pulmonary disease patients as controls. Subjects collected spontaneous sputum at home during nine consecutive days in three canisters I, II and III (ie, days 1–3, days 4–6, days 7–9, respectively). Quantitative methylation-specific PCR was performed to assess gene promoter methylation status of RASSF1A, APC and CYGB.

Results Analysis of each canister separately showed hypermethylation of RASSF1A, APC and/or CYGB in samples I, II and III, in 43%, 40% and 47% of cases, respectively. In control samples, these numbers were 4%, 2% and 4%, respectively. Cumulative analysis for days 1–6 and days 1–9 revealed an increase in sensitivity to 53% and 64%, and specificity of 94% and 91%, respectively.

Conclusion Sputum collected over multiple successive days results in a gain in sensitivity for the detection of lung cancer, at the expense of a small loss in specificity.

Condensed abstract Assessment of hypermethylation sensitivity of biomarkers in sputum collected over a prolonged period for the detection of lung cancer resulted in a promising gain in sensitivity, at the expense of a small loss in specificity.

  • Lung cancer
  • methylation
  • biomarkers
  • sensitivity
  • sputum

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  • Funding This study was funded by the Dutch Cancer Society (grant VU2008-4220).

  • Competing interests None declared.

  • Patient consent Patients signed an informed consent form not derived from BMJ, but according to the study protocol. The informed consent forms were approved by the Institutional Review Boards of all participating hospitals.

  • Ethics approval The ethics approval was provided by Institutional Review Boards of participating hospitals.

  • Provenance and peer review Not commissioned; externally peer reviewed.