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Purpose: Offering self-sampling of cervico-vaginal material for high-risk human papillomavirus (hrHPV) testing is an effective method to increase the coverage in cervical screening programs. Molecular triage directly on hrHPV-positive self-samples for colposcopy referral opens the way to full molecular cervical screening. Here, we set out to identify a DNA methylation classifier for detection of cervical precancer (CIN3) and cancer, applicable to lavage and brush self-samples. Experimental Design: We determined genome-wide DNA methylation profiles of 72 hrHPV-positive self-samples, using the Infinium Methylation 450K Array. The selected DNA methylation markers were evaluated by multiplex quantitative methylation-specific PCR (qMSP) in both hrHPV-positive lavage (n = 245) and brush (n = 246) self-samples from screening cohorts. Subsequently, logistic regression analysis was performed to build a DNA methylation classifier for CIN3 detection applicable to self-samples of both devices. For validation, an independent set of hrHPV-positive lavage (n = 199) and brush (n = 287) self-samples was analyzed. Results: Genome-wide DNA methylation profiling revealed 12 DNA methylation markers for CIN3 detection. Multiplex qMSP analysis of these markers in large series of lavage and brush self-samples yielded a 3-gene methylation classifier (ASCL1, LHX8, and ST6GALNAC5). This classifier showed a very good clinical performance for CIN3 detection in both lavage (AUC = 0.88; sensitivity = 74%; specificity = 79%) and brush (AUC = 0.90; sensitivity = 88%; specificity = 81%) self-samples in the validation set. Importantly, all self-samples from women with cervical cancer scored DNA methylation–positive. Conclusions: By genome-wide DNA methylation profiling on self-samples, we identified a highly effective 3-gene methylation classifier for direct triage on hrHPV-positive self-samples, which is superior to currently available methods. Clin Cancer Res; 24(14); 3456–64. ©2018 AACR.

Purpose: Epigenetic host cell changes involved in cervical cancer development following a persistent high-risk human papillomavirus (hrHPV) infection, provide promising markers for the management of hrHPV-positive women. In particular, markers based on DNA methylation of tumor suppressor gene promoters are valuable. These markers ideally identify hrHPV-positive women with precancer (CIN2/3) in need of treatment. Here, we set out to identify biologically relevant methylation markers by genome-wide methylation analysis of both hrHPV-transformed cell lines and cervical tissue specimens. Experimental Design and Results: Genome-wide discovery by next-generation sequencing (NGS) of methyl-binding domain–enriched DNA (MBD-Seq) yielded 20 candidate methylation target genes. Further verification and validation by multiplex-targeted bisulfite NGS and (quantitative) methylation-specific PCR (MSP) resulted in 3 genes (GHSR, SST, and ZIC1) that showed a significant increase in methylation with severity of disease in both tissue specimens and cervical scrapes (P < 0.005). The area under the ROC curve for CIN3 or worse varied between 0.86 and 0.89. Within the group of CIN2/3, methylation levels of all 3 genes increased with duration of lesion existence (P < 0.0005), characterized by duration of preceding hrHPV infection, and were significantly higher in the presence of a 3q gain (P < 0.05) in the corresponding tissue biopsy. Conclusions: By unbiased genome-wide DNA methylation profiling and comprehensive stepwise verification and validation studies using in vitro and patient-derived samples, we identified 3 promising methylation markers (GHSR, SST, and ZIC1) associated with a 3q gain for the detection of cervical (pre)cancer. Clin Cancer Res; 23(14); 3813–22. ©2017 AACR.

Purpose: To evaluate whether, in patients with non–small cell lung carcinoma (NSCLC), tumor uptake of [11C]erlotinib can be quantified and imaged using positron emission tomography and to assess whether the level of tracer uptake corresponds with the presence of activating tumor EGF receptor (EGFR) mutations. Experimental Design: Ten patients with NSCLCs, five with an EGFR exon 19 deletion, and five without were scanned twice (test retest) on the same day with an interval of at least 4 hours. Each scanning procedure included a low-dose computed tomographic scan, a 10-minute dynamic [15O]H2O scan, and a 1-hour dynamic [11C]erlotinib scan. Data were analyzed using full tracer kinetic modeling. EGFR expression was evaluated using immunohistochemistry. Results: The quantitative measure of [11C]erlotinib uptake, that is, volume of distribution (VT), was significantly higher in tumors with activating mutations, that is, all with exon 19 deletions (median VT, 1.76; range, 1.25–2.93), than in those without activating mutations (median VT, 1.06; range, 0.67–1.22) for both test and retest data (P = 0.014 and P = 0.009, respectively). Good reproducibility of [11C]erlotinib VT was seen (intraclass correlation coefficient = 0.88). Intergroup differences in [11C]erlotinib uptake were not correlated with EGFR expression levels, nor tumor blood flow. Conclusion: [11C]erlotinib VT was significantly higher in NSCLCs tumors with EGFR exon 19 deletions. Clin Cancer Res; 19(1); 183–93. ©2012 AACR.

Introduction: K-Ras mutation is thought to be related with poor outcome of NSCLC patients with an advanced stage of disease. The role of K-Ras mutation as a predictor of response for these patients treated only with chemotherapy is poorly understood. Patients and methods: From a retrospective database from 2 university hospitals all patients with advanced stage, nonsquamous, NSCLC treated with palliative platinum containing chemotherapy were selected. Patients were included when archive tumour tissue was available for DNA extraction. High resolution melting followed by PCR sequencing was performed for KRAS exon1 and 2. Response to treatment was assessed by RECIST. The databases were matched by treatment. Results: A total of 179 patients were included in this study. All patients were treated with chemotherapy and were no candidate for treatment with curative intent. There were 59 patients with a K-Ras mutation (32.9%). There was no significant difference in basic characteristics between the group with a K-Ras mutation and the group with K-Ras wild-type. Mean age 59 years (range 32–83 years), 108 males and 71 females. ECOG performance score 0/1/2/3 73/77/13/2. Thirty patients with stage IIIb disease, 149 patients with stage IV disease. Progression as best response to treatment was reported in 21/59 patients with a K-ras mutation (35.6%) and 34/120 patients (28.3%) with a K-Ras wild type, this was not significant (p=0.611). The median progression free survival (PFS) in the group with a K-Ras mutation was 4.1 months (95% CI 3.12–5.0 months) compared to 5.4 months (95% CI 4.4–6.4 months) in patients with K-Ras wild type (p=0.202). The overall survival (OS) in the group with a K-Ras mutation, was 8.3 months (95% CI 5.2–11.4 months) compared to 12.5 months (95% CI 10.2–14–7 months) in patients with K-Ras wild type (p=0.037). Conclusion: In our series K-Ras mutation was not predictive for response to platinum containing chemotherapy in patients with advanced NSCLC. K-Ras mutational status was prognostic for overall survival.

Background: In a pilot study (1) we found sorafenib to display clinical activity against patients with K-Ras positive NSCLC, sufficient for formal phase II testing. Methods: Patients with K-Ras mutated NSCLC that progressed after at least 1 platinum containing regimen with adequate organ reserve, ECOG 0–2, who provided written informed consent according to local IRB regulations were eligible. A tumor biopsy confirming the presence of a K-Ras mutation was mandatory. Treatment consisted of sorafenib 400 mg BID until disease progression or unacceptable toxicity. Dose reductions and delays were specified per protocol in the face of CTC toxicities grade 3 and 4. Primary endpoint: Rate of No Progression (NPR) at 6 weeks. Secondary endpoints: duration of response, progression free survival (PFS), overall survival and treatment related toxicities. A 2-stage design was implemented (Simon's optimal design; p0=40%, p1=60%, alpha=0.05, beta=0.20) for a total number of 48 pts. Results: 59 patients were entered between May 1st 2010 and February 18 2011. Median age was 58 (range 46–79) years, 17 Male/42 Female, ECOG PS 0/1/2 23/32/4. 57 patients started treatment. At 6 weeks 5 PR, 25 SD, 27 PD were observed; NPR 52.6%. Fifteen patients stopped treatment before 6 weeks, of which 10 patients stopped due to (clinical) progression. Median duration of treatment was 9 (range 0–62) weeks, 2 patients are still on treatment. Median duration of response was 32 (range 5–58) weeks. Median PFS was 2.3 months, median OS was 4.9 months, 14 patients still alive. Dose modifications were realized in 21 patients, of whom 4 discontinued treatment. Most reported adverse events were fatigue (6.4%), hand-foot reaction (5.7%), dyspnea (5.6%), anorexia (3.7%) diarrhea (3.6%) and cough (3.6%). Sorafenib related grade 3-4 toxicity was reported in 10 patients. Five patients with grade 3 skin toxicity, 4 patients with grade 3 gastrointestinal toxicity and 1 patient with both grade 3 metabolic abnormalities and a grade 3 pneumonitis. Conclusion: Treatment with sorafenib has relevant clinical activity in patients with K-Ras mutational status. Further randomized study with this agent is warranted. This abstract is also presented as Poster B18.