Afra Zaal

Combined CADM1 and MAL promoter methylation analysis to detect (pre‐)malignant cervical lesions in high‐risk HPV‐positive women

Given the lower specificity for high‐grade cervical lesions of high‐risk human papillomavirus (hrHPV) testing compared to cytology, additional triage testing for hrHPV test‐positive women is needed to detect high‐grade cervical lesions. Here, we tested whether combined methylation analysis for cell adhesion molecule 1 (CADM1) and T‐lymphocyte maturation associated protein (MAL), both functionally involved in cervical carcinogenesis, could serve as such a triage marker. Four quantitative methylation‐specific PCRs (qMSP), two for CADM1 (regions M12 and M18) and MAL (regions M1 and M2) each, were applied to 261 cervical tissue specimens ranging from no neoplasia to carcinoma. When qMSPs were combined and positivity for at least one of the qMSPs in the combination was taken into account, the highest positivity rates for cervical intraepithelial neoplasia grade 3 (CIN3) lesions (97%) and squamous cell‐ and adeno‐carcinomas (99%) were obtained by combining a single CADM1 marker with a single MAL marker. Subsequent qMSP analysis of 70 GP5+/6+‐PCR hrHPV‐positive scrapings revealed that a two‐marker panel consisting of CADM1‐M18 and MAL‐M1 was most discriminative, detecting 90% of women with CIN3 (n = 30), whereas it showed a positive result in only 13.5% of women without cervical disease (n = 40). Finally, we applied hrHPV GP5+/6+‐PCR testing followed by CADM1‐M18/MAL‐M1 methylation analysis to a cohort of 79 women visiting the outpatient colposcopy clinic. hrHPV testing revealed a sensitivity of 97% and a specificity of 33% for CIN3+. Additional CADM1‐M18/MAL‐M1 methylation analysis on the hrHPV‐positive women increased the specificity to 78% with a sensitivity of 70%. In conclusion, the methylation marker panel CADM1‐M18 and MAL‐M1 may serve as an alternative molecular triage tool for hrHPV‐positive women.

Prognostic significance of low volume sentinel lymph node disease in early-stage cervical cancer

OBJECTIVE Evaluate prognostic significance of low volume disease detected in sentinel nodes (SN) of patients with early stages cervical cancer. Although pathologic ultrastaging of SN allows for identification of low volume disease, including micro-metastasis and isolated tumor cells (ITC), in up to 15% of cases, prognostic significance of these findings is unknown. METHODS A total of 645 records from 8 centers were retrospectively reviewed. Enrolled in our study were patients with early-stage cervical cancer who had undergone surgical treatment including SN biopsy followed by pelvic lymphadenectomy and pathologic ultrastaging of SN. RESULTS Macrometastasis, micrometastasis, and ITC were detected by SN ultrastaging in 14.7%, 10.1%, and 4.5% patients respectively. False negativity of SN ultrastaging reached 2.8%. The presence of ITC was not associated with significant risk, both for recurrence free survival and overall survival. Overall survival was significantly reduced in patients with macrometastasis and micrometastasis; hazard ratio for overall survival reached 6.85 (95% CI, 2.59-18.05) and 6.86 (95% CI, 2.09-22.61) respectively. Presence of micrometastasis was an independent prognostic factor for overall survival in a multivariable model. CONCLUSION Presence of micrometastasis in SN in patients with early stage cervical cancer was associated with significant reduction of overall survival, which was equivalent to patients with macrometastasis. No prognostic significance was found for ITC. These data highlight the importance of SN biopsy and pathologic ultrastaging for the management of cervical cancer.

Bilateral ultrastaging of sentinel lymph node in cervical cancer: Lowering the false-negative rate and improving the detection of micrometastasis

OBJECTIVE To evaluate the sensitivity of sentinel node (SN) ultrastaging and to define parameters that may reduce the overall false-negative rate in women with early-stage cervical cancer. METHODS We analyzed data from a large retrospective multicenter cohort group with FIGO stages IA-IIB cervical cancer in whom at least one SN was identified and systematic pelvic lymphadenectomy was uniformly performed. All who were SN negative by initial evaluation were subjected to ultrastaging. RESULTS In all, 645 patients were evaluable. SN were detected bilaterally in 72% of cases and unilaterally in 28%. Patients with optimal bilateral SN detection were significantly more likely to have any metastasis detected (33.3% vs. 19.2%; P<0.001) as well as micrometastasis detected in their SN (39.6% vs. 11.4%). SN ultrastaging resulted in a low overall false-negative rate of 2.8% (whole group) and an even lower false-negative rate of 1.3% for patients with optimal bilateral mapping. Patients with false-negative SN after ultrastaging had a higher prevalence of LVSI and more frequent unilateral SN detection. Sensitivity of SN ultrastaging was 91% (95% CI: 86%-95%) for the whole group and 97% (95% CI: 91%-99%) in the subgroup with bilateral SN detection. CONCLUSION These data confirm previous observations that optimal bilateral SN detection substantially decreases the false negative rate of SN ultrastaging and increases detection of micrometastasis. In patients with bilateral SN detection, the sensitivity of SN ultrastaging is not reduced in more advanced stages of the disease. SN mapping and ultrastaging should become standard practice in the surgical management of early-stage cervical cancer.

Agreement between colposcopic impression and histological diagnosis among human papillomavirus type 16‐positive women: a clinical trial using dynamic spectral imaging colposcopy

Please cite this paper as: Zaal A, Louwers J, Berkhof J, Kocken M, ter Harmsel W, Graziosi G, Spruijt J, Balas C, Papagiannakis E, Snijders P, Meijer C, van Kemenade F, Verheijen R. Agreement between colposcopic impression and histological diagnosis among human papillomavirus type 16‐positive women: a clinical trial using dynamic spectral imaging colposcopy. BJOG 2012;119:537–544.

Detection of cervical cancer recurrence during follow-up: A multivariable comparison of 9 frequently investigated serum biomarkers

OBJECTIVE To assess the diagnostic accuracy and model the optimal combination of commonly studied serum biomarkers aimed at identifying recurrence in cervical cancer patients. METHODS From a systematic literature search, nine biomarkers (CA-15.3, CA-125, CEA, CYFRA 21-1, hsCRP, IL-6, SCC-Ag, TNF-α and VEGF) were selected for a serum analysis. Samples were derived from a historical cervical cancer cohort. Subjects with serum samples stored in a biobank were included when quality criteria were met, and one sample preceding and at least one following primary treatment were available. In case of recurrence, two additional post-recurrence samples were analyzed. Biomarker serum levels were quantified by enzyme linked or chemiluminescence microparticle immunoassays. Logistic regression and receiver operating curve analysis were employed for selection, modeling and comparison on the diagnostic accuracy of the tested biomarkers. RESULTS 205 samples were analyzed from 75 subjects, of whom 19 (25.3%) had a recurrence. The area under the curve (AUC) of CA-15.3, CA-125, CEA, CYFRA 21-1, IL-6, TNF-α and VEGF were all <0.750. Only SCC-Ag and hsCRP were included in the final model with an AUC of 0.822 (95% CI: 0.744-0.900) and 0.831 (95% CI: 0.758-0.905) respectively. Combined AUC was 0.870 (95% CI: 0.805-0.935). Rises in SCC-Ag and hsCRP significantly increased the odds for recurrence. Each ng/ml of SCC-Ag increase, related to an odds ratio (OR) of 1.117 (95% CI: 1.039-1.200). Comparably, the OR for hsCRP (in mg/ml) was 1.025 (95% CI: 1.012-1.038). CONCLUSION Combined testing of SCC-Ag and hsCRP yields the highest detection rate of disease recurrence during cervical cancer follow-up.

Pelvic lymphadenectomy improves survival in patients with cervical cancer with low-volume disease in the sentinel node: a retrospective multicenter cohort study.

Objective In this study, we aimed to describe the value of pelvic lymph node dissection (LND) after sentinel lymph node (SN) biopsy in early-stage cervical cancer. Methods We performed a retrospective multicenter cohort study in 8 gynecological oncology departments. In total, 645 women with International Federation of Gynecology and Obstetrics stage IA to IIB cervical cancer of squamous, adeno, or adenosquamous histologic type who underwent SN biopsy followed by pelvic LND were enrolled in this study. Radioisotope tracers and blue dye were used to localize the sentinel node, and pathologic ultrastaging was performed. Results Among the patients with low-volume disease (micrometastases and isolated tumor cells) in the sentinel node, the overall survival was significantly better (P = 0.046) if more than 16 non-SNs were removed. No such significant difference in survival was detected in patients with negative or macrometastatic sentinel nodes. Conclusions Our findings indicate that in patients with negative or macrometastatic disease in the sentinel nodes, an additional LND did not alter survival. Conversely, our data suggest that the survival of patients with low-volume disease is improved when more than 16 additional lymph nodes are removed. If in a prospective trial our data are confirmed, we would suggest a 2-stage operation.

Long-term CIN3 risk in women with abnormal cytology; Role of hrHPV testing

Background:Many studies have examined the short-term value of high-risk human papillomavirus (hrHPV) testing in predicting cumulative risk of cervical intraepithelial neoplasia grade 3 or cancer (CIN3+). This study focuses on long-term CIN3+ risk after initial wait and see policy.Methods:A total of 342 women with abnormal cytology of borderline/mild dyskaryosis (BMD) or worse (>BMD), included between 1990 and 1992, were followed-up by cytology and hrHPV testing until 1996 and monitored by cytology thereafter. Primary endpoint was cumulative CIN3+ risk by December 2009.Results:Women with BMD had a 5-year CIN3+ risk of 22.5% (95% confidence interval (CI) 17.0–29.1) and of 0.7% (0.1–4.5) in the subsequent 5 years. High-risk human papillomavirus-negative women with BMD had a 5-year risk of <0.01% (95% CI 0.0–5.1) and of <0.01% (0.0–5.7) in the following 5 years, while for hrHPV-positive women these risks were 37.5% (29.0–46.9) and 1.6% (0.2–9.5), respectively. Women with >BMD had a 5-year risk of 45.1% (36.4–54.1) and of 3.5% (0.9–12.2) in the subsequent 5 years. High-risk human papillomavirus-negative women with >BMD had a 5-year risk of 7.3% (2.0–23.6) and hrHPV-positive women of 56.6% (46.4–66.3).Conclusion:Women with BMD have an elevated CIN3+ risk for 5 years only; afterwards their risk is similar to the general population. High-risk human papillomavirus-negative women with BMD may return to regular screening directly. All other women with ⩾BMD should be referred for additional testing and/or colposcopy.

Genomic aberrations relate early and advanced stage ovarian cancer

BackgroundBecause of the distinct clinical presentation of early and advanced stage ovarian cancer, we aim to clarify whether these disease entities are solely separated by time of diagnosis or whether they arise from distinct molecular events.MethodsSixteen early and sixteen advanced stage ovarian carcinomas, matched for histological subtype and differentiation grade, were included. Genomic aberrations were compared for each early and advanced stage ovarian cancer by array comparative genomic hybridization. To study how the aberrations correlate to the clinical characteristics of the tumors we clustered tumors based on the genomic aberrations.ResultsThe genomic aberration patterns in advanced stage cancer equalled those in early stage, but were more frequent in advanced stage (p = 0.012). Unsupervised clustering based on genomic aberrations yielded two clusters that significantly discriminated early from advanced stage (p = 0.001), and that did differ significantly in survival (p = 0.002). These clusters however did give a more accurate prognosis than histological subtype or differentiation grade.ConclusionThis study indicates that advanced stage ovarian cancer either progresses from early stage or from a common precursor lesion but that they do not arise from distinct carcinogenic molecular events. Furthermore, we show that array comparative genomic hybridization has the potential to identify clinically distinct patients.

The diagnostic process of cervical cancer; areas of good practice, and windows of opportunity

OBJECTIVE Despite an extensive screening programme in The Netherlands, some cases of cervical cancer are still diagnosed in late stages of disease. The aim of the present study was to investigate which elements in the diagnostic process of cervical cancer may be improved. METHODS This is a retrospective study of 120 patients with cervical cancer diagnosed between January 1st 2008 and June 1st 2010 at the University Medical Center Utrecht. Patient charts, referral information, and pathology results were analyzed. RESULTS 39.1% of cancer cases were screen or interval detected; the other 60.9% of patients had not been screened, either due to non-attendance or because they fell outside the age range for screening. The final diagnosis of cervical cancer was established by biopsy in 77 (64.2%) and by excision of the cervical transformation zone in 35 (29.2%) of the patients. Fifteen (43%) of these excisions could have been avoided if biopsies would have been taken at the first examination, and had shown invasive cancer. CONCLUSIONS Cervical cancer screening aims at early detection of precursor lesions to decrease the incidence of cancer. This in-depth analysis suggests that improvement of quality of care is to be expected from correct recognition of cervical cancer by physicians and adjustments of the screening programme to reach younger women and non-responders.

The performance of Dynamic Spectral Imaging colposcopy depends on indication for referrals

OBJECTIVE. A previous study has shown that Dynamic Spectral Imaging (DSI) colposcopy increases the sensitivity of the colposcopic examination in women referred with abnormal cytology. In this study we have reanalyzed the performance of DSI and conventional colposcopy for new referral conditions and for low-grade cytology referrals versus high-grade cytology referrals. METHOD. Data from a previous validation trial was used to assess the performance of DSI in different cytology groups:Women referred with BMD (borderline and mild dyskaryosis) cytology and women referred with NBMD cytology either hrHPV positive or negative were separately analyzed. Furthermore, we tried to assess the clinical performance by appropriate filtering of patients to replicate two different referral strategies. RESULTS. The sensitivity of DSI and conventional colposcopy to detect CIN2+ lesions in women referred with BMD cytology is 82% and 44% respectively (p= 0.001) and in the NBMD group 77% and 64% respectively (p= 0.24). If the two techniques are combined the sensitivity is 85%.When the conditions of new screening strategies are applied DSI colposcopy has a higher sensitivity to detect CIN2+ than conventional colposcopy. Findings are similar when CIN3+ is used as a threshold. CONCLUSION. We found that in most cases DSI colposcopy has a higher sensitivity than conventional colposcopy, even when referral criteria are changed. Unlike conventional colposcopy, the sensitivity of colposcopy with DSI in low-grade cytology referrals was found similar to the sensitivity in high-grade cytology referrals. This suggests that a baseline colposcopy sensitivity may be possible with the adjunctive use of the DSI map, irrespective of referral cytology.