Miriam Reuschenbach

A systematic review of humoral immune responses against tumor antigens

This review summarizes studies on humoral immune responses against tumor-associated antigens (TAAs) with a focus on antibody frequencies and the potential diagnostic, prognostic, and etiologic relevance of antibodies against TAAs. We performed a systematic literature search in Medline and identified 3,619 articles on humoral immune responses and TAAs. In 145 studies, meeting the inclusion criteria, humoral immune responses in cancer patients have been analyzed against over 100 different TAAs. The most frequently analyzed antigens were p53, MUC1, NY-ESO-1, c-myc, survivin, p62, cyclin B1, and Her2/neu. Antibodies against these TAAs were detected in 0–69% (median 14%) of analyzed tumor patients. Antibody frequencies were generally very low in healthy individuals, with the exception of few TAAs, especially MUC1. For several TAAs, including p53, Her2/neu, and NY-ESO-1, higher antibody frequencies were reported when tumors expressed the respective TAA. Antibodies against MUC1 were associated with a favorable prognosis while antibodies against p53 were associated with poor disease outcome. These data suggest different functional roles of endogenous antibodies against TAAs. Although data on prediagnostic antibody levels are scarce and antibody frequencies for most TAAs are at levels precluding use in diagnostic assays for cancer early detection, there is some promising data on achieving higher sensitivity for cancer detection using panels of TAAs.

The APTIMA HPV assay versus the hybrid capture 2 test in triage of women with ASC-US or LSIL cervical cytology: A meta-analysis of the diagnostic accuracy†

Testing for DNA of 13 high‐risk HPV types with the Hybrid Capture 2 (HC2) test has consistently been shown to perform better in triage of women with cervical cytology results showing atypical squamous cells of undetermined significance (ASC‐US) but often not in triage of low‐grade squamous intraepithelial lesions (LSIL) detected in cervical cancer screening. In a meta‐analysis, we compared the accuracy of the APTIMA HPV test, which identifies RNA of 14 high‐risk HPV types, to HC2 for the triage of women with ASC‐US or LSIL. Literature search‐targeted studies where the accuracy of APTIMA HPV and HC2 for detection of underlying CIN2/3+ was assessed concomitantly including verification of all cases of ASC‐US and LSIL. HSROC (Hierarchical Summary ROC) curve regression was used to compute the pooled absolute and relative sensitivity and specificity. Eight studies, comprising 1,839 ASC‐US and 1,887 LSIL cases, were retrieved. The pooled sensitivity and specificity of APTIMA to triage ASC‐US to detect underlying CIN3 or worse was 96.2% (95% CI = 91.7–98.3%) and 54.9% (95% CI = 43.5–65.9%), respectively. APTIMA and HC2 showed similar pooled sensitivity; however, the specificity of the former was significantly higher (ratio: 1.19; 95% CI = 1.08–1.31 for CIN2+). The pooled sensitivity and specificity of APTIMA to triage LSIL were 96.7% (95% CI = 91.4–98.9%) and 38.7% (95% CI = 30.5–47.6%) for CIN3+. APTIMA was as sensitive as HC2 but more specific (ratio: 1.35; 95% CI = 1.11–1.66). Results were similar for detection of CIN2 or worse. In both triage of ASC‐US and LSIL, APTIMA is as sensitive but more specific than HC2 for detecting cervical precancer.

Performance of the Aptima High-Risk Human Papillomavirus mRNA Assay in a Referral Population in Comparison with Hybrid Capture 2 and Cytology

ABSTRACT This study compared the Aptima human papillomavirus (HPV) (AHPV; Gen-Probe Incorporated) assay, which detects E6/E7 mRNA from 14 high-risk types, the Hybrid Capture 2 HPV DNA (HC2; Qiagen Incorporated) test, and repeat cytology for their ability to detect high-grade cervical lesions (cervical intraepithelial neoplasia grade 2+ [CIN2+]) in women referred to colposcopy due to an abnormal Papanicolaou (Pap) smear. A total of 424 clinical specimens, stored in liquid-based cytology (LBC) vials at room temperature for up to 3 years, were tested by repeat cytology, the AHPV assay, and the HC2 test. Assay results were compared to each other and to histology results. The overall agreement between the AHPV assay and the HC2 test was 88.4%. The sensitivity (specificity) of cytology, the HC2 test, and the AHPV assay for the detection of CIN2+ was 84.9% (66.3%), 91.3% (61.0%), and 91.7% (75.0%) and for the detection of CIN3+ was 93.9% (54.4%), 95.7% (46.0%), and 98.2% (56.3%), respectively. Of the disease-positive specimens containing high-risk HPV (HR HPV) DNA as determined by Linear Array (Roche Diagnostics), the AHPV assay missed 3 CIN2 and 1 microfocal CIN3 specimen, while the HC2 test missed 6 CIN2, 4 CIN3, and 1 cervical carcinoma specimen. The AHPV assay had a sensitivity similar to but a specificity significantly higher (P < 0.0001) than the HC2 test for the detection of CIN2+. The AHPV assay was significantly more sensitive (P = 0.0041) and significantly more specific (P = 0.0163) than cytology for the detection of disease (CIN2+).

Performance of p16INK4a-cytology, HPV mRNA, and HPV DNA testing to identify high grade cervical dysplasia in women with abnormal screening results

OBJECTIVE The prognostic value of dysplastic lesions of the uterine cervix cannot be adequately determined by Pap cytology alone. Detection of HPV DNA increases the diagnostic sensitivity. However, due to the very high prevalence of transient HPV infections, HPV DNA testing suffers from poor diagnostic specificity. Biomarkers that highlight the shift from self limited transient to potentially dangerous transforming HPV infections may improve the accuracy of cervical cancer screening. We evaluated HPV E6/E7 mRNA detection (APTIMA), p16(INK4a)-immunocytology (CINtec), and HPV DNA testing (HC2) to identify women with high grade cervical neoplasia in a disease-enriched cross-sectional cohort. METHODS Liquid based cytology specimens were collected from 275 patients. All assays were performed from these vials. Detection rates of each test were evaluated against conventional H&E based histopathology alone and stratified by p16(INK4a)-immunohistochemistry (IHC). RESULTS All assays yielded a high sensitivity for the detection of CIN3+ (96.4% (95% CI, 90.4-98.8) for HC2, 95.5% (89.2-98.3) for APTIMA and CINtec) and CIN2+ (91.5% (85.8-95.1) for HC2, 88.4% (82.3-92.7) for APTIMA, 86.6% (80.2-91.2) for CINtec). The specificity to detect high grade dysplasia was highest for CINtec p16(INK4a)-cytology (60.6% (52.7-68.0) in CIN3+ and 74.8% (65.5-82.3) in CIN2+), followed by APTIMA (56.4% (48.4-64.0) in CIN3+ and 71.2% (61.7-79.2) in CIN2+) and HC2 (49.1% (41.3-56.9) in CIN3+ and 63.4% (53.7-72.1) in CIN2+). All tests had higher sensitivity using p16(INK4a)-IHC-positive CIN2+ lesions as endpoint. CONCLUSIONS Biomarkers that detect HPV induced dysplastic changes in the transforming stage are promising tools to overcome the current limitations of cervical cancer screening.

Biomarkers for cervical cancer screening: the role of p16(INK4a) to highlight transforming HPV infections.

Biomarkers indicating the initiation of neoplastic transformation processes in human papillomavirus (HPV)-infected epithelial cells are moving into the focus of cancer prevention research, particularly for anogenital cancer, including cancer of the uterine cervix. Based on the in-depth understanding of the molecular events leading to neoplastic transformation of HPV-infected human cells, the cyclin-dependent kinase inhibitor p16INK4a turned out to be substantially overexpressed in virtually all HPV-transformed cells. This finding opened novel avenues in diagnostic histopathology to substantially improve the diagnostic accuracy of cervical cancer and its precursor lesions. Furthermore, it provides a novel technical platform to substantially improve the accuracy of cytology-based cancer early-detection programs. Here, we review the molecular background and the current evidence for the clinical utility of the p16INK4a biomarker for HPV-related cancers, and cervical cancer prevention in particular.

Differential methylation of E2 binding sites in episomal and integrated HPV 16 genomes in preinvasive and invasive cervical lesions

Enhanced expression of the HPV 16 E6‐E7 oncogenes may trigger neoplastic transformation of the squamous epithelial cells at the uterine cervix. The HPV E2 protein is a key transcriptional regulator of the E6‐E7 genes. It binds to four E2 binding sites (E2BSs 1–4) in the viral upstream regulatory region (URR). Modification of E2 functions, for example, by methylation of E2BSs is hypothesized to trigger enhanced expression of the viral E6‐E7 oncogenes. In the majority of HPV‐transformed premalignant lesions and about half of cervical carcinomas HPV genomes persist in an extra‐chromosomal, episomal state, whereas they are integrated into host cells chromosomes in the remaining lesions. Here we compared the methylation profile of E2BSs 1–4 of the HPV 16 URR in a series of 18 HPV16‐positive premalignant lesions and 33 invasive cervical cancers. CpGs within the E2BSs 1, 3, and 4 were higher methylated in all lesions with only episomal HPV16 genomes compared with lesions displaying single integrated copies. Samples with multiple HPV16 integrated copies displayed high methylation levels for all CpGs suggesting that the majority of multiple copies were silenced by extensive methylation. These data support the hypothesis that differential methylation of the E2BSs 1, 3 and 4 is related to the activation of viral oncogene expression in cervical lesions as long as the viral genome remains in the episomal state. Once the virus becomes integrated into host cell chromosomes these methylation patterns may be substantially altered due to complex epigenetic changes of integrated HPV genomes.

Evaluation of cervical cone biopsies for coexpression of p16INK4a and Ki‐67 in epithelial cells

Diffuse overexpression of p16INK4a in basal and parabasal cells of cervical epithelium is a hallmark of human papillomavirus‐mediated transformation. Focal p16INK4a expression is occasionally observed in nondysplastic epithelium. In normal cells, expression of p16INK4a triggers cell cycle arrest. However, cells undergoing transformation in intraepithelial lesions actively proliferate. To prove that the different expression patterns of p16INK4a, i.e., focal versus diffuse, reflect biologically different entities, we hypothesized that p16INK4a‐positive cells in epithelia displaying focal p16INK4a expression pattern do not coexpress proliferation‐associated Ki‐67 protein, while p16INK4a‐positive cells in lesions with diffuse p16INK4a expression may do. A total of 138 cervical cone biopsies were stained for the expression of p16INK4a and Ki‐67 using a primary antibody cocktail. All metaplastic lesions (n = 21) displayed focal staining for p16INK4a, and in all of these lesions p16INK4a‐positive cells were found to be negative for Ki‐67 expression. Diffuse expression of p16INK4a was observed in 12/21 (57.1%) cervical intraepithelial neoplasia (CIN) 1 lesions, all of them simultaneously showed Ki‐67 immunoreactivity in a large proportion of p16INK4a‐positive cells. Seventeen of 23 (73.9%) CIN2 lesions and all 27 (100%) CIN3/carcinoma in situ (CIS) as well as all 46 (100%) carcinoma cases displayed diffuse and combined expression of p16INK4a and Ki‐67. Coexpression of Ki‐67 and p16INK4a in the same cell is entirely restricted to cervical lesions displaying diffuse p16INK4a expression, whereas in lesions with focal p16INK4a expression, p16INK4a‐expressing cells are negative for Ki‐67. Thus, diffuse expression of p16INK4a reflects lesions with proliferation‐competent cells, while p16INK4a‐expressing cells associated with focal expression patterns are cell cycle arrested.

Chromosomal gains and losses in human papillomavirus-associated neoplasia of the lower genital tract – A systematic review and meta-analysis

BACKGROUND Overexpression of the human papillomavirus (HPV) oncogenes E6 and E7 is necessary for the development of distinct lower genital tract cancers. However, secondary cellular genomic alterations are mandatory to promote progression of HPV-induced premalignant stages. We aimed at identifying the chromosomal regions most frequently gained and lost and the disease stage at which the latter occurs. These regions might be relevant for carcinogenesis and could serve as diagnostic markers to identify premalignant lesions with high progression risk towards invasive cancer. METHODS We performed a systematic literature review and meta-analysis of studies listed in PubMed that analysed chromosomal copy number alterations by comparative genomic hybridisation (CGH) in HPV-positive and -negative cancers or premalignant lesions of the anogenital tract (cervix, anus, vagina, penis and vulva). FINDINGS Data were extracted and analysed from 32 studies. The most common alterations in cervical squamous cell carcinoma (SCC) (12 studies, 293 samples) were gains at 3q with a rate of 0.55 (95% confidence interval (CI) 0.43-0.70), losses at 3p (0.36, 95%CI 0.27-0.48) and losses at 11q (0.33, 95%CI 0.26-0.43). Gains at 3q were particularly frequent in HPV16-positive cervical SCC (0.84, 95%CI 0.78-0.90). Also more than one quarter of high grade cervical intraepithelial neoplasia (CIN) harboured gains of 3q (0.27, 95%CI 0.20-0.36), but the rate in low grade CIN was low (0.02, 95%CI 0.00-0.09). For HPV-associated vulvar SCC (four studies, 30 samples) the same common alterations as in cervical SCC were reported. Studies on non-cervical and non-vulvar SCC and premalignant lesions of the lower genital tract are scarce. INTERPRETATION 3q gains were most frequently found in HPV16-positive cervical SCC. The results suggest the selection of HPV-transformed cell clones harbouring 3q gains in high grade premalignant lesions, while alterations in low grade lesions are rare.

Characterization of humoral immune responses against p16, p53, HPV16 E6 and HPV16 E7 in patients with HPV-associated cancers

The cellular tumor suppressor p16 is strongly overexpressed in cervical cancers and precancers. We have previously demonstrated that infiltrating T lymphocytes reactive against p16 can be found in cervical cancer patients. Here, we analyzed whether p16 induces humoral immune responses. Sera of patients with cervical cancer, oropharyngeal cancer, colorectal cancer and autoimmune disease were included. A total of 919 sera were analyzed, including 486 matched sera from a cervical cancer case control study. p16 antibodies were analyzed in Western blot and a newly developed peptide ELISA covering the complete p16 protein. In addition, a Luminex‐based multiplex assay was used for simultaneous detection of antibodies directed against p16, p53, HPV16 E6 and HPV16 E7. In all entities, only low p16 antibody reactivity was observed. Epitope mapping revealed 2 predominant epitope regions of the p16 protein. No significant difference in p16 antibody frequency (OR = 0.9; 95% CI = 0.6–1.3) and p53 antibody frequency (OR = 0.6; 95% CI = 0.3–1.2) was found between patients and healthy controls in the cervical cancer case control study. Antibodies against the HPV16 oncoproteins E6 and E7 were detected more frequently in cervical cancer patients when compared with healthy controls (E6 OR = 27.8; 95% CI = 11.1–69.7, E7 OR = 5.7; 95% CI = 2.9–11.1). In conclusion, despite the strong expression of p16 and the observed induction of cellular immune responses, antibody reactivity against p16 was observed only at very low levels independent of the disease background.

p16INK4a immunocytochemistry versus human papillomavirus testing for triage of women with minor cytologic abnormalities

The best method for identifying women who have minor cervical lesions that require diagnostic workup remains unclear. The authors of this report performed a meta‐analysis to assess the accuracy of cyclin‐dependent kinase inhibitor 2A (p16INK4a) immunocytochemistry compared with high‐risk human papillomavirus DNA testing with Hybrid Capture 2 (HC2) to detect grade 2 or greater cervical intraepithelial neoplasia (CIN2+) and CIN3+ among women who had cervical cytology indicating atypical squamous cells of undetermined significance (ASC‐US) or low‐grade cervical lesions (LSIL). A literature search was performed in 3 electronic databases to identify studies that were eligible for this meta‐analysis. Seventeen studies were included in the meta‐analysis. The pooled sensitivity of p16INK4a to detect CIN2+ was 83.2% (95% confidence interval [CI], 76.8%‐88.2%) and 83.8% (95% CI, 73.5%‐90.6%) in ASC‐US and LSIL cervical cytology, respectively, and the pooled specificities were 71% (95% CI, 65%‐76.4%) and 65.7% (95% CI, 54.2%‐75.6%), respectively. Eight studies provided both HC2 and p16INK4a triage data. p16INK4a and HC2 had similar sensitivity, and p16INK4a has significantly higher specificity in the triage of women with ASC‐US (relative sensitivity, 0.95 [95% CI, 0.89‐1.01]; relative specificity, 1.82 [95% CI, 1.57‐2.12]). In the triage of LSIL, p16INK4a had significantly lower sensitivity but higher specificity compared with HC2 (relative sensitivity, 0.87 [95% CI, 0.81‐0.94]; relative specificity, 2.74 [95% CI, 1.99‐3.76]). The published literature indicated the improved accuracy of p16INK4a compared with HC2 testing in the triage of women with ASC‐US. In LSIL triage, p16INK4a was more specific but less sensitive. Cancer (Cancer Cytopathol) 2012.