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IFN- Produced by Human Papilloma Virus-18 E6-Specific CD4⁺ T Cells Predicts the Clinical Outcome after Surgery in Patients with High-Grade Cervical Lesions¹

Samantha Seresini^*,¶,||, Massimo Origoni^2,, Flavia Lillo^2,3,, Luigi Caputo, Anna Maria Paganoni^#, Simone Vantini^#, Renato Longhi^**, Gianluca Taccagni, Augusto Ferrari, Claudio Doglioni, Piercesare Secchi^# and Maria Pia Protti^4,*,¶,||

^* Tumor Immunology Unit, Clinica Ostetrica e Ginecologica Università Vita-Salute San Raffaele, Laboratory of Virology, Unità Operativa Anatomia Patologica, ^¶ Cancer Immunotherapy and Gene Therapy Program, and ^|| Department of Oncology, Scientific Institute H. San Raffaele, Milan, Italy; ^# MOX–Dipartimento di Matematica, Politecnico di Milano, Milan, Italy; and ^** CNR-Istituto di Chimica del Riconoscimento Molecolare, Milan, Italy

	Abstract

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Cervical neoplastic lesions are associated with infection by high-risk human papilloma viruses (HPVs). HPV-16 and HPV-18 are the most common genotypes. It has been proposed that development of HPV-16-positive cervical lesions is associated with impaired CD4⁺ T cell immunity against early Ags. The aim of the study was to evaluate whether this impairment also applies to HPV-18. We investigated the presence and the quality of anti-HPV-18 E6 CD4⁺ T cell responses in the blood of 37 consecutive patients with high-grade cervical lesions, 25 normal donors, and 20 cord bloods. The immune infiltrate in the cervical lesions was also evaluated. The characteristics of the responses were correlated to the clinical outcome. We found that one or more HPV-18 E6 peptides, containing naturally processed epitopes, were able to induce a response in 40–50% of the patients, depending on the effector function tested. Importantly, these percentages rose to 80–100% when HPV-18-positive patients were considered. HPV-18 E6-specific CD4⁺ T cells produced mixed Th1/Th2 responses and statistical analysis of the cytokines produced revealed that the amount of IFN- released could predict infection persistence and/or disease relapse after surgery. Finally, we found that a higher number of infiltrating CD4⁺ and T-bet⁺ T cells in the lesions correlated with a favorable clinical outcome. Our results strongly suggest a relevant role for CD4⁺ T cells in the control of the HPV-18 compared with HPV-16 infections in patients with high-grade cervical lesions and identify an immunologic parameter potentially useful for patients’ stratification.

	Introduction

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Cervical cancer is the second most common cancer in women worldwide, and is the consequence of persistent infection with human papillomaviruses (HPVs)⁵ (1, 2). Development of HPV-related neoplastic lesions, from cervical intraepithelial neoplasia (CIN) to cervical carcinoma, is due to infection by high-risk HPV genotypes in which integration of the viral episome into host-cell DNA occurs. The oncogenic effects are mediated by the binding of the viral early proteins E6 and E7 to the products of the tumor suppressor genes p53 and retinoblastoma, respectively (1, 2).

The immune system plays an important role in the control of HPV infections. Immunosuppressed women have increased incidence of HPV infections, CIN lesions, and prolonged persistence of intraepithelial lesions (3). Abs against HPV proteins can be found in the sera of patients (4) and regressing lesions are infiltrated by immune cells (5). Although development of HPV-derived tumors has been related to immune escape mechanisms (6, 7), little is known on the role of T cell immunity against HPV in the different stages of disease progression. Even less is known about the ability to induce immune responses among the different high-risk HPVs genotypes.

Studies have primarily focused on HPV-16 because it is the most common genotype found in neoplastic lesions. HPV-18 is the second most common incident HPV infection, and the most strongly associated with adenocarcinoma of the cervix (8, 9).

Previous studies have shown in healthy subjects high frequencies of circulating memory CD4⁺ T cells reacting with HPV-16 E2 and E6, but not E7, sequences (10, 11). More conflicting results have been reported when CIN or cervical carcinoma patients were studied (12, 13, 14). In one study (12), CD4⁺ T cells from women with CIN did not react with HPV-16 peptides, while CD4⁺ T cells from cervical cancer patients proliferated without cytokine production. In another study (13), CD4⁺ T cells from patients with low-grade CIN displayed Th1-type responses, while patients with cervical cancer displayed Th2 type.

Much less is known on HPV-18. By in vitro studies, we previously identified promiscuous HPV-18 E6 CD4 epitopes recognized by 18% of healthy donors (15). Similar frequencies were found in another study (16), in which no reactivity in cervical cancer patients was found.

In the present study, we evaluated the anti-HPV-18 E6 CD4⁺ immunity in the blood of women suffering from high-grade cervical lesions and found a high percentage of responders. Importantly, the level of IFN- produced by HPV-18 E6-specific CD4⁺ T cells predicted infection persistence and/or disease relapse after surgery. Immunohistochemical analysis also verified that the number of CD4⁺ and T-bet⁺ cells was significantly higher in patients with favorable clinical outcome.

	Materials and Methods

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Subjects

PBMC were obtained from 20 cord bloods, 25 healthy donors, and 37 patients with cervical lesions. The institutional ethics committee approved the study protocol and informed consent was obtained from all donors before blood sampling. Mean age distribution was as follows: healthy donors (36 ± 8), CIN2–3 patients (37 ± 8), stage I cervical cancer patients (42 ± 13) (Table I).

Cervical brushes were collected in a DIGENE cervical sampler. DNA extraction was performed using the QIAmp DNA minikit (Qiagen). Paraffin-embedded samples (5–10 slices) were rinsed in xylene washed in ethanol, digested in proteinase K, and extracted as above. The HPV-DNA screening was performed by the Hybrid Capture 2 (HC2) System (Digene), following the manufacturer’s instruction as described in Ref. 17 . HPV genotyping was obtained by a reverse hybridization assay (INNOLIPA HPV; Innogenetics) as described in Ref. 17 .

Selection and synthesis of HPV 18 E6 peptides

Five sequences (E6_27–41, E6_52–66, E6_97–111, E6_105–119, and E6*_32–47) of the HPV-18 E6 and E6* (truncated form derived by alternatively spliced mRNA (18)) proteins were selected by the TEPITOPE algorithm (19) as promiscuous HLA-DR binders, as previously described (15). Peptides corresponding to selected sequences were synthesized by the stepwise solid-phase method as in Ref. 20 . The peptides were lyophilized, reconstituted in DMSO at 10 mg/ml, and diluted in RPMI 1640 (BioWhittaker) as needed.

Short-term culture and cytokine release assay

CD4⁺ T cells were purified from total PBMC (Miltenyi Biotec). CD4⁺ T cells (3 x 10⁴/well) were cultured, in RPMI 1640 (BioWhittaker) supplemented with L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (50 mg/ml; BioWhittaker), and 8% heat-inactivated autologous serum (tissue-culture medium (TCM)), in the presence of irradiated CD4⁺-depleted PBMC as APC, at a 1:3 ratio in 96-well plates in six replicates for each condition. Stimuli were: PHA (10 µg/ml), as positive control; CD4⁺ T cells in the presence of the APC only, as baseline (blank); and each single peptide (10 µg/ml). At day 7, half medium from each well was removed and replenished with fresh TCM containing IL-2 (25 U/ml), without any further Ag stimulation. At day 13, 100 µl of supernatant was removed from each well for cytokine detection and the cultures were pulsed for 16 h with [³H]TdR (1 µCi/well, 6.7 Ci/M; Amersham). The cells were collected with a FilterMat Universal Harvester (Packard Instrument) in specific plates (Unifilter GF/C; Packard Instrument) and the thymidine incorporated was measured in a liquid scintillation counter TopCount NXT; Packard Instrument). Supernatant collected from each well of the six replicates of each condition were pooled and 50 µl was used for detection of cytokines released using the cytometric bead array kit (BD Biosciences), according to the manufacturer’s instructions.

Immunohistochemical analysis

Surgical specimens were fixed in buffered formalin and embedded in paraffin. Immunohistochemistry was performed on 5-µm tissue sections. All cases were immunostained with a sensitive non-biotin detection system (NovoLink polymer; Novocastra), with diaminobenzidine development. Heat-induced Ag retrieval was performed using citrate buffer 0.01 M (pH 6.0) or Tris-EDTA (pH 9.0) in a water bath for 30 min. The following Abs were used: anti-CD4 mAb (clone 4B12; Novocastra), anti-CD8 mAb (clone 1A5; Novocastra), anti-T-bet mAb (clone 4B10; Santa Cruz Biotechnology), and anti-GATA3 polyclonal Ab (AF2605; R&D Systems). All stainings were performed with an automatic immunostainer (Biogenex Optimax). Immunostaining was scored semiquantitatively as follows: 0, absence of positive cells; 1, scattered positive lymphoid cells in the stroma and 10 positive lymphoid cells per 100 dysplastic cells in the epithelium; 2, confluent lymphoid infiltrates in the stroma and 20 lymphoid cells x 100 dysplastic cells in the epithelium; 3, dense lymphoid infiltrate in the stroma and >20 lymphoid cells x 100 dysplastic cells in the epithelium.

Statistical analysis

Cytokine release data were categorized by labeling each observed expression, say d, as V_L (below the minimum standard value) if d < 20 pg/ml, L (low) if 20 pg/ml d < 200 pg/ml, H (high) if 200 pg/ml d < 1000 pg/ml, and V_H (very high) if d 1000 pg/ml. A random forest analysis (21) applied to CART predictors (22) was performed to assess the discriminatory power of the variables (four cytokines for each of the five peptides). The analysis showed that variables related to IFN- and IL-5 were among those that most discriminate between the two groups. To test the effects due to production of IFN- and IL-5, the measurements of these variables along the five peptides were aggregated. Thus, each patient was represented by a vector of two variables: the arithmetic means of the production of IFN- (IFN-_m) and of IL-5 (IL-5_m) along the five peptides. A bivariate Shapiro-Wilk normality test (23) on the samples (IFN-_m, IL-5_m) in the two groups questioned the assumption of normality for the distributions generating sample data. Therefore, data were transformed into (IFN-_m^1/2, IL-5_m^1/2) according to the following:

A bivariate Shapiro-Wilk normality test (p value = 0.7467 for group A and p value = 0.2522 for group B) and a Bartlett test (23) for homogeneity of covariances (p value = 0.1737) performed on the transformed data showed that the assumptions for a MANOVA (24) were satisfied. Hence, a Hotelling’s T2 test (24) was implemented to verify the null hypothesis that the mean vector of the distribution (IFN-_m^1/2 and IL-5_m^1/2) for group A is equal to the mean vector of the distribution (IFN-_m^1/2 and IL-5_m^1/2) for group B, against the alternative hypothesis that the two mean vectors are different. T2 and Bonferroni’s simultaneous confidence intervals (25) for the differences of the marginal means of IFN-_m^1/2 and IL-5_m^1/2 were also computed (Table II) along with one-at-a-time t tests that ignore the covariance structure (IFN-_m^1/2 and IL-5_m^1/2) (Table III). Finally, a logistic regression (26) was fitted on the transformed data to quantify the discriminatory power of IFN-_m^1/2 for classifying patients as belonging to group A or to group B.

	Results

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Twenty-five healthy donors, 31 CIN2–3 patients, and 6 stage 1 cervical cancer patients were studied. Patients’ characteristics are reported in Table I. HPV detection and typing were performed on cervical brushes and/or surgical specimens. We confirmed HPV-16 as the most prevalent type, followed by HPV-18, -31, -45, -53, -51, -58, and -66. Comparison between percentages of HPV types found in cervical brushes and surgical specimens revealed that HPV-16, -31, -66, and -51 were comparable in the two tissues, while HPV-18, -45, -53, and -58 were five to seven times more represented in surgical samples than in cervical brushes. Surgical specimens contained a statistically significant higher number of HPV types than cervical brushes.

Normal donors and patients, irrespective of the HPV type carried, were tested for the presence of CD4⁺ T cells against the previously described HPV-18 E6 sequences (15). Naive CD4⁺ T cells purified from 20 cord bloods were also tested to verify whether the culture system could induce in vitro priming.

We considered as responders subjects with specific reactivity of CD4⁺ T cells in proliferation and/or cytokine release assays in the presence of one or more HPV-18 E6 peptides (Table IV). When considering the percentages of responders in both testes, we found no responders in cord bloods, 12% in normal donors, and almost 40% in HPV patients. Importantly, this percentage reached almost 80% when only HPV-18-positive patients were considered. Cytokine release was more sensitive compared with proliferation in detecting responders, reaching a 100% of responders among HPV-18-positive patients.

The level IFN- by CD4⁺ T cells against HPV-18 E6 epitopes predicts disease relapse and/or infection persistence after surgery

Thirteen of the 19 responsive patients reported in Table V could be followed for clinical outcome after surgery. Follow-up was performed at 3, 6, 9, and 12 mo and subsequently every 6–8 mo, and consisted of clinical inspection, cytology, colposcopy, and whenever indicated HPV detection, depending on suspected relapse. Minimum follow-up for identification of infection persistence or disease relapse was 6 mo. As reported in Table VI, the 13 patients were divided into two categories (groups A and B) based on their clinical outcome. Favorable clinical outcome (group A) was defined based on clinical inspection and when both cytology and colposcopy were negative; unfavorable clinical outcome (group B) when either one of cytology, colposcopy or HPV detection tests were positive. Eight of the 13 patients (62%) belonged to group A, 5 (38%) belonged to group B. Within patients of group B, 3 patients (23%) had disease progression while 2 (15%) had infection persistence.

View larger version (18K): [in this window] [in a new window]   FIGURE 1. MANOVA and logistic regression analyses. Lower panel, Patients belonging to group A (blue dots) and to group B (red dots) are represented in terms of IFN-m1/2 and of IL-5m1/2 values. Mean values for the two groups are shown with a blue and a red cross, respectively. Green ellipse represents 95% confidence region for the difference of the group mean values. Dashed green and orange lines represent T2 and Bonferroni’s 95% simultaneous confidence intervals for the differences of the marginal means. The green in-box cross represents the difference of the mean values for the two groups. Upper panel, The probability of membership to group A (blue line) and to group B (red line), estimated by means of a logistic regression, is plotted as functions of IFN-m1/2.

Systemic anti-HPV-18 CD4⁺ T cell response associates with immune infiltrates in cervical lesions

Immunohistochemical analysis was performed to characterize the immune infiltrate in the cervical lesions. We tested the presence of CD4⁺ and CD8⁺ T cells, and, because responsive CD4⁺ T cells in the blood produced both Th1 and Th2 cytokines, we evaluated the expression of transcription factors T-bet and GATA-3 by lymphoid cells expressed by Th1 and Th2 cells, respectively (27). Cervical lesions from 12 responsive patients could be evaluated (Table VIII). CD4⁺ and CD8⁺ T cells as well as T-bet⁺ cells were present within both the stroma and the epithelium. GATA-3⁺ cells were significantly more abundant within the stroma compared with the epithelium.

View larger version (58K): [in this window] [in a new window]   FIGURE 2. Anti-HPV CD4+ T cell responses in the blood correlate with immune infiltrates in the lesions. Proliferation (upper panel) and IFN- release (lower panel) by CD4+ T cells from patients 43 (A) and 25 (C). Analysis of the immune infiltrate (B–D); arrows indicate examples of positive lymphoid cells. Responses significantly higher than the blanks are indicated as: *, p < 0.05; **, 0.001< p < 0.05 (determined by unpaired, one-tailed Student’s t test).

	Discussion

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In this study, we report that a high percentage of patients with high-grade cervical lesions have circulating CD4⁺ T cells specific for the HPV-18 E6 protein. The percentage of response is as high as almost 80%, considering both effector functions tested, for patients in whom we detected the HPV-18. However, when considering all HPV patients tested, we found almost 40% of responders. This percentage highly exceeded the 12% found within the healthy population, suggesting that all responsive patients, irrespective of the HPV found in the lesions, have encountered the HPV-18. We believe that cross-recognition by CD4⁺ T cells of sequences from other HPV genotypes is not supported by similarity searches, which showed a low degree of sequence homology among HPV E6 proteins, except for sequences HPV-18 E6_97–111 and HPV-45 E6_98–111, for which cross-recognition might have occurred.

To explain why only about half of the patients with anti-HPV-18 immune responses had HPV-18-positive lesions (Tables I and VI), we hypothesized that either HPV-18 is cleared in a large majority of infected women or that HPV-18 is present in the lesions but not easily detectable. We believe that both hypotheses can apply to our finding.

A strong HPV-18 immunogenicity is supported by the report that in HIV-infected women the percentage of HPV-18-positive high-grade CIN reach almost 90% (17). In contrast, the percentage of HPV-16-positive lesions in the HIV-infected or immunocompetent women does not change substantially (17), suggesting a major role for immunocompetence in the clearance of HPV-18 compared with HPV-16 viruses. Preliminary results in 10 HPV-16-positive patients of our cohort in which we tested the CD4⁺ T cell response to HPV-16 E6 and E7 promiscuous epitopes support this hypothesis. Indeed, no patients responded both in proliferation and cytokine release assays, 3 of the 10 (30%) showed cytokine release only and one (10%) proliferation only (data not shown). These numbers are much lower than the ones we obtained with HPV-18 (Table IV).

On the other hand, lack of HPV-18 detection in surgical specimens in high-grade lesions might be attributed also to a deeper localization (28) or to viral latency (29). It is interesting to note that in two (nos. 29 and 38) of the patients of group B, we could detect the HPV-18 only during the follow-up after surgery. Detection of new HPV types could be the result of sequential infections; however, alternatively these two patients could have mounted an anti-HPV-18 immune response early on in the presence of simultaneous infections, which comprised HPV-18 and, as a consequence of surgery, HPV-18 had became accessible to the cervical brush or reactivated.

We found that HPV-18 E6-specific CD4⁺ T cells secrete both Th1 and Th2 cytokines. Most importantly, a favorable clinical outcome was associated to the level of IFN- release. The importance of CD4⁺ T cells and IFN--producing cells in determining the clinical outcome is also suggested by the features of the lesions’ infiltrate where the numbers of CD4⁺ and T-bet⁺ cells in the epithelium were significantly higher for patients of group A (Table VIII).

Five to 25% of patients with high-grade cervical lesions experience relapse after surgery (30). In our cohort, 38% of patients were classified with an unfavorable clinical outcome (Table VI). This percentage is slightly higher than what was reported in the literature because we considered, as unfavorable outcome, patients with disease relapse (23%) as well as infection persistence (15%). Much effort is currently put to identify suitable markers of residual/recurrent disease because of the risk of progression to invasive carcinoma (30, 31, 32). An important finding of our study is the identification of an immunologic parameter (i.e., the level of IFN- produced by HPV-18-specific CD4⁺ T cells) that could be applied to discriminate, at the time of surgery, between patients with favorable or unfavorable clinical outcome.

Future studies are needed to define in a larger cohort of patients the exact discriminatory power of the logistic regression and its actual error rate as well as the rates of false positive or false negative. The application of the validated immunologic parameter identified will be useful for patients’ stratification and follow up. Studies are also needed to evaluate the immunogenicity of other genotypes frequently found in the lesions and the role of Th2 CD4⁺ T cells infiltrating stromal cells.

	Acknowledgments

 
We are grateful to all donors for giving their blood samples for these experiments. We thank Matteo Bellone, Catia Traversari, Angelo Manfredi, Paolo Dellabona, and Vincenzo Russo for critical reading of the manuscript.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by the Cancer Research Institute (Clinical Investigation grant), the Italian Association for Cancer Research, the European Community (DC-THERA), the Compagnia di San Paolo, the Fondazione CARIPLO, and the Italian Ministry of Health.

² M.O. and F.L. equally contributed to the work.

³ Current address: Fondazione Istituto San Raffaele G. Giglio di Cefalù, Palermo, Italy.

⁴ Address correspondence and reprint requests to Dr. Maria Pia Protti, Cancer Immunotherapy and Gene Therapy Program, DIBIT, Scientific Institute H. San Raffaele, Via Olgettina 58, 20132 Milan, Italy. E-mail address: m.protti{at}hsr.it

⁵ Abbreviations used in this paper: HPV, human papilloma virus; CIN, cervical intraepithelial neoplasia; HC2, hybrid capture 2; TCM, tissue-culture medium.

Received for publication May 31, 2007. Accepted for publication September 6, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

1. Zur Hausen, H.. 2002. Papillomaviruses and cancer: from basic studies to clinical application. Nat. Rev. Cancer 2: 342-350. [Medline]
2. Bosch, F. X., A. Lorincz, N. Munoz, C. J. Mejer, K. V. Shah. 2002. The casual relation between human papillomavirus and cervical cancer. J. Clin. Pathol. 55: 244-265. [Abstract/Free Full Text]
3. Serraino, D., P. Carrieri, C. Pradier, E. Bidoli, M. Dorrucci, E. Ghetti, A. Schiesari, R. Zucconi, P. Pezzotti, P. Dellamonica, et al 1999. Risk of invasive cervical cancer among women with, or at risk for, HIV infection. Int. J. Cancer 82: 334-337. [Medline]
4. Rosales, R., M. Lopez-Contreras, R. R. Cortes. 2001. Antibodies against human papillomavirus (HPV) type 16 and 18 E2, E6 and E7 proteins in sera: correlation with presence of papillomavirus DNA. J. Med. Virol. 65: 736-744. [Medline]
5. Coleman, N., H. D. Birley, A. M. Renton, N. F. Hanna, B. K. Ryait, M. Byrne, D. Taylor-Robinson, M. A. Stanley. 1994. Immunological events in regressing genital warts. Am. J. Clin. Pathol. 02: 768-774.
6. Tindle, R. W.. 2001. Immune evasion in human papillomavirus-associated cervical cancer. Nat. Rev. Cancer 2: 1-7. [Medline]
7. Kanodia, S., L. M. Fahey, M. W. Kast. 2007. Mechanisms used by human papillomaviruses to escape the host immune response. Curr. Cancer Drug Targets 7: 79-89. [Medline]
8. Woodman, C. B., S. Collins, H. Winter, A. Bailey, J. Ellis, P. Prior, M. Yates, T. P. Rollason, L. S. Young. 2001. Natural history of cervical human papillomavirus infection in young women: a longitudinal cohort study. Lancet 357: 1831-1836. [Medline]
9. Nakagawa, S., H. Yoshikawa, T. Onda, T. Kawana, A. Iwamoto, Y. Taketani. 1996. Type of human papillomavirus is related to clinical features of cervical carcinoma. Cancer 78: 1935-1941. [Medline]
10. de Jong, A., S. H. van der Burg, K. M. Kwappenberg, J. M. van der Hulst, K. L. Franken, A. Geluk, K. E. van Meijgaarden, J. W. Drijfhout, G. Kenter, P. Vermeij, et al 2002. Frequent detection of human papillomavirus 16 E2-specific T-helper immunity in healthy subjects. Cancer Res. 62: 472-479. [Abstract/Free Full Text]
11. Welters, M. J., A. de Jong, S. J. van den Eeden, J. M. van der Hulst, K. M. Kwappenberg, S. Hassane, K. L. Franken, J. W. Drijfhout, G. J. Fleuren, G. Kenter, et al 2003. Frequent display of human papillomavirus type 16 E6-specific memory T-helper cells in the healthy population as witness of previous viral encounter. Cancer Res. 63: 636-641. [Abstract/Free Full Text]
12. de Jong, A., M. I. van Poelgeest, J. M. van der Hulst, J. W. Drijfhout, G. J. Fleuren, C. J. Melief, G. Kenter, R. Offringa, S. H. van der Burg. 2004. Human papillomavirus type 16-positive cervical cancer is associated with impaired CD4⁺ T-cell immunity against early antigens E2 and E6. Cancer Res. 64: 5449-5455. [Abstract/Free Full Text]
13. Warrino, D. E., W. C. Olson, W. T. Knapp, M. I. Scarrow, L. J. D’Ambrosio-Brennan, R. S. Guido, R. P. Edwards, W. M. Kast, W. J. Storkus. 2004. Disease-stage variance in functional CD4⁺ T-cell responses against novel pan-human leukocyte antigen-D region presented human papillomavirus-16 E7 epitopes. Clin. Cancer Res. 10: 3301-3308. [Abstract/Free Full Text]
14. Steele, J. C., C. H. Mann, S. Rookes, T. Rollason, D. Murphy, M. G. Freeth, P. H. Gallimore, S. Roberts. 2005. T-cell responses to human papillomavirus type 16 among women with different grades of cervical neoplasia. Br. J. Cancer 93: 248-259. [Medline]
15. Facchinetti, V., S. Seresini, R. Longhi, C. Garavaglia, G. Casorati, M. P. Protti. 2005. CD4⁺ T cell immunity against the human papillomavirus-18 E6 transforming protein in healthy donors: identification of promiscuous naturally processed epitopes. Eur. J. Immunol. 35: 806-815. [Medline]
16. Welters, M. J., P. van der Logt, S. J. van den Eeden, K. M. Kwappenberg, J. W. Drijfhout, G. J. Fleuren, G. G. Kenter, C. J. Melief, S. H. van der Burg, R. Offringa. 2006. Detection of human papillomavirus type 18 E6 and E7-specific CD4⁺ T-helper 1 immunity in relation to health and disease. Int. J. Cancer 118: 950-956. [Medline]
17. Lillo, F. B., S. Lodini, D. Ferrari, C. Stayton, G. Taccagni, L. Galli, A. Lazzarin, C. Uberti-Foppa. 2005. Determination of human papillomavirus (HPV) load and type in high-grade cervical lesions surgically resected from HIV-infected women during follow up of HPV infection. Clin. Infect. Dis. 40: 451-457. [Medline]
18. Pim, D., P. Massimi, L. Banks. 1997. Alternatively spliced HPV-18 E6* protein inhibits E6 mediated degradation of p53 and suppresses transformed cell growth. Oncogene 15: 257-264. [Medline]
19. Sturniolo, T., E. Bono, J. Ding, L. Raddrizzani, O. Tuereci, U. Sahin, M. Braxenthaler, F. Gallazzi, M. P. Protti, F. Sinigaglia, J. Hammer. 1999. Generation of tissue-specific and promiscuous HLA ligand databases using DNA microarrays and virtual HLA class II matrices. Nat. Biotechnol. 17: 555-561. [Medline]
20. Curnis, F., A. Gasparri, A. Sacchi, R. Longhi, A. Corti. 2004. Coupling tumor necrosis factor- with _V integrin ligands improves its antineoplastic activity. Cancer Res. 64: 565-571. [Abstract/Free Full Text]
21. Breiman, L.. 2001. Random forests. Machine Learning 45: 5-32.
22. Breiman, L., J. Fredman, R. Olshen, C. Stone. 1994. Classification and Regression Trees Wadsworth, Belmont.
23. Royston, P.. 1092. Algorithm AS 181: the W test for normality. Applied Stat. 31: 176-180.
24. Anderson, T. W.. 2003. An Introduction to Multivariate Statistical Analysis Ed. 3. John Wiley & Sons, London.
25. Johnson, R. A., D. W. Wichern. 2002. Applied Multivariate Statistical Analysis Prentice Hall, Upper Saddle River.
26. Hand, D. J.. 1981. Discrimination and Classification John Wiley & Sons, London.
27. Dong, C., R. A. Flavell. 2000. Control of T helper cell differentiation–in search of master genes. Sci. STKE 2000: PE1[Medline]
28. Im, S. S., S. P. Wilczynski, R. A. Burger, B. J. Monk. 2003. Early stage cervical cancers containing human papillomavirus type 18 DNA have more nodal metastasis and deeper stromal invasion. Clin. Cancer Res. 9: 4145-4150. [Abstract/Free Full Text]
29. Woodman, C. B., S. I. Collins, L. S. Young. 2007. The natural history of cervical HPV infection: unresolved issues. Nat. Rev. Cancer 7: 11-22. [Medline]
30. Zielinski, G. D., A. G. Bais, T. J. Helmerhorst, R. H. Verheijen, F. A. de Schipper, P. J. Snijders, F. J. Voorhorst, F. J. van Kemenade, L. Rozendaal, C. J. Meijer. 2004. HPV testing and monitoring of women after treatment of CIN 3: review of the literature and meta-analysis. Obstet. Gynecol. Surv. 59: 543-553. [Medline]
31. Nobbenhuis, M. A., C. J. Meijer, A. J. van den Brule, L. Rozendaal, F. J. Voorhorst, E. K. Risse, R. H. Verheijen, T. J. Helmerhorst. 2001. Addition of high-risk HPV testing improves the current guidelines on follow-up after treatment for cervical intraepithelial neoplasia. Br. J. Cancer 84: 796-801. [Medline]
32. Alonso, I., A. Torne, L. M. Puig-Tintore, R. Esteve, L. Quinto, E. Campo, J. Pahisa, J. Ordi. 2006. Pre- and post-conization high-risk HPV testing predicts residual/recurrent disease in patients treated for CIN 2–3. Gynecol. Oncol. 103: 631-636. [Medline]

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