Protocol. This trial protocol has been provided by the authors to give readers additional information about their work.

Transcription

1 Protocol This trial protocol has been provided by the authors to give readers additional information about their work. Protocol for: Palefsky JM, Giuliano AR, Goldstone S, et al. HPV vaccine against anal HPV infection and anal intraepithelial neoplasia. N Engl J Med 2011;365:

2 PROTOCOL 020 VACCINE/PLACEBO ADMINISTRATION 1) Preparation for Administration Both vaccines and placebo should be used as supplied. No dilution is required before administration. Prior to withdrawing and use, mix the contents of the vial/pre-filled syringe thoroughly by rolling the vial/pre-filled syringe between the palms of both hands. The 0.5-mL dose for the GARDASIL vaccine/placebo should be withdrawn from the vial containing 0.75 ml of injectable material. The pre-filled syringe will contain 0.62 ml. The GARDASIL vaccine and the placebo are whitish, semi-translucent suspensions when thoroughly mixed. If the appearance is otherwise, do not administer, and call the SPONSOR (see SPONSOR Contact Information page) and the IVRS. The preferred site for the intramuscular injections is in the deltoid muscle. A needle long enough to ensure intramuscular deposition of vaccine should be used for the injections. 2) Guidelines for Vaccinations The 0.5-mL injection of vaccine or placebo will be administered intramuscularly at Day 1 and at Months 2 and 6. Injections should be administered at a 90º angle into the deltoid muscle of the nondominant arm using a 1.0-mL syringe with the following needle length and gauge specifications: 1-inch needle, 22 to 23 gauge, for men weighing <200 pounds (90.9 kg), 1½-inch needle, 22 to 23 gauge for men weighing 200 pounds (90.9 kg). If the injection is given in the thigh, a 1½-inch needle, 22 to 23 gauge, should be used. The vaccination may be given in the thigh if this is the subject s preference. Subjects who do not complete the series of 3 vaccinations will be allowed to continue participation in the study. ERROR! REFERENCE SOURCE NOT FOUND. DEFINITION 1. Inclusion Criteria

3 Candidate subjects must meet ALL of the following: a. For HM: healthy, males between the ages 16 years and 0 days and 23 years and 364 days. a.1 For MSM: healthy, males between the ages 16 years and 0 days and 26 years and 364 days. b. No clinical evidence of gross genital lesion suggesting sexually-transmitted disease, and no clinically present anogenital warts. c. No temperature 100 F or 37.8 C (oral) within 24 hours prior to vaccinations (vaccinations can be scheduled at a later date when the temperature falls into normal range). d. Must agree to refrain from sexual activity (including vaginal and anal penetration and any genital contact) for 2 calendar days prior to any scheduled visit that includes sample collection, to avoid detection of viral DNA which has been deposited in the male genital area during sexual intercourse and is not the result of ongoing infection. e. HM who have experienced sexual debut but have had no more than 5 lifetime sexual partners. For protocol purposes, a female sexual partner is defined as a woman with whom the subject has engaged in vaginal intercourse. For protocol purposes, a male sexual partner is defined as a man with whom the subject engaged in insertive or receptive anal intercourse. e.1 MSM subjects may have fewer than one lifetime sexual partner but no greater than 5 lifetime sexual partners. For MSM subjects with fewer than one lifetime sexual partner, they must identify themselves as a man who has sex with men and must have engaged in oral sex with another man within the past year. For protocol purposes, a female sexual partner is defined as a woman with whom the subject has engaged in vaginal intercourse. For protocol purposes, a male sexual partner is defined as a man with whom the subject engaged in insertive or receptive anal intercourse. f. Must agree to provide study personnel with a primary telephone number as well as an alternate telephone number for follow-up purposes.

4 In FINLAND only, an alternate telephone number is not necessary as the national population register provides a second safety net for contact information. g.1. Additional inclusion criteria for heterosexual male subjects Subjects must be a heterosexual male, who has had exclusively female sexual partners. g.2. Additional inclusion criteria for MSM subjects Subjects must identify themselves as a man who has sex with men and must have engaged in either insertive or receptive anal intercourse or oral sex with another male sexual partner within the past year. 2. Exclusion Criteria Candidate subjects who manifest ANY of the following exclusion criteria at the time of randomization will NOT be eligible for the study: a. Individuals concurrently enrolled in clinical studies of investigational agents or studies involving collection of genital specimens. b. History of known prior vaccination with an HPV vaccine. c. Receipt of inactivated vaccines within 14 days prior to enrollment or receipt of live virus vaccines within 21 days prior to enrollment. d. Individuals who have history of anogenital warts, or who have clinically present anogenital warts at Day 1. e. History of severe allergic reaction (e.g., swelling of the mouth and throat, difficulty breathing, hypotension or shock) that required medical intervention. f. Individuals allergic to any vaccine component, including aluminum, yeast, or BENZONASE (nuclease, Nycomed [used to remove residual nucleic acids from this and other vaccines]). g. Individuals who have received any immune globulin or blood derived products within the 6 months prior to the first injection, or plan to receive any through Month 7 of the study. h. Individuals with history of splenectomy, known immune disorders (e.g., systemic lupus erythematosus, rheumatoid arthritis), or receiving immunosuppressives

5 (e.g., substances or treatments known to diminish the immune response such as radiation therapy, administration of antimetabolites, antilymphocytic sera, systemic corticosteroids). Individuals who have received periodic treatments with immunosuppressives, defined as at least 3 courses of oral corticosteroids each lasting at least 1 week in duration for the year prior to enrollment, will be excluded. Subjects using topical steroids (i.e., inhaled or nasal) will be eligible for vaccination. i. Individuals who are immunocompromised or have been diagnosed as having Human Immunodeficiency Virus (HIV) infection. j. Individuals with known thrombocytopenia or any coagulation disorder that would contraindicate intramuscular injections. k. History of recent (within the last 12 months) or ongoing alcohol or drug abuse. Alcohol and drug abusers are defined as those who drink or use drugs despite recurrent social, interpersonal, and legal problems as results of alcohol or drug use. l. Any condition which in the opinion of the investigator might interfere with the evaluation of the study objectives. m. Any plan to permanently relocate from the area prior to the completion of the study or to leave for an extended period of time when study visits would need to be scheduled. n. HM with fewer than one or greater than 5 lifetime sexual partners. n.1 MSM subjects with greater than 5 lifetime sexual partners. o. Inability to give informed consent/assent. In Finland ONLY: local regulations concerning clinical conduct and subject s own right to decide of his/her participation in a clinical study, give the right to decide oneself at the age of 15. However, before the subject s 18 th birthday it is obligatory to inform the guardian of his/her child s decision in writing. This needs to be done before Visit 1 and is typically done by a study coordinator after giving an appointment to the subject. In these cases, no assent form will be signed. The Consent form will be signed by the study participant. In all other countries, local regulations will govern the age of consent.

6 MEASUREMENTS 1. Measurements Primary Endpoint The primary endpoint includes HPV 6-, 11-, 16-, 18-related external genital warts, penile/perianal/perineal intraepithelial neoplasia (PIN), penile, perianal or perineal cancer. These endpoints will occur if there is: Pathology panel consensus diagnosis of condylomata acuminata (genital warts), PIN 1, PIN 2/3, penile, perianal, or perineal cancer; AND Detection of HPV 6, 11, 16, 18 DNA by Thinsection PCR in an adjacent section of the same tissue block. It is expected that, of the 32 primary endpoint cases required for the primary analysis, ~3 will occur in the intensive evaluation of MSM substudy, and ~29 will occur in the remaining study sites. MSM Substudy Endpoint The endpoint of HPV 6-, 11-, 16-, 18-related AIN or Anal cancer is only used in the MSM substudy. This endpoint will occur if both conditions are met: Pathology panel consensus diagnosis of AIN 1, AIN 2, AIN 3 or Cancer; AND Detection of HPV 6, 11, 16, 18 DNA by Thinsection PCR in an adjacent section of the same tissue block. To avoid ascertainment bias, any cases of AIN/anal cancer detected via HRA performed as a result of external genital lesions in the perianal region will not be counted in the per-protocol analysis of the MSM substudy. Secondary Endpoints 1) Persistent HPV Infection This secondary endpoint is the combined incidence of persistent HPV 6, 11, 16, or 18 infection. This endpoint will occur if at least one of the following conditions occur:

7 Detection of HPV 6, 11, 16 or 18 DNA (at least one common gene) by PCR in anogenital specimens collected on at least 2 consecutive visits spaced at least 4 months apart; OR Pathology panel consensus diagnosis of condylomata acuminata (genital warts), penile/perianal/perineal intraepithelial neoplasia (PIN), penile, perianal or perineal cancer, AND detection of HPV 6, 11, 16 or 18 DNA by Thinsection PCR in adjacent sections from the same tissue block; AND positivity for the same HPV type to at least one common gene in the sample obtained at a separate adjacent visit, prior to or following the biopsy showing HPV disease. 2) HPV Detection This secondary endpoint is the combined incidence of HPV 6, 11, 16 or 18 detection at one or more visits. This endpoint will occur if the following condition occurs: Detection of HPV 6, 11, 16 or 18 DNA by PCR in anogenital specimens collected on at least one visit. Other Efficacy Measurements In addition to the primary endpoint definition, the incidence of all external anogenital lesions will be measured, however intra-anal warts detected in the MSM substudy will not count as cases toward the primary endpoint. For biopsies in which HPV 6, 11, 16, and 18 are not detected by Thinsection PCR, type-specific HPV PCR assays will be used to evaluate non-vaccine HPV types in the biopsy lesion. 2. Immunogenicity Competitive Luminex Immunoassay (clia) Assay for Serum Antibody Response to HPV The purpose of the quadrivalent human papillomavirus (HPV) competitive Luminex immunoassay (clia) is to detect antibodies to HPV virus-like particles (VLPs), types 6, 11, 16, 18 before and after vaccination with the HPV quadrivalent vaccine. This is the primary assay used by the Vaccines and Biologics Research Serology Laboratory of Merck Research Laboratories (MRL)

8 to evaluate the serological response to the vaccine, to measure HPV infection induced antibody, and to exclude subjects with evidence of a current or past HPV infection from the primary analysis. Yeast-derived VLPs are coupled to a set of 4 distinct fluorescent Luminex microspheres. Antibody titers are determined in a competitive format in which known, type-specific phycoerythrin (PE)-labeled, neutralizing monoclonal antibodies (mabs) compete with the subject s serum antibodies for binding to conformationally sensitive, neutralizing epitopes on the VLPs. The fluorescent signals from the bound HPV-specific detection mabs are inversely proportional to the subject s neutralizing antibody titers. Results for the assay are reported as concentration of antibody in milli- Merck Units per milliliter (mmu/ml). Any sample with a value less than the serostatus cutoff is considered serostatus negative. Samples with values equal to or greater than the serostatus cutoff are considered serostatus positive. The cutoffs for the HPV 6, 11, 16, and 18 clias are 20 mmu/ml, 16 mmu/ml, 20 mmu/ml, and 24 mmu/ml, respectively [28]. This test is an experimental assay, and it is performed in a non-clia-certified laboratory. The test results are merely used for the purpose of conducting clinical trials, not for making medical decisions. Reporting serology test results to the study participants will be conducted in accordance with the local IRB regulations and the interpretations of CLIA exemptions. 3. PCR Assays to Detect HPV in Clinical Specimens a. Multiplex PCR Assays The following procedures will be done at Merck Research Laboratories (MRL), West Point, PA or a central laboratory designated by SPONSOR. For the detection of HPV types 6-, 11-, 16-, 18-swab samples and Thinsection microtomy specimens are received and then prepared for multiplex PCR using a DNA purification method (Qiagen Technology Kit). Multiplex PCR (based on real-time fluorescent PCR) allows the simultaneous detection of 3 gene products (L1, E6, and E7) for a given HPV type in one reaction. The HPV type-specific primer pairs based on the published HPV L1, E6, and E7 sequences, are used to specifically amplify a portion of each gene simultaneously. The specific amplicons are detected in real-time by fluorescently-labeled oligonucleotide probes. The gene-specific oligonucleotide probes are each labeled with a different fluorescent label and the fluorescent emission is captured during PCR cycling. After analysis of the raw fluorescent data by the Sequence Detection Software, a

9 threshold cycle (Ct), which represents the PCR cycle at which an increase in reporter fluorescence above a baseline signal can first be detected, is determined. Each gene-specific assay (i.e., gene-specific dye layer) for HPV types 6, 16, and 18 is considered positive if the Ct is <45 cycles. The gene-specific assay for 6, 16, and 18 is considered negative if the Ct = 45 or "No Ct". For HPV 11, the gene specific assay is considered positive if the Ct is <50 cycles. The HPV 11 gene-specific assay is considered negative if the Ct=50 or "No Ct". A sample is called positive when 2 or 3 genes are positive or when the same single gene scores positive on consecutive tests. b. Quantitative PCR Assays To further assess HPV viral load in the infected men, the optional quantitative PCR assay may be performed at Merck Research Laboratories, West Point, PA or a central laboratory designated by the SPONSOR. Swabs are received and prepared for PCR using a DNA purification method (Qiagen Technology Kit). Samples previously determined to be positive by multiplex PCR (see above) may be further analyzed using HPV type-specific primers based on the HPV E7 genes only (see above) to determine the HPV genome copy number. Copy number determination is by standard curve using qualified plasmid standards for the HPV E7 genes. c. Type-Specific PCR to Detect High-Risk HPV Types Other Than Vaccine Types PCR assays to ascertain the presence of HPV types not included in the vaccine will be performed on biopsy Thinsections at a central laboratory designated by the SPONSOR or at MRL, West Point, PA. Thinsections are prepared for PCR using a DNA purification method (Qiagen Technology Kit). HPV 6, 11, 16, 18 multiplex PCR (see above) will then be performed on a fraction of the DNA prepared from each Thinsection to determine the HPV type in the Thinsection. Thinsections positive by HPV multiplex PCR for HPV 16, 18 may be further analyzed by HPV type-specific primers based on the HPV E7 genes only to quantify viral load (see above). All Thinsections will be further analyzed by typespecific multiplex (for HPV types 31, 33 45, 52, and 58) or duplex (for HPV types 35, 39, 51, 56, and 59) PCR assays for multiple HPV types. For description of multiplex assays see Section I.F.3.a.). Duplex assays for the additional types are essentially the same assays as multiplex PCR assays (see above Section I.F.3.a), but will only detect the HPV E6 and E7 genes.