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CAP Home > CAP Committees and Leadership > Technology Assessment Committee > POET Reports > Human Papillomavirus DNA Testing (HPV)

  Human Papillomavirus DNA Testing (HPV)


Updated December 17, 2010

The Pathologist’s Message

High risk HPV (hrHPV) DNA testing is a mainstay component of cervical cancer screening and a cost-effective management tool for equivocal cervical cytology results. The presence of hrHPV DNA is a necessary agent for the development of cervical cancer. Infection with hrHPV is common and, in the vast majority of cases, is self-limited - clearing within 2 years.1 In a small percentage of cases, the infection becomes persistent and oncogenic portions of the hrHPV genome can interact with host cells in ways that lead to genetic and cell regulatory changes. These events may, in turn, lead to carcinogenesis. Therefore, identification of hrHPV DNA is important in the context of screening and triage.

Currently the use of hrHPV testing is FDA-approved in the following three scenarios: (1) Triage of patients having equivocal (ASC-US) Pap tests; (2) Use in conjunction with the Pap test in primary screening for women over the age of 30 years; (3) Use of HPV 16/18 testing for triage in women in the second scenario above who have positive hrHPV and negative Pap tests. Additional consensus management guidelines also include hrHPV testing for post-colposcopy and post-treatment management of patients, including those with histologic cervical intraepithelial neoplasia (CIN) and endocervical adenocarcinoma in situ (AIS), and for the triage of postmenopausal women with low grade squamous intraepithelial lesion (LSIL) on cytology.

Because of the high volume of tests performed and the significance of the results for cancer risk and high cost interventions, well-validated tests - with known operating characteristics - are vital to maintain the robust nature of the tests’ prediction of risk. The FDA has approved three tests for hrHPV DNA detection. FDA-approval indicates that the performance characteristics of these tests are well validated and, therefore, predictive for the indications of excluding risk of high-grade cervical dysplasia or carcinoma (screening), and the triage of equivocal cervical cytology tests.

  • Qiagen Hybrid Capture 2 assay2: approved for use with Hologic ThinPrep Pap Test media (PreservCyt) and with Qiagen Standard Transport Media. It detects 13 of the most prevalent hrHPV types.
  • Hologic/Third Wave Cervista HR assay3: approved for use with the Hologic ThinPrep Pap Test (PreservCyt). It detects 14 of the most prevalent HPV types and has a cellular control incorporated in the procedure to insure that the specimen is adequate.
  • Hologic/ThirdWave Cervista HPV 16/18 assay4: approved by the FDA and detects only hrHPV types 16 and 18, the two most prevalent HPV types in cervical cancers.

Use of these tests with other types of transport media requires self-validation as per the regulations of CLIA 88. Other methods of testing such as polymerase chain or in situ hybridization assays have been developed for the detection of hrHPV, however at the time of this POET; none of these methodologies has been approved by the FDA and as such require self-validation as per CLIA 88.

At the present time, there are no clinical indications for the use of kits for low risk HPV DNA detection and testing for this analyte is strongly discouraged.5 hrHPV testing is not currently recommended for use as a single test in primary screening for cervical cancer, however, studies have suggested that due to its high sensitivity for cervical cancer and its precursor lesions, hrHPV testing may be an effective first step.6,7,8 hrHPV’s lack of specificity for cancer and high grade lesions is a limiting characteristic; any use of hrHPV alone in a primary screening scenario would require a more specific secondary triage method for hrHPV positive cases (e.g., Pap tests or other markers of high grade disease – to be discussed in a future POET)

Clinical Context

High risk types of human papillomavirus have been shown to be associated with virtually all forms of cervical cancer and their precursor lesions. As such, testing for the presence of high risk HPV DNA has become an important tool in cervical cancer screening. Currently available hrHPV DNA detection systems have been FDA-approved for use in 3 clinical applications:

1. Triage of patients having equivocal (ASC-US) Pap tests – reflex hrHPV testing.9

In this context, patients having ASC-US interpretations can receive a hrHPV test. A positive hrHPV result would triage the patient to colposcopic examination whereas a negative result would triage a patient back to annual Pap screening. Data from the National Cancer Institute’s ASC-US/Low Grade Squamous Intraepithelial Lesion Triage Study (ALTS) show that use of this triage method identified as many patients having cervical intraepithelial neoplasia grade 2 and higher (CIN2+) lesions as direct referral to colposcopic examination, with overall referral of about half the number of patients. In this study, when compared to cytologic followup only, use of hrHPV testing identified more CIN2+ lesions with no need for repeat cytology specimens requiring costly followup office visits.10 HPV testing should not be performed in women less than 21 years of age with ASC-US on the Pap test9.

2. Use in conjunction with the Pap test in women over the age of 30 years.9

In this context, a negative Pap in conjunction with a negative hrHPV test has a nearly perfect (100%) negative predictive value for the presence of CIN2+. In women having negative results in both of these tests, the very low risk of the presence of a neoplastic process allows the screening interval to be safely increased to 3 years. In women having a negative Pap test with a positive hrHPV test, the followup recommendation is to rescreen with both tests at 12 months. If the Pap test is negative, but the hrHPV test remains positive at 12 months, the patient should be sent for colposcopic examination at that point.

3. The use of specific testing for hrHPV types 16 and 18 (distinct from the pooled tests for the 13 or 14 of the most prevalent hrHPV types)11

In this context, testing for hrHPV types 16 and 18 has been FDA-approved for adjunctive use only at the time of this POET. Adjunctive use indicates that the test should only be used in addition to the hrHPV tests approved for the 2 clinical indications noted above. The American Society for Colposcopy and Cervical Pathology (ASCCP) has issued a recommendation for the use of specific type 16/18 testing only in the circumstance of screening using Pap and hrHPV testing in the over 30 years of age population.7 In this circumstance, women having a positive test for hrHPV in addition to a negative Pap test could be triaged to immediate colposcopy if the 16/18 test were positive and triaged to 12 month repeats of both tests if the 16/18 test were negative.

At present, there is no recommendation for triage using the 16/18 test alone in women having ASC-US Pap tests. Approximately 25% of women who are positive for hrHPV in the circumstance of an ASC-US Pap test were found to be positive for hrHPV types 16 and 18.12 Those ASC-US patients who were found to be HPV types 16/18 positive had a 40% cumulative risk or CIN2+, while those found to be 16/18 negative still had a 20% cumulative risk of CIN2+. Hence, although some risk stratification was possible, the rate in the 16/18 negative group was still substantial, warranting immediate referral to colposcopy. Some studies have suggested that long term follow-up of patients having HPV 16/18 positive tests versus all other hrHPV positive tests might be useful, as these patients appear to be at significantly higher risk for the development of CIN3+ lesions and hence risk stratification may be clinically useful at this level.13 At the present time, confirmatory studies will need to be performed to determine if this holds true.

In addition to FDA-approved indications, recent consensus management guidelines (American Society for Colposcopy and Cervical Pathology (ASCCP)) have indicated that hrHPV testing can also be used as part of the post-colposcopic management of histologic diagnoses of CIN 1/2/3 and endocervical adenocarcinoma in situ. In addition, hrHPV testing can be used to triage postmenopausal women having LSIL cytology results. ASCCP consensus management guidelines stipulate that hrHPV testing should not be performed in women under the age of 21 years and, in general, should not be performed on any patient more than once per calendar year.9,14

Non-FDA approved off-label methods for hrHPV DNA detection are commonly used in laboratories, and are acceptable under CLIA ’88 assuming appropriate self-validation studies are performed. Such off-label use most commonly consists of approved methods (Qiagen or Hologic) used with different non-FDA approved transport medias ( e.g., BD SurePath).15,16 In addition, non-FDA approved methods such as polymerase chain reaction (PCR) and in situ hybridization have been used for high risk HPV DNA detection. Because of the high volume of hrHPV testing performed and the implications for clinical management of patients based on the results, guidelines for validations have recently been published and include both analytic and clinical components.1

These guidelines include the following:

  • Capable of detecting the 13 most common high risk HPV types
  • Clinical specificity of at least 85% - adequate PPV for CIN 3+
  • Clinical sensitivity of 92% +/- 3% for CIN 3+
  • Sensitivity data is available for peer review
  • Interlaboratory reproducibility kappa > 0.7
  • Determination of specimen adequacy

Technology Overview

FDA Approved Methods

  • Qiagen Hybrid Capture 2: This method is FDA approved for use with Qiagen Standard Transport Media (STM) and the ThinPrep Pap Test (Hologic). Published self-validation studies have shown similar utility with BD SurePath media.15,16 The method uses whole genomic RNA probe cocktail directed toward 13 hr HPV types (16/18/31/33/35/39/45/ 51/52/56/58/59/68). Probes are reacted with denatured sample DNA, which form complementary DNA-RNA “hybrids” when hrHPV types are present. The hybrids are stabilized on the walls of the testing vial or plate by antibodies directed against hybrids. Once stabilized the hybrids are identified via additional anti-hybrid antibodies which are labeled with alkaline phosphatase. Multiple alkaline phosphatase molecules are present on each antibody and multiple antibodies attach to each hybrid leading to substantial final signal amplification. A positive specimen is detected by a chemiluminescent reaction caused when a substrate molecule is cleaved by the alkaline phosphatase. The intensity of the light emitted is measured as relative light units (RLU) which is compared to known negative and positive controls in each specimen run. A cutoff value is obtained from these control specimens which determines the positive and negative results for clinical samples. The RLU level is proportional to the amount of hybrid (and hence the amount of hrHPV DNA in the tested sample), however the assay is only semi-quantitative as there is no control for the number of cells present in the sample. (e.g., high signal may denote large numbers of cells containing low copy numbers of hrHPV DNA or low numbers of cells containing high copy numbers of hrHPV DNA).

    Automation procedures are available for the Hybrid Capture 2 assay (Rapid Capture) that may allow for significant improvement in laboratory productivity. This method is well-validated, both analytically and clinically, based on the results of the ALTS and FDA approval. Limitations of the test include issues of cross reactivity with some non-hrHPV types (types 6 and 13), which may account for small numbers of false-positive results. In addition, the test does not account for low- or non-cellular samples which may yield false-negative results, because no probe recognizing normal cellular components is included with the assay.

  • Hologic/Third Wave Cervista HPV HR and Cervista HPV 16/18: These 2 tests utilize the same method and are FDA approved for use only with the ThinPrep Pap Test PreservCyt Solution (Hologic). There have been a number of studies evaluating the use of Third Wave Cervista in BD SurePath samples and results have shown comparability with FDA-approved methods; however, as per the regulations of CLIA ’88, use of non-FDA approved methods require in-house validation studies.18,19

    The Third Wave Cervista HPV assay is a qualitative test for the detection of 14 hrHPV types (16/18/31/33/35/39/45/51/52/ 56/58/59/66/68). The test uses the Invader chemistry technology, which is a 2 stage signal amplification method. In the first stage specific probe oligonucleotides directed toward hrHPV DNA sequences and Invader oligonucleotides are reacted with the target mixture. When these oligonucleotides overlap by at least one base pair at the target sequence, an invasive structure forms that acts as a substrate for a proprietary enzyme (Cleavase). This enzyme cleaves the 5’ portion (flap) of the probe at the position of the overlap. The oligonucleotide probes are in large excess in the mixture and continually cycle on and off the target sequence such that many cleaved 5’ flaps are generated at each target sequence – the basis of the amplification. In the second stage, the cleaved flaps bind to a universal hairpin fluorescence resonance energy transfer (FRET) oligonucleotide creating another invasive structure that the Cleavase enzyme recognizes as a substrate. This enzyme cleaves the FRET oligonucleotides between the fluorophore and quencher molecule and produces a fluorescence signal as the cleaved flaps cycle on and off. For each copy of the HPV DNA target, the combined primary and secondary reactions result in 106 – 107 fold signal amplification per hour.

    In addition, an oligonucleotide probe directed toward human histone 2 gene is included in the mix, serving as an internal control. Reaction of this probe with complementary cellular DNA is detected simultaneously using a different flap sequence and different FRET oligonucleotide, each with a different fluorophore. Positive reactions for each of the analytes are determined by levels of fluorescence above an empirically derived cut-off value. The control probe (human histone 2) guards against negative HPV results being caused by inadequate numbers of cells in the sample and as an overall quality control check on the testing procedure. The Cervista HPV 16/18 assay uses identical technology but contains probes only directed at HPV types 16 and 18. Automation procedures are available for the Cervista assays that may allow for significant improvement in laboratory productivity. FDA trials of the Cervista assay showed 92.8 and 100% sensitivity for the detection of colposcopically confirmed CIN 2+ and CIN 3+, respectively. When compared to the data derived from ALTS, the results of the trial were comparable. Reproducibility studies showed 99% agreement for positive tests and 100% agreement for negative tests. No cross reactivity for the most common low risk HPV types (6/11) was identified in analytic studies.

    Post-approval studies have shown high sensitivities for CIN 2+ detection in the ASC-US triage testing application and direct comparisons with Hybrid Capture 2 have shown good concordance and fewer false positive results, with a slight majority of discordant cases being verified by sequencing as correct in the Third Wave method.20,21,22

Non-FDA Approved Methods

  • Polymerase Chain Reaction (PCR): Identification of HPV DNA by polymerase chain-based assays has been in widespread use for many years. Many assays have been laboratory-developed tests and a number of tests are commercially available. The Roche Amplicor method is one widely used commercial product for PCR detection of HPV. This assay detects the 13 most common high risk HPV types and extension of the procedure with the Roche Linear Array Genotyping kit allows for specific typing of the HPV once it is found to be present. Although not FDA approved at present, PCR detection of HPV has been used as a “gold-standard” for the presence of HPV in numerous studies and clinical trials, including ALTS.

    Interestingly when compared directly to Hybrid Capture 2 in ALTS, PCR was found to be slightly less sensitive for detection of clinical disease;23 however numerous additional studies have shown PCR clinical sensitivity to be comparable for use in screening tasks.24-27 In addition, specific typing of hrHPV provides utility in the identification of simultaneous infection by multiple HPV types and investigation of persistent hrHPV infections to determine if the same hrHPV types are implicated.

  • In Situ Hybridization (ISH): ISH refers to direct visualization on tissues or cells of a hybridization reaction between nucleotide probes and target DNA. ISH for HPV has been in wide use as a research tool for many years. The only commercial assay developed is the Ventana INFORM HPV. Use of this assay has not been shown to be sensitive enough for routine use in clinical situations of ASC-US triage or primary screening.20,28 Its potential advantage however, is higher specificity for high grade lesions and the ability to directly identify signal and its association with cells/tissues of interest under the microscope. Some investigators have advocated ISH for HPV in circumstances of equivocal morphology for high grade dysplasia in biopsy specimens.

Impact on Current Pathology Practice

The use of hrHPV testing has become routine in most cervical cancer screening programs at present. Its use in triage of ASC-US cases to colposcopic examination or followup is the primary application, and its use as an adjunct to the Pap test in screening the over 30 years of age population is growing. Use of HPV 16/18 typing has not become a mainstream procedure at present, although its use may increase over time as refinements to clinical triage algorithms mature.

Acceleration/Deceleration Triggers to Adoption

Although hrHPV testing is not currently approved for use as the primary cervical cancer screening test, there have been a number of studies that suggest that hrHPV testing may be more sensitive than the Pap test for the detection of high grade cervical disease and hence might be a more attractive first screen, followed by the Pap (or other more specific) test in positive cases.30 If ultimately found to be a cost-effective method of cervical cancer screening, HPV testing would balloon in use, to the detriment of Pap testing. In addition, other markers (cell cycle related, gene amplifications) have the potential to provide sensitive and specific information about the potential for the presence or risk of development of a high grade dysplasia or carcinoma. Such markers have the potential to serve as either primary screening probes or as downstream triage mechanisms following either positive hrHPV or Pap tests. (These new markers will be the topic of a future POET analysis.)


The TAC would like to thank Teresa Darragh, MD, FCAP for critical review and suggestions for the paper.

For More Information/References:

  1. Schiffman M, Castle PE, Jeronimo J, Rodriguez AC, Wacholder S. Human Papillomavirus and cervical cancer. Lancet 2007;370:890-907.
  2. Hybrid Capture 2, REF 5198-1220, US Food and Drug Administration Package Insert. L01002 Rev .0, 2007.
  3. Cervista™ HPV HR, REF 95-438, US Food and Drug Administration Package Insert. PIN 15-3100, Revision A, 2009.
  4. Cervista™ HPV 16/18, REF 95-438, US Food and Drug Administration Package Insert. PIN 15-3101, Revision A, 2009.
  5. Solomon D, Papillo JL, Davey DD; Cytopathology Education and Technology Consortium. Statement on human papillomavirus DNA test utilization. Arch Pathol Lab Med. 2009;133:1276-7.
  6. Mayrand MH, Duarte-Franco E, Rodrigues I, Walter SD, Hanley J, Ferenczy A, Ratnam S, Coutlée F, Franco EL; Canadian Cervical Cancer Screening Trial Study Group. Human papillomavirus DNA versus Papanicolaou screening tests for cervical cancer. N Engl J Med. 2007;357:1579-88.
  7. Naucler P, Ryd W, Törnberg S, Strand A, Wadell G, Elfgren K, Rådberg T, Strander B, Johansson B, Forslund O, Hansson BG, Rylander E, Dillner J. Human papillomavirus and Papanicolaou tests to screen for cervical cancer. N Engl J Med. 2007;357:1589-97.
  8. Kitchener HC, Almonte M, Thomson C, Wheeler P, Sargent A, Stoykova B, Gilham C, Baysson H, Roberts C, Dowie R, Desai M, Mather J, Bailey A, Turner A, Moss S, Peto J. HPV testing in combination with liquid-based cytology in primary cervical screening (ARTISTIC): a randomised controlled trial. Lancet Oncol. 2009;10:672-82. Epub 2009 Jun 17.
  9. Wright Jr TC, Massad LS, Dunton CJ, Spitzer M, Wilkinson EJ, Solomon D. 2006 Consensus Guidelines for the Management of Women with Abnormal Cervical Cancer Screening Tests. Am J Obstet Gynecol 2007;197:346-55.
  10. Solomon D, Schiffman M, Tarone R. Comparison of three management strategies for patients with atypical squamous cells of undetermined significance: baseline results from a randomized trial. J Natl Cancer Inst 2001;93:293-9.
  12. Wheeler CM, Hunt WC, Schiffman M, Castle PE; Atypical Squamous Cells of Undetermined Significance/Low-Grade Squamous Intraepithelial Lesions Triage Study Group. Human papillomavirus genotypes and the cumulative 2-year risk of cervical precancer. J Infect Dis. 2006;194:1291-9. Epub 2006 Sep 27.
  13. Khan MJ, Casle PE, Lorincz AT, et al. The elevated 10-year risk of cervical precancer and cancer in women with human papillomavirus (HPV) type 16 or 18 and the possible utility of type-specific HPV testing in clinical practice. J Natl Cancer Inst 2005;97:1072-9.
  14. Wright Jr TM, Massad LS, Dunton CJ, Spitzer M, Wilkinson EJ, Solomon D. 2006 consensus guidelines for the management of women with cervical intraepithelial neoplasia or adenocarcinoma in situ. Am J Ob Gynecol 2007;197:340-5.
  15. Ko V, Tambouret RH, Kuebler DL, Black-Schaffer WS, Wilbur DC. Human Papillomavirus Testing using Hybrid Capture II with SurePath Collection: Initial Evaluation and Longitudinal Data Provides Clinical Validation for this Method. Cancer (Cancer Cytopath) 2006;108:468-74.
  16. Siddiqi A, Spataro M, McIntire H, Akhtar I, Baliga M, Flowers R, Lin E, Guo M. Hybrid capture 2 human papillomavirus DNA testing for women with atypical squamous cells of undetermined significance papanicolaou results in SurePath and ThinPrep specimens. Cancer Cytopathol. 2009;117:318-25. [Epub ahead of print]
  17. Stoler MH, Castle PE, Solomon D, Schiffman M; American Society for Colposcopy and Cervical Pathology. The expanded use of HPV testing in gynecologic practice per ASCCP-guided management requires the use of well-validated assays. Am J Clin Pathol. 2007;127:335-7.
  18. Wong AK, Chan RC-K, Nichols WS, Bose S. Human Papillomavirus (HPV) in Atypical Squamous Cervical Cytology: the Invader HPV Test as a New Screening Assay. J Clin Microbiol 2008;46:869-875.
  19. Harvey M, Stout S, Starkey CR, Hendren R, Holt S, Miller GC. The clinical performance of Invader technology and Surepath when detecting the presence of high-risk HPV cervical infection. J Clin Virol 2009;45:S79-83.
  20. Kurtycz DFI, Smith M, He R, Miyazaki K, Shalkham J. Comparison of Methods Trial for High-Risk HPV. Diagn Cytopathol 2009 Aug 17 (Epub ahead of print).
  21. Johnson LR, Starkey CR, Palmer J, Taylor J, Stout S, Holt S, Hendren R, Bock B, Waibel E, Tyree G, Miller GC. A Comparison of Two Methods to Determine the Presence of High-Risk HPV Cervical Infections. Am J Clin Pathol 2008;130:401-8.
  22. Ginocchio CC, Barth D, Zhang F. Comparison of the Third Wave Invader human papillomavirus (HPV) assay and the digene HPV hybrid capture 2 assay for detection of high-risk HPV DNA. J Clin Microbiol. 2008;46:1641-6. Epub 2008 Mar 26.
  23. Schiffman M, Wheeler CM, Dasgupta A, Solomon D, Castle PE; ALTS Group. A comparison of a prototype PCR assay and hybrid capture 2 for detection of carcinogenic human papillomavirus DNA in women with equivocal or mildly abnormal Papanicolaou smears. Am J Clin Pathol. 2005;124:722-32.
  24. Wahlström C, Iftner T, Dillner J, Dillner L; Swedescreen study group. Population-based study of screening test performance indices of three human papillomavirus DNA tests. J Med Virol. 2007;79:1169-75.
  25. Stevens MP, Garland SM, Rudland E, Tan J, Quinn MA, Tabrizi SN. Comparison of the Digene Hybrid Capture 2 assay and Roche AMPLICOR and LINEAR ARRAY human papillomavirus (HPV) tests in detecting high-risk HPV genotypes in specimens from women with previous abnormal Pap smear results. J Clin Microbiol. 2007;45:2130-7. Epub 2007 May 9.
  26. Carozzi F, Bisanzi S, Sani C, Zappa M, Cecchini S, Ciatto S, Confortini M. Agreement between the AMPLICOR Human Papillomavirus Test and the Hybrid Capture 2 assay in detection of high-risk human papillomavirus and diagnosis of biopsy-confirmed high-grade cervical disease. J Clin Microbiol. 2007;45:364-9. Epub 2006 Nov 22.
  27. Sandri MT, Lentati P, Benini E, Dell'Orto P, Zorzino L, Carozzi FM, Maisonneuve P, Passerini R, Salvatici M, Casadio C, Boveri S, Sideri M. Comparison of the Digene HC2 assay and the Roche AMPLICOR human papillomavirus (HPV) test for detection of high-risk HPV genotypes in cervical samples. J Clin Microbiol. 2006;44:2141-6.
  28. Hesselink AT, van den Brule AJ, Brink AA, Berkhof J, van Kemenade FJ, Verheijen RH, Snijders PJ. Comparison of hybrid capture 2 with in situ hybridization for the detection of high-risk human papillomavirus in liquid-based cervical samples. Cancer. 2004;102:11-8.
  29. Kong CS, Balzer BL, Troxell ML, Patterson BK, Longacre TA. p16INK4A immunohistochemistry is superior to HPV in situ hybridization for the detection of high-risk HPV in atypical squamous metaplasia. Am J Surg Pathol. 2007;31:33-43.
  30. Naucler P, Ryd W, Tornberg S, et al. Efficacy of HPV DNA testing with cytology triage and/or repeat HPV DNA testing in primary cervical cancer screening. J Nathl Cancer Inst 2009;101:88-99.

Technology Assessment Committee (TAC) Members at the time of original POET publication:
John W. Turner, MD, FCAP, Chair
Frederick L. Baehner, MD, FCAP
Kenneth J. Bloom, MD, FCAP
Samuel K. Caughron, MD, FCAP
Thomas J. Cooper, MD, FCAP
Richard C. Friedberg, MD, PhD, FCAP
Jonhan Ho, MD, FCAP
Federico A. Monzon, MD, FCAP
David C. Wilbur, MD, FCAP
Crystal Palmatier Jenkins, MD, Junior Member

This POET was developed by the Technology Assessment Committee (TAC) with input from the Council on Scientific Affairs. Opinions expressed herein are solely those of the authors and do not represent those of the College of American Pathologists (CAP). No endorsement of any proprietary technology or product referenced is implied by the TAC or CAP. This report is provided for educational purposes only. None of the contents of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means (electronic, mechanical, photocopying, recording, or otherwise) without prior written permission of the publisher.


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