Diagnostic Cytology
Fig. 7.60 Human papillomavirus (HPV) infection cervix, LSIL.
(A) LBGS (Papanicolaou 60 x MP). (B) In situ hybridization on the same specimen reveals
intranuclear HPV high-risk localization (in situ hybridization with DAB changes x MP).
prolonged immunosuppression, as after a kidney transplant,
and chemotherapy may cause clinical HPV disease. In fact, the
first case reported by Gupta and co-workers of cervical dysplasia
after azathioprine (Imuran) therapy we believe now to represent
a case with condylomatous changes in the cervical epithelium
following immunosuppression.111
Increased incidence of con-
dyloma in patients with acquired immunodeficiency syndrome
(AIDS) is also well documented.
Molecular testing and ancillary techniques:
As noted earlier, HPV
HR testing is commonly used as an adjunctive tool in cervical
cancer screening for ASC-US triage/reflex testing, and in women
aged 30 and older with negative cytology to assess overall cer-
vical cancer risk. As the 2002 ASCCP management guidelines
noted: "HPV testing has matured, appears clinically validated
and should become integral to both screening and clinical man-
There are a variety of molecular techniques available for
detecting and quantifying HPV in clinical specimens, including
cytology and histology. Historically, tests for HPV using nuclei
acid probes have been commercially available since the late
1980s, but were cumbersome and involved radioactive labeling.
Nucleic acid ISH was also commercially available. Currently,
available HPV tests include three major types of nucleic acid
hybridization methodologies: direct probe methods, hybridiza-
tion signal amplification, and target amplification.112
Direct probe methods include Southern blot, the gold stand-
ard for HPV genomic analysis, and ISH. The latter provides the
advantage of correlating the presence of HPV with morphology
(cytologic or histological). Disadvantages of direct probe meth-
ods include low sensitivity, labor intensity, and potential need
for highly purified DNA, which is a challenge in formalin-fixed
tissue. Signal amplification methods are proprietary technologies
that boost sensitivity of direct probe methods by detection inno-
vations. Examples of signal amplification include multimeric
layering of reporter molecules on DNA probes (hybrid capture),
branched DNA (bDNA), and isothermal signal amplification
(invader technology). Target amplification methods include
PCR and can be home brew or commercially available. Target
amplification is highly sensitive and can be used for detection,
viral load quantitation, DNA sequencing, and mutation analy-
sis, and can be performed in multiplex assays. Patented HPV
sequences limits the applicability of PCR as an in vitro diagnos-
tic test due to legal and/or proprietary restrictions. Home-brew
assays also lack interlaboratory standardization.112,113
At the time of this writing, there is only one FDA-approved
HPV test for in vitro diagnostic use/clinical testing, Digene
Hybrid Capture 2 (HC2), a signal amplification methodol-
ogy. HC2 utilizes a cocktail of specific RNA probes directed
toward individual DNA sequences of 13 HR HPV genotypes to
be detected. A proprietary antibody directed toward DNA-RNA
hybrids in the capture and detection steps is developed by a
chemiluminescence system.100,101 This test also has the most
clinical experience to date, having been utilized in ALTS with
well-documented positive and negative predictive values and
has formed the basis of current cervical cancer screening guide-
lines incorporating HPV HR testing. Despite its success, this
assay is time-consuming and labor intensive, contains no inter-
nal control to evaluate for adequate cellular material, and can
cross-react between low-risk and probable high-risk HPV types.
Although clinically sensitive, improvement in clinical specifi-
city and positive predictive value is also desirable.106 The ability
to correlate HPV presence with cytomorphology and histol-
ogy provided by ISH is preferred by some. Automated ISH is
available for HPV detection in liquid cytology and histology
specimens (Ventana), but variable reports of sensitivity and
specificity which do not allow advocating for widespread clini-
cal use at present have been documented in the literature. In
some ISH platforms, episomal versus integrated HPV patterns
can be distinguished, but whether this distinction is predictive
of significant concurrent or future disease is still for investiga-
tive purposes only (Figs. 7.60, 7.61). In our experience using
Ventana probes, the results for the detection, specificity, sensi-
tivity, and predictive values are comparable to the HC2 detec-
tion. There is slightly increased detection among the normal
and HSIL groups, and somewhat decreased detection in ASC-
US cases (unpublished data).
Despite the fact that emergence of other HPV assays is expected
and desired, utilization of non-FDA approved tests for HPV HR
testing has raised considerable concern in the literature.106
The future of HPV testing is continuing to evolve and in the
USA, additional FDA-approved assays are anticipated following
well-controlled, statistically powered clinical validation trials.
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