UTILITY OF HUMAN PAPILLOMAVIRUS

CAPSID PROTEIN L1 AND p16 IN THE

ASSESSMENT AND ACCURATE
CLASSIFICATION OF ANAL SQUAMOUS
INTRAEPITHELIAL LESIONS

Patil D T, Yang B. Am J Clin Pathol. 2015; 144:113-21

Department of Anatomic Pathology, Cleveland Clinic,

Cleveland, OH, United States of America
INTRODUCTION
 ANAL SQUAMOUS LESIONS
• HPV is etiologically associated with anal
squamous carcinogenesis
• Oncogenic/hrHPV infection has been
documented in 84% to 95% of anal squamous
cell carcinomas*

1. Frisch M, Fenger C, van den Brule AJ, et al. Variants of squamous cell carcinoma of the
anal canal and perianal skin and their relation to human papillomaviruses. Cancer Res.
1999;59:753-757
2. Wong AK, Chan RC, Aggarwal N, et al. Human papillomavirus genotypes in anal
intraepithelial neoplasia and anal carcinoma as detected in tissue biopsies. Mod Pathol.
2010;23:144-150
3. Centers for Disease Control and Prevention. HPV-associated cancers statistics. 2014.
http://www.cdc.gov/cancer/hpv/statistics
• Orchestrated molecular events that morphologically
manifest as progressively worsening grades of dysplasia

• Morphologic interpretation of dysplasia suffers from

significant inter-observer and intra-observer variability

• Limited biopsy samples, tangential sectioning artifact,

marked reactive atypia of the transitional zone, and
thermal artifact are some of the factors that contribute
to diagnostic discrepancies

• As a result, ancillary markers, such as Ki-67 and p16,

have gained popularity
 HUMAN PAPILLOMA VIRUS
• 55nm non-enveloped virus
• Contains a double-stranded closed
circular DNA genome, associated
with histone-like proteins
• Protected by a capsid formed by
two late proteins, L1 and L2
• L1 pentamers join to copies of L2
that occludes the center of each
pentavalent capsomere
• Each virion contains 72 copies of
the L1, the major component of the
capsid, and a variable number of
copies of L2, a secondary
component of the viral capsid
 HPV CAPSID PROTEIN L1
• Major capsid protein of HPV
• Composed of 72 capsomeres
• Forms pentamer and combines with L2 capsid
proteins
• Production is highest in LSIL/CIN1 &
Condylomas
• Progressively decreases in CIN2 & almost
entirely lost in CIN3
 p16/ CYCLIN DEPENDANT KINASE
INHIBITOR 2A
• 148 amino acids tumour suppressor protein
coded by CDKN2A
• Inhibits cyclin dependant kinases viz CDK4 &
CDK6
• Decelerates cell cycle progression from G1 to S
phase
• Implicated in prevention of melanomas,
oropharyngeal SCC, cervical carcinomas, anal
carcinomas and oesophageal carcinomas.
 VIRAL CARCINOGENESIS

Transforming effects of HPV E6 and E7 proteins. The net effect of HPV E6 and E7
proteins is to immortalize cells and remove the restraints on cell proliferation
 Early genes (E1, E2, Episomal /
E6, and E7) – Under Transient Stage
transcriptional control
of LCR

Persistent
Infection

Upregulation of E7 leads
to inactivation of RB
protein, which in turn
causes reciprocal
overexpression of p16
 AIM

TO EVALUATE UTILITY OF HPV CAPSID PROTEIN

L1 AND p16 IN THE ASSESSMENT AND
ACCURATE CLASSIFICATION OF ANAL SILs
MATERIALS AND METHODS
• Type of study: Cross sectional
• Study group: All anal SILs diagnosed between January
2004 and December 2011
– Total no. of cases – 145
– Location of the biopsies were classified as perianal vs anal
– Indications for obtaining these biopsies
• H/o condylomas/recurrent condylomas – 13
• Follow up of previous diagnosis of anal dysplasia – 15
• H/o cervical/penile/vulvar dysplasia – 17
• Follow up for remote H/o SCC in patients with H/o ulcerative colitis
or renal transplant – 9
• Atypical cytological brushing – 13
• Grossly identifiable lesions on high-resolution anoscopy – 16
• Unknown – 17
– Total of 145 lesions from 100 patients
 HISTOLOGIC REVIEW
• Condyloma
– squamoproliferative lesion showed papillomatosis, acanthosis, and hyperkeratosis, with
or without parakeratosis
• LSIL
– cytologic atypia and expansion of the immature basal cell layer was limited to the lower
one-third
• HSIL
– atypical squamous proliferation involved more than one-third the thickness of the
squamous epithelium
• In case the biopsy showed both low-grade and high-grade dysplastic areas,
each lesion was analyzed separately with respect to histologic features
and the tested markers
• Growth pattern was further categorized
– Warty: Acanthotic squamous proliferation with an undulating or “spiked” surface
– Basaloid: Relatively smooth and flat surface; epithelium lacked cellular maturation;
relatively small, uniform cells with hyperchromatic nuclei and coarse chromatin
– Combined warty/basaloid: warty growth pattern at the surface with basaloid cells at the
basal aspect of the lesion
METHODOLOGY
 p16 IHC
• 30 min EDTA antigen retrieval
• 4µm thick deparaffinized tissue sections incubated with p16
antibody (clone E6H4, prediluted; Ventana Medical Systems,
Tucson, AZ) for 1hr at 37°C
• Incubated with a secondary antibody (OmniMap Anti–Rabbit
HRP; Ventana Medical Systems) for 32 minutes at 37°C
• Chromogenic substrate (ChromoMap DAB; Ventana Medical
Systems) for 8 minutes at 37°C
• Counterstained
• Scoring (Cytoplasmic and/or nuclear immunoreactivity)
– Negative (No staining)
– Patchy/focal (cluster of at least five immunostained cells in any layer of
squamous epithelium or immunoreactivity restricted to the lower one-
third of the thickness of squamous epithelium)
– Diffuse (contiguous nuclear and cytoplasmic immunoreactivity
involving more than two-thirds of the thickness of squamous
epithelium)
 HPV L1
• CytoReact Cell/Tissue HPV Broad Spectrum L1 Kit (Advanced
Medical Science and Technology, Sparks, MD)
• Slides were rehydrated and antigen retrieval was performed for 15
minutes at 95°C
• Primary antibody was applied for 1 hour at room temperature
• 30-minute incubation with CytoReact Labelled Polymer and DNA
Probe reagent
• Slides were then incubated in CytoReact Peroxidase and RNA
Enzyme for 15 minutes at room temperature
• CytoReact AEC Chromogen and ALP were added to each slide for
visualisation
• Covered with a glass coverslip and incubated for 25 minutes
• Staining with aqueous-based hematoxylin
• Slides were rinsed with water and mounted using Aqua-Mount
• Scoring
– Nuclear immunoreactivity was considered a positive reaction
 STATISTICAL ANALYSIS
• Dichotomous measures were compared using
the chi-square test
• p value <0.05 was considered statistically
significant
RESULTS
 DEMOGRAPHIC DATA
• Total no. of patients – 100 (58 Men & 42 Women)
• Mean age of patient – 46 years (20-88 years)
• Immunocompromised – 46 patients
– HIV = 27
– Organ transplantation = 4
– Idiopathic IBD = 6 (UC=2; CD=4)
– HBV = 2
– Malignancy = 5
– AI disorder = 2
 HISTOPATHOLOGICAL FINDINGS
• 145 lesions from 100 patients
• Condylomas = 34 (23%)
• LSILs = 64 (44%)
• HSILs = 47 (32%)
HSV L1 EXPRESSION
 p16 EXPRESSION

Some condylomas displayed a singly scattered p16

immunostaining pattern, with a stellate appearance,
resembling dendritic cells. However, further immunostaining
with antibodies to CD1a and CD68 failed to confirm their
dendritic cell origin
 CO-EXPRESSION OF HPV L1 & p16

Co-expression of HPV L1 and diffuse p16 staining pattern

was mutually exclusive in almost all except four (12%) cases
- focal HSILs in a background of extensive LSILs
DISCUSSION
• Histological assessment of anal/perianal lesions is still
considered the definitive way of assessing squamous
dysplasia
• Inter-observer variability - processing artifact, limited
biopsy samples, thermal artifact, and inflammatory atypia
• Inter-observer agreement is especially low when
distinguishing reactive atypia from LSIL
• HPV L1 is an early capsid protein whose production peaks
during the productive phase of the viral life cycle. This
phase correlates with the development of proliferative
lesions, such as LSILs
• Loss of L1 expression is seen with neoplastic progression
• L1 evaluation may serve as a risk stratification tool to
identify patients with SCC-IS who have more aggressive
disease*
*Hernandez J, Elahi A, Siegel E, et al. HPV L1 capsid protein
detection and progression of anal squamous neoplasia. Am J
Clin Pathol. 2011;135:436-441
• There is a reciprocal relationship of L1 and p16
protein expression in anal precursor lesions
– Loss of HPV L1 and gain of diffuse p16 expression are
the hallmark of HSILs

• Co-expression was seen in 4 cases

– HSIL was arising in a background of an extensive LSIL

• Given the differences in clinical management of

LSILs and HSILs, the combined evaluation of HPV
L1 and p16 expression may serve as a potential
triage point for therapy
– LSILs that lose L1 expression could be an indication for
ablation therapy
 MERITS
• One of the first to study the combined
immunostaining of HPV L1 & p16 in anal SILs

• Results are very similar to previous studies

performed in cervical precursor lesions, which
have shown loss of L1 expression with neoplastic
progression along with diffuse p16 staining*

• Mutually exclusive staining properties provides a

synergistic conclusive effect
*Yemelyanova A, Gravitt PE, Ronnett BM, et al. Immunohistochemical detection of
human papillomavirus capsid proteins L1 and L2 in squamous intraepithelial lesions:
potential utility in diagnosis and management. Mod Pathol. 2013;26:268-274
 DEMERITS
• Mathematical errors while tabulating

• Not mentioned as to why only 31/64 LSILs &

34/47 HSILs were taken into account while
analysing the co-expression of both the
markers
 CONCLUSION

HPV L1 and diffuse p16 expression is mutually

exclusive in most anal SILs & helps separate LSIL
& HSIL cases. Application of both HPV L1 & p16
can not only facilitate accurate grading but also
contribute to risk assessment in anal neoplasia