Evolution of Pap Cytology : It
was not discovered my grandson –believe me!!!
History & Geography of Pap:-In the 1920s Aurel
Babes and Georgiou Papanicolaou described the use of exfoliative cytology to
identify women with cervical cancer. The
technique commonly referred to as a conventional or Papanicolaou smear gained popularity in the 1940s . It involved placing a sample
of fluid from the posteriorvaginal
fornix on a slide which was then fixed with a Papanicoloau stain. Ayres
refined the technique with a spatula to take a scrape of cells in the cervix
increasing the yield of cervical cells
which are then reviewed by a cytologist. This remained the standard
screening tool for over 50 years until
the advent of liquid based cytology. Studies had been reported showing a wide
range of sensitivities and specificities for the Pap smear False negative rates
could be as high as 50%
Liquid based
cytology
Liquid based
cytology was developed to produce a more representative sample of the specimen
than a conventional smear and to reduce contamination by blood cells pus and
mucus. It has been shown that LBC reduces the inadequate rate from 9 to 1.6%
return for repeat cytology.
The two most widely studied technologies for
LBC preparation are SurePath and ThinPrep . LBC involves taking a sample of
cells from the transformation zone of the cervix in a similar way to conventional
cytology. The material obtained on the broom collection device is dispersed in
a preservative fluid to generate a suspension of cells. In the lab the
suspension is centrifuged and passed through a filter to create a slide with a monolayer preparation of well
preserved cellular morphology .
The original
Papanicolaou classification of cytology
findings has largely been superseded Currently there are tow main
classification of cervical cytology. The Bethesda classification is
widely used in many countries. This incorporates both cytological and
histological change Until 2013 the British Society of Clinical cytology had a
different categorization. The latest revision is now more similar to the
Bethesda system
Cytological
abnormalities are described as dyskaryosis . this is an abnormal chromatin
pattern with the grade of dyskaryosis defined by determining th ratio of nuclear diameter to
cytoplasmic diameter.
Squamous
intraepithelial lesion
In the
Bethesda classification abnormalities are described as either low grade squamous intraepithelial lesion
equivalent to CINI or high grade squamous intraepithelial lesion equivalent to
CIN2 and CIN3 . In addition another term atypical squamous cells of unknown significance
is used. ASCUS has no direct equivalent
in the UK classification though
borderline abnormalities are a
close comparison. Both represent a class
of smears with a low risk of underlying CIN3.
Cervical
Screening What does it the word screening means in Good Clinical Practice ??
Cervical screening was first introduced in British
Columbia and Finland. It began in the Uk in the 1960s Implementation was opportunistic and did
not significantly reduced cancer rates
until 1988 when a systematic call and
recall programmes.
Cervical screening saves approximately 4500 lives per year in England and in other
countries with high population coverage
from screening a 60-70% reduction in
deaths has ben seen Mortality rates in 2008
were nearly 70% lower than 30 years earlier In 2011 there were 972 deaths from cervical
cancer in the UK A total of 3,6 million
women aged 25-64 years were tested I
2011-2012
Primary
Screening
Coverage
which refers to the proportion of eligible women being screened in the previous
5 years has remained fairly consistent
in the UK over the last 20 years. There has been a gradual decline from 82% in
the 1990s to 79% in the last 5 years.
In
England the first invitation occurs at
25 years of age. In other parts of the
UK cervical screening begins at the age of 20 years . the age range was changed
in England in 2003 because of concerns over treatm3nt in young women with low grade or small volume
premalignant lesions that would resolve spontaneously and because of fears
related to subsequent pregnancy morbidity Between the ages
of 25-49 years subsequent
invitations are distributed every 3 years. From 50-64 years women
are invited every 5 years Women over 65
years are only invited if they have not
been screened in the past or they have had recent abnormal cytology results tests. Women aged
65 years or over whose last three consecutive adequate tests have been reported
as negative are removed from the screening programme.
Secondary
screening colposcopy
Colposcoy
first describd in 1925 by Hams
Hinselmann is the examination of the cervix with a lighted low powered microscope.
Although colposcopy may be part of an annual general gynaecology assessment as
in much of continental Europe , it is predominantly a secondary screening tool
used to determine appropriate management for women referred with an abnormal
cytology result. A biopsy may be taken
from the cervix for diagnosis and /or the cervix may be treated. Alternatively
women may remain under surveillance and
have repeated testing every 6-12 months In the UK approximately 150 000 referrals to colposcopy
are made annually .
Human
papillomavirus
In 1983zur
Hausen first isolated human papillomavirus from cervical carvinoma which led to
the identification of HPV as a potential
causative organism for the development of cervical cancer It is predominantly
sexually transmitted and a reflection of sexual activity with a lifetime risk
of up to 80% . the prevalence of HPV declines with age . The infection is often
an asymptomatic and transient event persisting in 10-15% of women . Over 120
types of HPV have since been identified HPV types 16 and 18 are together responsible
for 70-80 % of cervical cancers and
50% of all premalignant change. They
also account for 40-50% of vulval and Oropharyngeal cancers and 70-80% of anal
cancer More recently an expert panel have classified HPV types into five
categories
Categorisation
of HPV subtypes and pathogenesis
Group 1-
Definition – Carcinogenic to humans –HPV types –
16,18,31,33,35,39,45,51,52,56,58,59
Group 2A-
Definition – Probably carcinogenic to humans –HPV types – 68
Group 2B –
Definition Possibly carcinogenic to humans – HPV- 53,64,65,66,67,69,70,73,82
Group
3-Definition – Not classifiable – HPV- 6,11
Group 4 –
Definition – Probably not carcinogenic – HPV Nil
Let us be
acquainted with the etiological factors associated with human papillomavious &
later ca Cx ?
Reduced HPV
risk
Increasing
age
Obtaining
tertiary education
White
ethnicity
Married / co
habiting status
Pregnancy
resulting in children
Increased HPV
risk
Lower age
Lower
educational level
Non white
ethnicity eg black African Indian Pakistani
Single or
divorced / Separated / Windowed
Current and previous oral contraceptive use
Current use of other
hormonal contraception
Current
smoking status
Methods of human papillomavirus testing
The first
clinically applied HPV detection methods
in the 1980s included in situ hybridization. HPV has a stable double stranded DNA genome that can be accessed easily from
exfoliated cells . Currently fie techniques
for detecting HPV are approved in
the US for clinical use including four DNA based assays digene Hybrid Capture 2
, Cervista HPV HR Cervista HPV 16/18
test , Cobas 4800 HPV and one RNA
assay APTIMA HPV assay
Qiagen
hybrid capture II is the gold standard HPV test and was the first reliable quality
standardized HPV DNA test. It works by hybridization in solution of one strand of template DNA to complementary RNA
probes of 13 different HPV types and these types are responsible for the
vast majority of cervical cancers. In
2003 FDA approval was extended to the use of HC 2 alongside routine Pap
testing for women over the age of 30 years . There is a simpler version care HPV
available for use in low resource settings.
The emergence
of mucleic acid amplification techniques
has allowed for the development of alternative HPV detection assays
beyond hybrid capture II.
Cervista is a
signal amplification method that uses
invader technology for the qualitative detection of 14 high risk HPV type.
Using a cleavase enzyme 14 HR HPVs can
be detected.
Role of human
papillomavirus testing
In
clinical practice there are three roles
for HPV DNA testing
1.
Primary screening fro cervical neoplasia
2.
In
triage of minimally abnormal and
inconclusive smears
3.
As
a test of cure post treatment
4.
Human
papillomavirus as a primary screening test for cervical cancer
In 2006 an overview of the European and North American
studies on primary cervical cancer screening reviewed over 60,000 women and demonstrated a
sensitivity of 96.1% ans specificity of 90.7% for HPV testing versus cytology which had a sensitivity
of 53% and specificity of 90.7% of HPV
testing versus cytology which had a sensitivity of 53% and specificity of
96.3% . A recent meta analysis demonstrated that primary screening with
HPV testing can allow for an increase in
the interval between screening episodes of up to 6 years which could offset the
increased costs of new technology with
less frequent screening . the high positive rates for HPV testing in young un immunized women limit the
clinical effectiveness of primary
HPV screening in the population
under the age of 30 years . However the
sentinel sites project of primary HPV screening is including women from the age
25 years to mirror the current English
cervical screening programme.
Its potential value in
a low resource setting was demonstrated by a study from rural India , which
showed that a single round of HR HPV testing by HC II was associated
with a significant decline in the rate of advanced cervical cancers and associated deaths compared with the
unscreened control group.
What is called triaging, say in GDM (50 Gm of glucoseàthen decide who warrants 100Gm full
CTT). Similarly all women don’t warrant Colposcopy. Human papillomavious triage
–by how?? It is costly !!! Ans:-HR- HPV
prevalence in women with borderline smears varies within the studies from 31% to 72% HPV testing of
women with low grade abnormal smears can
separate those HPV negative women who are unlikely to have high grade CIN from those who are HPV positive and therefore at higher risk .
Human papillomavirus
studies
Although many studies have been undertaken the following four
studies have provided the most robust evidence for the role of HPV in screening and triage. Following these
studies HPV triage has been introduced
in England.
ASCUS/ LSIL triage study
ASCUS/ LSIL triage study was a multicentre randomised clinical trial
designed to evaluate three alternative methods of management of ASCUS or LSIL
namely immediate colposcopy cytologic follow up and triage by HPV
HPV DNA testing showed the highest sensitivity and identified
96.3% of women with CIN3 which was at
least as sensitive as an immediate
colposcopy . High sensitivity combined
with a reasonable specificity for triage has made HPV DNA testing the recommended option for the
management of ASCUS cytology.
As 83% of patients
with LISL were positive for HPV , it was not considered useful for triage of this group. By contrast in England triage of this group has proved cost
effective as 17% of women can be returned to normal recall rather than be referred for colposcopy
The ALTS study also looked at
variation in the interpretation of cytology samples . Substantial
differences were found among expert pathologists in interpreting both thin layer
Pap tests and biopsies. Regarding the accuracy of colposcopy directed biopsy
this study found increased sensitivity when more than one biopsy was taken.
Trial of
management of borderline and other low
grade abnormal smears study
The aims of this 7 year multicentre trial were :
1.
To
assess whether conservative management or immediate colposcopy was more
effective in women with low grade smear abnormalities .
2.
To
determine whether women should have immediate treatment with large loop excision of the
transformation zone or a colposocopy and directed biopsy with recall for
treatment if the punch biopsy showed high grade disease.
3.
To
determine whether HPV testing added to the effectiveness of the current
management of low grade smear abnormalities.
The study population comprised 10 000 women between the ages of 20 and 59
years with an index borderline test followed by another test, 6 months later ,
which showed either borderline or mild
dyskaryosis. Cytological screening was performed every 6 months in primary care or patients were
referred for colposcopy and related interventions . All women were
followed for 3 years . Among women diagnosed. Of women randomised to
cytological surveillance a similar number 77% of cases were detected by
surveillance cytology and related interventions.
The trial recommended a policy of
surveillance rather than triage for
two reasons the first was that some CIN
2 will regress with time and the second was that they identified a high
proportion of HPV negative CIN2. The authors demonstrated the ability to return
to recall not only 50% of those referred to colposocpy but also the 35% of
women who were HPV negative based on HPV
triage .
How accurate is Liquid Based Cytology? Is it superior in detection &
prediction than slide
dried smear of good old age used
to be done at OPD?? The accuracy of LBC
has not improved from with the
addition of HPV testing . However an initial negative HPV test provided the same degree of protection over tow screening rounds as negative
cytology for one screening round. This
would allow the screening
interval to be increased to 5-6 years or longer in women over 30 years of age .
No significant adverse psychosocial
effects of HPV testing were detected .
The authors concluded that it would not
be cost effective to screen with cytology and HPV combined , but HPV testing , as either triage
or initial test triaged by cytology
would be cheaper than cytology without
HPV testing.
Other uses of human papillomavirus
testing
Human papillomavirus in the resolution of uncertainties
HR-HPV testing may be of use in the following clinical scenarios
1.
In
women under long term surveillance for low grade CIN
2.
Cervical
stenosis
3.
Colposcopy
is unsatisfactory
4.
Those with a persistent mismatch between high grade cytology and low
grade histology .
Human papillomavirus
and glandular abnormalities
The incidence of invasive and preinvasive glandular lesions
of the uterine cervix has been increasing . Cervical cytology was designed to
detect squamous rather than glandular abnormalities. Despite this glandular
abnormalities are an important category
as it may be a sign of non cervical malignant change including endometrial or ovarian cancer.
For women with a
glandular abnormality conisation with close surveillance including repeat
cytology colposcopy punch biopsy and endocervical curettage is frequently
recommended.
Unfortunately the conservative management of glandular lesions has a substantial false negative rate. Following treatment there is a
15% rate of recurrence compared with
5% for squamous abnormalities For this
reason hysterectomy is usually considered once a woman’s family is complete.
Costa and colleagues found that the combination of HC2 and cytology
reached 90% sensitivity in detecting persistent lesions at the first
follow up visit and 100% sensitivity at
the second among women treated
conservatively for adenocarcinoma in situ or adenocarcinoma. This may prove a
more robust method of follow up for these lesions.
How helpful is Genotyping of HPV?? As HPV positivity is so
common there is an increasing need to look for other prognostic markers to
assist management and identify those
most at risk of high grade pre malignancy. The American Society for Colposcopy and Cervical Cytology screening
guidelines include genotyping in
the management algorithm for cytology
negative and HR – HPV –DNA
positive patients . Women with
evidence of HPV 16 and / or 18 are
recommended for immediate colposcopy , whilst
those who have one of the other HR-HPV types are kept under annual
cytological and HPV surveillance.
