# Presentation: 045959

## HPV Testing and Its Impact on Cervical Cancer Screening

**John M. Douglas, Jr., MD**
**Denver Public Health**

## Genital HPV Infection

- Over 100 HPV types identified; > 30 genital types
- Low-risk types (6,11)
- genital warts and early Pap smear changes (atypia, LSIL)
- Higher-risk types (16,18,31,33,35,others)
- “flat lesions” (SIL) and both early/late Pap smear changes
- All types cause subclinical infection

## HPV and Anogenital Cancer

- Genital HPV
- found in 95% of cervical cancers (and majority of anal, vulvar, vaginal, penile cancers)
- In vitro studies
- interaction between “high-risk” HPV proteins and cellular oncogenes

## Steps in HPV Induced Cervical Cancer

**Notes:**
Next, we examine the progression of HPV infection to cervical cancer. After exposure to HPV, events in the viral life cycle are initiated, with specific activity regulated by the factors responsible for the host’s immune response. HPV infection alone is not sufficient to induce an immediate carcinoma because malignant tumors develop only after HPV-induced lesions persist for several months or even years.  There is a large reservoir of HPV infection in young women in their teens and twenties.  Only a few of these women go on to develop persistent HPV infection.  Progression depends on viral type, immunologic factors and possibly co-carcinogens.  Persistent high-risk infections can lead to cellular deregulation.  Over time, this cellular deregulation can cause high-grade cervical lesions and ultimately, cervical cancer.  

Oncogenic activity has been attributed to the viral oncoproteins E6 and E7, which are crucial for malignant transformation of HPV associated cancers.  In non-malignant cells, inter- and intra- cellular signals suppress these oncogenes at the transcriptional level thus limiting malignant progression.  The E6 and E7 viral proteins interfere with cellular proteins that are involved in cell cycle control.  They cause the human cell to experience disturbed physiologic function and genetic instability.  Mutations in cellular genes devoted to the intracellular surveillance of HPV infections, integration of viral DNA, and deletions or mutations of viral transcription control sequences lead to a significantly increased expression of the E6/E7 genes, which is a consistent characteristic of high-grade intraepithelial neoplasia and cancers.6

## HPV testing and cervical cancer prevention:  how to “leverage” the association?

- Triage
- Screening?
- Management?
- Other??

## HPV Testing Methods

- Non-amplified tests commercially available in  U.S. since late 80s
- Virapap/Viratype (dot blot assay for HPV 6/11, 16/18, 31/33/35)
- HPV Profile (dot blot assay for HPV 6/11/42/43/44, and 16/18/31/33/35/45/51/52/56)
- Hybrid Capture-I (tube-based solution hybridization assay for same HPV types as Profile, sensitivity threshhold, _>_10pg/ml)
- Hybrid Capture-II (microtiter-based solution hybridization assay; same HPV types as HC-I plus 39/58/59/68)
- Amplified tests (polymerase chain reaction--PCR) using various primers (most commonly MY09/MYll) are more sensitive but not available in standardized formats

## Bethesda System

- WNL - within normal limits; includes reactive and reparative changes
- ASC (ASCUS)--atypical squamous cells (of undetermined significance); borderline category of equivocal lesions
- LSIL--Low-grade squamous intraepithelial lesions; combines condylomatous atypia with mild dysplasia (CIN 1)
- HSIL--High-grade squamous intraepithelial lesions; combines moderate/severe dysplasia (CIN 2 & 3)
- Squamous Cell Carcinoma

**Notes:**
In an effort to standardize the approach to the management of cervical disease, The Bethesda System (TBS) was created in 1991.  This classification system changed the terminology used by U.S. labs to report cervical cytological abnormalities.  TBS was developed to reflect the advanced understanding of cervical neoplasia and to ensure correlation with histological terminology. 

Today, there is ongoing debate about the effectiveness of TBS.  Controversy is especially focused on the ASCUS category, which has led to under-classification and over-classification, unnecessary colposcopic examinations, and legal implications.

## HPV Testing for Triage of Low-grade Pap Smear Abnormalities

**HPV Testing for Triage of Low-grade Pap Smear Abnormalities**

- Low-grade abnormalities (ASCUS, AGUS, LSIL) occur in 5-10% of the  50 million Pap smears  in the U.S. annually
- Most represent processes which will revert to normal without therapy, but minority of smears with ASCUS (5-15%) and LSIL (10-20%) will have high-grade lesions (CIN2/3) on biopsy (accounting for the majority of CIN2/3 lesions in some settings)
- Cost of conservative management (colposcopy/biopsy) $ 3-4 billion per year.
- New generation commercial HPV tests have high sensitivity (90%) and specificity similar to follow-up Pap smear (65%)
- Use unlikely to prevent cancer, but could save money; cost-effectiveness analyses will be critical

## Hybrid Capture-II Assay in Triage of 973 Women with ASCUS--Kaiser Study

## Kaiser Study:  Conclusions

- HPV testing of ASCUS is highly sensitive and moderately specific for HSIL
- Similar results for AGUS
- HPV testing for LSIL reported to be less specific and maybe useful only in women 40+
- Testing for HPV with either STM or PreservCyt had similar results
- Cost-effectiveness analysis pending

## ALTS STUDY DESIGN

- Multi-center, NCI clinical trial to determine optimal management of ASCUS, LSIL
- Patients (_>_18,_ _not pregnant or immunocompromised, no previous abative therapy) with Pap smear with ASCUS or LSIL  in past 6 mos, enrolled at four clinical centers (n=3488)
- Randomization to 1 of 3 arms
- Immediate colposcopy (colpo, biopsy, RX if CIN2/3)
- HPV Triage (to colpo if HPV+ or HSIL on Pap)
- Conservative management (to colpo if HSIL)
- Baseline evaluation with Thin-prep, HPV test by HC-II, cervicography (to result in colposcopy if “suspicious for cancer”); 6-monthly FU for 2 yrs with Thin-prep Pap and other study tests; to colpo if HSIL on Pap and RX if CIN2/3 on biopsy

## Performance of HPV testing vs. Repeat Pap for ASCUS

- 				ALTS study*	Kaiser study**
- 			   (n=2324; 11.4% CIN 2/3)	 (n=973; 6.6% CIN 2/3)
- 		       HPV test   Repeat Pap	   HPV test    Repeat Pap
- Sensitivity	   96%		85%		    89%	   76%
- Specificity	    na		na		    64%	   64%
- PPV		   20%		17%		    15%	   13%
- NPV		   99%		96%		     99%	    97%
- % referred	   56%		58%		     40%	    39%
- *Modified from Solomon D et al. JNCI 2001: 93: 293-9
- **Modified from Manos MM et al. JAMA 1999; 281: 1605-10.

## ALTS Study:  LSIL

- 642 women with LSIL, 532 (83%) HPV+
- HPV detection rate same for HC-2 and PCR in 210 samples (86%); some evidence of non-specificity in HC-2 assay
- High HPV+ rate limits utility of testing for triage of LSIL and this part of ALTS prematurely ended
- ALTS Group. JNCI 2000; 92: 397

## HPV Testing for Triage of ASCUS:  Issues to Consider

- Logistics:  requires liquid-based Paps or collection of sample transport media, so most appealing in settings using liquid-based testing
- Cost-effectiveness:  will vary depending on local factors (costs, CIN 2/3 and HPV prevalence, FU issues)
- Counseling:  issues of diagnosing an STD for patients and partners

## HPV Testing in Cervical Cancer Screening

- Possible use in primary screening under evaluation, either with/instead of Pap smear, especially in older women
- to enhance sensitivity combined with Pap smear (of greatest value to reduce screening interval)
- as single method in resource-poor settings (eg, potentially cheaper and easier to QA)
- as a self-collected sample for outreach settings or clinical situations where screening speculum exam not feasible
- Proposal to FDA to use in combination with liquid-based Pap smear (“DNA Pap”) pending

## HPV Screening Studies

**Notes:**
Now that we have established HPV testing provides increased sensitivity in combination with cervical cytology and decreases the risk of missing significant cervical neoplasia, I want to acknowledge some of the findings from recent trials that demonstrate HPV testing may soon have other applications.

In cervical cancer primary screening, HPV testing combined with the Pap smear is being evaluated.  Determining Pap smear intervals in women over 35 years of age is being studied based on the risk according to the type of HPV DNA.  The high positive predictive value of such combined testing in women over 35 years may allow screening efforts to focus on the women at greatest risk and support longer intervals between screening Pap tests.    

In underdeveloped countries, the application of HPV testing is also being considered for primary screening programs.  Such use of the test is most notable in areas lacking a cervical cytology screening program due to costs or other attributing barriers.

As you can see from the findings of the 3 trials listed on the slide, it has been proven that the sensitivity of HPV testing even as a primary screener can exceed that of the conventional Pap smear.  In these 3 trials, performed by investigators in France, England, and Costa Rica, HPV testing had an average sensitivity of 90%, compared to the 78% sensitivity of the Pap smear.  While HPV testing in the United States is only FDA approved as an adjunct to the Pap smear, these trials suggest HPV testing may have expanded uses in the near future.

## HPV testing:  applications  for the future?	 

- CIN1:  assessment of those who need RX vs. FU without RX?
- CIN 2/3:  post-RX assessment of those who need earlier FU or colposcopy?
- Anal cancer screening??
- Screening exposed sexual partners?? Screening for infection????

## Top 10 Questions Among Patients with Genital HPV Infection (information from ASHA HPV line)

- 1. How, when, or from whom did I get HPV?
- 2. Will HPV affect a pregnancy or a baby?
- 3. Can a person get or give HPV through oral sex or from hands?
- 4. How can I get tested for HPV?
- 5. Will I always have HPV?
- 6. How can I prevent giving or getting HPV?
- 7. Can partners re-infect each other?
- 8. Does HPV cause cervical cancer?
- 9. What should I tell my partner about HPV?
- 10. What are best RX options?

## The 20 minute HPV curbside consult generated by HPV testing

- 34 yo grad student with LSIL and equivocal (+, -, -, +) HPV test; wants individual typing, referred to public health by private GYN
- worries: cancer>STD>perinatal
- long conversations with fiancee who is fortunately very understanding
- has been all over web:  still confused
- “Has HPV testing helped your life?”:  (long pause)  “I think so...”
- “How?”:  (longer pause) “...made me more likely to take good care of my health (eg, exercise if exams)...”

## Triage of ASCUS in a Public Hospital Setting (Shlay et al. ObGyn 2000; (96:410) 

- 			  	 Patients < 30 	   Patients _> _30 				     (n=76) 	                (n=119)
- CIN 2-3 %	   	   10.5%		       5.9%
- % referred		    49%		       20%
- Sensitivity		    100%		       86%
- Specificity		     57%		        84%
- PPV			     22%		        25%
- NPV			    100%		        99%

## Triage of ASCUS in a Public Hospital Setting:  Cost Comparisons (Shlay et al. ObGyn 2000; (96:410)

**Strategy****	****	****	****Cost/CIN2-3 case detected**
- Colpo all				$4875
- HPV triage (STM vial)		$4809
- HPV triage (liquid Pap)		$4308
- Costs: 	Colpo/biopsy $375; HPV test $48.50; STM/storage $7.50; Liquid Pap extra $5.60

## Hybrid Capture-II Assay in Triage of ASCUS, by Age--Kaiser study

## Progress in Diagnostics

## Hybrid Capture® 2 HPV DNA Test Cervical SamplerTM

**Notes:**
Next, we have a visual of the contents of a Digene Cervical Sampler kit, which includes a cervical brush and specimen transport medium.

The specimen collection steps:
1)   Remove excess mucus from the cervical os and surrounding ectocervix using a cotton or dacron swab.  Discard this swab.
2)   Insert the brush 1-1.5 cm into the os of the cervix until the largest bristles    of the brush touch the ectocervix.
3)   Rotate the brush 3 full turns in a counterclockwise direction

DO NOT INSERT BRUSH COMPLETELY INTO THE CERVICAL CANAL

## Hybrid Capture-II Assay in the Triage of ASCUS Pap Smears:  Kaiser Study

- 46,000 routine Pap smears from four N. CA Kaiser centers
- Cervical sample with broom for conventional Pap smear; residual rinsed into PreservCyt fluid for HPV testing and Thin-Prep smear
- Cervical sample with conical brush into STM for HPV testing
- HPVtesting by Hybrid Capture-II assay (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58; cut-off _>_ 1.0 pg/ml);  89% agreement between STM and PreservCyt
- Study nurses attempted to contact women with ASCUS (n=1632) or greater:  1340 (82%) returned a mean of 67 days after initial Pap, 995 (61% enrolled), and 973 (60%) had follow-up Pap smear, biopsy, and HPV result.  CIN 2/3 detected in 64 (6.6%) and HPV in 384 (39.5%)
- Unpublished data. Manos et al

## Psychosocial Impact of HPV Infection (Clarke. Int J STD AIDS 1996; 7:197)

**Psychosocial Impact of HPV Infection** (Clarke. Int J STD AIDS 1996; 7:197)

## Epidemiology of Genital HPV Infection

- True incidence/prevalence difficult to define
- Incidence estimated to be 5 million/year (ASHA) and approximately 500,000 initial office visits for genital warts in U.S. annually (NDTI)
- HPV detectable in 20-50% young sexually active women, rates appear to be similar in men
- Infection chronic and of variable course, with an estimated prevalence of 24-40 million in U.S.

## Association of Cervical Cancer and STDs

- Positive association with:
- 	a)	Age of onset of sexual activity
- 	b)	Multiple sexual partners
- 	c)	Unstable marital relationships
- 	d)	Male sexual partners with penile cancer or 	with prior consorts with cervical cancer
- 	e)	Presence of STD’s: syphillis, gonorrhea, 	trichomonas, HSV-2, chlamydia, CMV,           	HPV

## HPV-Associated Squamous Intraepithelial Lesions (SIL)

- A synonym for dysplasia/CIN** **
- Categorized as
- low-grade SIL (LSIL)--HPV-associated changes, CIN 1
- high-grade SIL (HSIL)--CIN 2/3, CIS
- Can involve anus, vulva, vagina, penis as well as cervix

## Diagnosis of Cervical SIL

- Cytology
- non-invasive, inexpensive
- sensitivity low with single smear, enhanced with repeat
- liquid media approach may enhance sensitivity, but reduce specificity
- Colposcopy/biopsy
- recommended to follow-up HSIL
- management of ASCUS and LSIL controversial (e.g., colposcopy, repeat Pap, HPV testing)

## New Developments in HHPV testing	

- Availability:  HPV testing for ASCUS covered by over 30 BCBS plans in US (55 million members)

## Hybrid Capture® 2 HPV DNA Test Specimen Collection

- Cervical samplerTM specimen storage and transport
- Transport medium
- Digene specimen collection kit, or
- Cytyc preservcyt®  transport medium
- Cervical samples
- Storage and shipment up to 2 weeks at 2-30o C
- Remaining storage at 2-8o C for up to 1 week
- Long term storage at - 20o C for up to 3 months

**Notes:**
The shipment and storage requirements for cervical samples are shown here.

Some clinicians choose to collect an HPV Testing specimen at the time of colposcopy. The HPV sample should be collected prior to the application of acetic acid or any other type of solution. Cervical biopsies should be between 2-5mm in diameter and the  proper shipment and storage requirements listed on this slide should be followed.  

## HPV Prevalence by Referral Pap Smear and Age--Kaiser Study