1. Downgrading -IN 2, Diagnoses and Predicting Higher-grade Lesions
Dr.Soheila Sarmadi
Professor of Pathology
Tehran university of Medical Sciences

2. Cervical cancer and its precursor lesions, induced by persistent infection with human papillomavirus (HPV), represent a significant public health problem.
Low-grade squamous intraepithelial lesion/cervical intraepithelial neoplasia grade 1 (LSIL/CIN1) are usually followed up without treatment because around 80% regress spontaneously and only 10% progress to high-grade squamous intraepithelial lesion (HSIL) or cervical cancer.

3. In contrast, CIN grades 2 and 3 are collectively classified as HSIL and excisional treatment is the standard way to manage these lesions.
Although HSIL/CIN3 has been shown to carry a higher risk of subsequent cancer, HSIL/CIN2 is not so obviously precancerous as the spontaneous regression and progression rates are around 40 and 20%, respectively.
Unfortunately HSIL/CIN2 commonly occurs in women who desire preservation of fertility and their overtreatment may increase the risk of subsequent obstetric complications.

4. In 2012, the College of American Pathologists and American Society for Colposcopy and Cervical Pathology published the “LAST” recommendations for histopathology reporting of human papilloma virus–related squamous lesions of the lower anogenital tract, including the use of a 2-tier nomenclature (low-grade squamous intraepithelial lesion (LSIL)/human papilloma virus changes and high-grade squamous intraepithelial lesion (HSIL)

5. More recently, it has been recognized that the biology of HPV infection is characterized by 2 dichotomous pathways rather than a stepwise progression from grade 1 to grade 3 preneoplasia. Specifically, HPV causes either transient productive infection of the squamous epithelium or persistent infection with potential for progression (precancer).

6. In addition, it has been demonstrated that the reproducibility of the intermediate grade (-IN 2) category is poor, and some have questioned whether this category represents a true biological entity.
The authors of LAST recommended switching to a 2-tiered reporting system of low-grade squamous intraepithelial lesion (LSIL; representing transient productive infection) and high-grade squamous intraepithelial lesion (HSIL; representing precancer).

7. The distinction between grade 1 lesions (-IN 1) and higher-grade lesions (-IN 2/3) is most crucial for management decisions, as current guidelines recommend observation for -IN 1 and excision for -IN 3.
The management of HSIL/CIN2 lesions is still a dilemma for a gynecologist. Several reports demonstrating HSIL/CIN2 spontaneous regression in young women have been published during the past decades , but predictors of this are poorly understood.

8. The LAST project recommended use of the biomarker p16ink4.
Strong and diffuse immunohistochemical (IHC) staining by antibodies to p16 is significantly associated with high risk HPV and lesion grade, with the majority of -IN 2 (75.5% to 100%) and -IN 3 (100%) lesions being associated with this staining pattern.
The use of p16 as an adjunct to morphologic diagnosis significantly improves interobserver diagnostic agreement.

9. Thus, p16 immunohistochemical staining may have an important role as an adjunctive stain to improve the reliability of HSIL/CIN2 histopathological diagnosis and could be useful for planning the clinical management of patients with HSIL/CIN2.

10. If the pathologist is entertaining an H&E morphologic interpretation of -IN 2 (under the old terminology , which is a biologically equivocal lesion falling between the morphologic changes of HPV infection [low-grade lesion] and precancer).
As an adjunct to morphologic assessment for biopsy specimens interpreted as LSIL (-IN 1) or less that are at high risk for missed high-grade disease, which is defined as a prior cytologic interpretation of HSIL, ASC-H, ASC-US/HPV 16+,or atypical glandular cells (not otherwise specified).

11. p16 is a commonly used IHC marker; however, its interpretation is unique in the context of HPV-related lower anogenital lesions. Both quality and quantity of p16 immunoreactivity affect its specificity in predicting high-risk HPV and HSIL outcomes.
One cannot simply designate it as positive or negative; rather, pathologists must consider multiple parameters such as staining intensity, extent, continuity, and location.
When using p16 to triage CIN 2 lesions, pathologists need to be aware of the ramifications of an ambiguous p16 result.

12. In ideal circumstances, HSILs demonstrate strong positive p16 staining in a block pattern defined as nuclear ± cytoplasmic staining that extends from basal layers upward at least one-third of the epithelial thickness as well as laterally over a significant distance.
It is worth noting that exceptions to these observations occur frequently in daily practice.
Multiple technical variables in the IHC protocol among diagnostic laboratories (antibody clone, antigen retrieval,etc) significantly influence test results .
As for interpretation, the threshold for p16 positivity is still imprecise and needs to be standardized. Some authors consider 5% staining of lesional cells as positive, whereas others require at least 25%or as much as 50%. Additionally, variations in staining distribution and intensity pose great interpretive challenges

14. Study Design and Patient Selection
This prospective observational study included consecutive women of first-time HSIL/CIN2 colposcopy directed biopsy diagnosis from December 2011 to October 2013 who attending the Cervical Pathology Unit of the Hospital del Mar, Barcelona.
All HSIL/CIN2 patients were evaluated by gynecologic colposcopists who proposed conservative management in the framework of this study versus immediate excisional treatment according to local guidelines.

15. Inclusion criteria:
(1) had an exocervical histological diagnosis of HSIL/CIN2
(2)the entire squamocolumnar junction of the cervix was visible
(3) could be followed-up every 4 months during 1 year
(4) signed the consent form.
Patients with unsatisfactory colposcopy, endocervical histological diagnosis of HSIL/CIN2, previous cervical treatment, or immunosuppressive treatment/immunodeficiency disease were excluded.

16. The gynecologic colposcopists followed patients every 4 months with cervical cytology and colposcopy.
A biopsy was performed when colposcopy showed a worsening of suspicious lesion in relation to previous examination.
When the biopsy revealed HSIL/CIN2 or less, patients continued on surveillance; conversely when the biopsy revealed HSIL/CIN3, patients underwent immediate treatment using loop electrosurgical excision procedure.

17. At 12 months of follow-up, all women who still showed abnormal liquid-based cytology or colposcopy abnormalities, a colposcopy-directed biopsy was taken from the most severe and suspicious areas.
The final outcome of HSIL/CIN2 was classified as complete regression when two consecutive cervical cytology, colposcopy and biopsy results showed no lesion at 12 months of follow-up; partial regression if final biopsy showed LSIL/CIN1; persistence when biopsy showed HSIL/CIN2; or progression if a HSIL/CIN3 biopsy was detected at any time during the follow-up.

18. Among the 96 patients included, 81 (84%) were p16 positive and 15 (16%) were p16 negative.
All 15 patients with p16 negative status regressed, of which 8 presented complete regression and 7 partial regression (P=0.008).
All 35 patients who had persistence or progression of HSIL/CIN2 lesion (HSIL/CIN2+) were p16 positive in the initial biopsy.
Nevertheless, 46 of 81 (57%) patients positive for p16 regressed.

19. The results of the present study are in agreement with most of the published studies, showing 64% of HSIL/CIN2 spontaneous regression rate during the first 12 months of follow-up in patients with satisfactory colposcopy and not immunosuppressed , independent of their age.
Therefore, a conservative approach could be considered in these cases to reduce unnecessary cone excision.

20. This is the largest prospective study that evaluates p16 status as a predictor of HSIL/CIN2 behavior.
The key findings from this study were that the regression rates are highest in the setting of p16 INK4a negative HSIL/CIN2 biopsies, and that it supports the safety of conservative management of HSIL/CIN2 in selected patients.

21. Studies on p16 and intermediate-grade lesion behavior are few and have demonstrated varied results.
The current study demonstrates that adherence to these recommendations will also result in the downgrading of a significant proportion of cases (approximately one third), which would otherwise have been diagnosed as HSIL and thus excised.
However, on the basis of the significantly higher proportion of definitive HSIL (-IN 3) on subsequent specimens following p16-positive biopsies compared with p16-negative biopsies , this downgrading seems appropriate and may prevent the overtreatment of these patients.

22. It should be pointed out that, although HSIL/CIN2 lesions positive for p16 showed a significantly higher tendency to persist or progress than p16 negative, 57% of them regressed at first 12 months of follow-up.
Given that, if we considered all HSIL/CIN2 lesions positive for p16 as high-grade lesions, we will overtreat 57% of HSIL/CIN2 that could regress spontaneously.
This suggests that further biomarkers and other factors may be required to distinguish those HSIL/CIN2 p16 positive lesions that are more likely to progress from those will regress.

23. Conclusion
All patients with HSIL/CIN2 negative for p16 regressed in the first 12 months of follow-up.
Conversely, HSIL/CIN2 lesions positive for p16 have higher tendency to persist or progress to HSIL/CIN3, and might require closer follow-up. Thus, p16 immunohistochemical staining may have an important role as an adjunctive stain to improve the reliability of HSIL/CIN2 histopathological diagnosis and could be useful for planning the clinical management of patients with HSIL/CIN2.
This study supports the safety of conservative management of HSIL/CIN2 in select patients; however, further larger follow-up studies are encouraged to identify other biomarkers to predict the evolution of HSIL/CIN2 patients, in particular looking for predictors of HSIL/CIN2 patients with p16 positive staining.

24. Some authors that evaluated the prognostic value of p16 in low-grade lesions.
In these studies, p16 positive was associated with significantly higher rates of progression and, in contrast, those low-grade lesions negative for p16 had a higher tendency to regress.
Thus, at present, although p16 is a promising biomarker for predicting the outcome of low-grade lesions, there is insufficient evidence to recommend clinical management of low-grade lesions based on p16 staining.