Effects of Mycophenolate Mofetil, Methotrexate and Pimecrolimus on cdk4 and p16 in Erosive Oral Lichen Planus.

BACKGROUND: This study evaluated the effect of mycophenolate mofetil, methotrexate, and pimecrolimus on the expression of cdk4 and p16, important proteins implicated in hyperproliferation and arrest in the G1 phase, in oral lichen planus (OLP).
MATERIALS AND METHODS: In this randomized clinical trial, 60 patients were randomly assigned in three equal groups to receive either pimecrolimus cream, or mycophenolate mofetil or methotrexate, both supplemented with pimecrolimus. Pretreatment and post-treatment specimens were immunohistochemically stained for detecting cdk4 and p16.
RESULTS: A significant decrease in cdk4 cytoplasmic positivity was noted in all three treatment groups and was especially more significant in the MTX group (P < 0.0001) than in the other two groups (P < .001). However, a significant decrease in the cdk4 nuclear staining was noted with only systemic treatment groups of MMF (P < 0.05) and MTX (P < 0.01), both supplemented with pimecrolimus. No significant decrease in cytoplasmic p16 levels was noted in the MTX group while a significant decrease in cytoplasmic p16 levels was noted in the other two groups; however, no significant decrease in p16 nuclear staining was noted with any treatment.
CONCLUSION: By decreasing the expression of cdk4 and p16, pimecrolimus, methotrexate, and mycophenolate mofetil decrease the malignant potential of OLP lesions. However, methorexate can be a better alternative in cases showing high cdk4 expression. Copyright:

Introduction

Lichen planus (LP) is a mucocutaneous chronic inflammatory disease.[1] The oral form of LP is more common, recalcitrant, and chronic than cutaneous variety, causing significant impairment of quality of life due to associated pain and discomfort.[2]

It has been associated with a low but clinically relevant increased risk of oral squamous cell carcinoma (OSCC).[3] There are many studies showing cdk4 (cyclin-dependent kinases 4) and p16 changed levels being associated with OSCC.[4] A recent study has shown overexpression of cdk4 and p16 in oral lichen planus (OLP), supporting the concept of premalignancy.[5] Hence, these can be considered as a reliable indicator for therapeutic efficacy of various treatment modalities in overcoming the malignant potential and correlate with their clinical improvement efficacy.

Keratinocytes in OLP have been shown to express increased cdk4,[5] which by forming a complex with cyclin D helps in G1 to S phase progression, which is a mechanism to preserve epithelial architecture in response to lymphocytic infiltration.[567] With increased cdk4, keratinocytes, in turn, express p16, which forms complexes with cdk4 and arrests the cell in the G1 phase, which has been demonstrated to result in basal cell liquefactive degeneration.[46] These changes in the cell cycle have been proposed to contribute to cancer formation.[48]

In the present study, we assessed the effect of pimecrolimus ointment, mycophenolate mofetil (mmf), and methotrexate (both supplemented with pimecrolimus) on cdk4 and p16 expression. Pimecrolimus belongs to the ascomycin class of macrolactam immunosuppressives, which inhibits T cell activation by the calcineurin pathway and release of numerous inflammatory cytokines, hence preventing the cascade of inflammatory signals.[9] MMF is an immunosuppressive agent, which specifically and reversibly inhibits the proliferation of activated T cells. MTX is also an immunosuppressive agent that acts by inhibiting DNA and RNA synthesis and thus affects proliferating lymphocytes and malignant cells.[10]

Although corticosteroids have been the mainstay of treatment for OLP since long, there is no evidence of long-term remission being established. Further corticosteroids are also associated with their own side effects.[11]

Use of calcineurin inhibitors in cases that are resistant to corticosteroids has proven good results and they have a safer side-effect profile.[91213] FDA had approved pimecrolimus 1% cream for atopic dermatitis.[14] It has been found to be effective in the management of symptoms and erosions of OLP.[91516] Pimecrolimus has a similar mode of action to that of tacrolimus but is more selective with no effect on dendritic cells[14] and has been associated with better efficacy and side-effect profile than tacrolimus.[1213]

High success rates with MMF have been seen in recalcitrant cases of erosive LP and long-term remission. MMF has a safer side-effect profile compared to other immunosuppressive drugs such as corticosteroids, cyclosporine, and azathioprine.[17] Methotrexate as a supplement with topical steroids also has shown success in resolving erosive OLP cases recalcitrant to other common treatment modalities.[1819]

As of now, most of the studies[912131719202122] with these drugs in OLP have focused on the clinical aspects of the disease only. But broad-based interventions are required at the tissue level to assess the pathways involved in the pathogenesis of OLP and to evaluate the effects of these treatments on, since a long time debated malignant potential of OLP.

To date, there are few published studies regarding the efficacy of pimecrolimus[91213] and its comparison with other therapies in OLP[13], and fewer are available for methotrexate[1920] and mycophenolate in OLP[172122] and have a limited sample size. We have conducted a large-scale study with 60 OLP patients, of which 47 were erosive OLP (EOLP) cases. So, we intended to compare the various therapeutic modalities for EOLP and their effects on various pathogenic aspects of the disease (i.e. cdk4 and p16 markers) to manage the disease on a long-term basis and to further establish the association of these molecular markers with the disease pathogenesis. Thus we evaluated the effects of mycophenolate mofetil, methotrexate, and pimecrolimus on cdk4 and p16 in EOLP.

Materials and Methods

The proposed study had been approved by the ethical committee of Maulana Azad Institute of Dental Sciences (MAIDS); and was conducted in the department of Oral Medicine and Radiology, MAIDS, New Delhi, in association with the Department of General Pathology and Department of Dermatology, Maulana Azad Medical College along with Lok Nayak Jaiprakash Hospital, New Delhi. The study was approved by ethical committee of institute as well as accepted for thesis grant award by ICMR and registered under ctri (Regn no. submitted), the clinical trial registry.

This study was a prospective, double-blind, randomized clinical trial. Patients were selected from the outpatient department of MAIDS, and those with histologically proven EOLP were included in the study. Patients older than 19 years with histologically proven EOLP cases were included in the study. Patients were excluded from the study if they had lichenoid contact reaction; history of therapy for LP or drugs associated with lichenoid reaction within past 8 weeks; any malignant or viral involvement intraorally, pregnant as well as nursing women, patients whose complete follow up was lacking, and patients not willing to participate in the study were also excluded.

Patient selection was being done based on the characteristic clinical features of OLP (bilateral reticular striae with or without atrophic erosive areas). Informed consent regarding the procedures being performed was taken from all the patients selected. Pretreatment biopsy from the representative area without loss of epithelium for confirmation of the diagnosis with the characteristic findings on histopathological examination of subepithelial dense lymphocytic infiltrate, basilar vacuolization with apoptosis leading to homogeneous eosinophilic civette bodies formation.

In this study, 42 erosive OLP patients were enrolled in the study group. The mean age at presentation was 46.95 ± 11.96 years. The mean duration since when the lesions have been present was 21.293 ± 20.77 months. The male to female ratio was 8:7. All the oral lichen planus cases were randomized based on treatment into three groups, each with 14 subjects. Patients intended to be subjected to MMF and MTX underwent baseline serum investigations for liver function and kidney function profiling. Complete blood count with hemoglobin and platelet counts were evaluated every week. Methotrexate was used at lower doses in patients with an abnormal serological profile, or blood counts and similar caution was taken for MMF in case of abnormal liver profile. These groups were subjected to pimecrolimus, mycophenlate mofetil, and methotrexate along with pimecrolimus. All patients were advised to stop any other drug treatment if taking for at least 8 weeks. Pimecrolimus was prescribed as 1% (w/w) topical cream formulation, four times/day, for 9 months, after meals and the patient was advised not to drink and eat for ½ hour after topical application. MMF was prescribed as oral 1 g daily in two equally divided doses for 6 months and the dose was titrated in each case depending upon the clinical response and 0.5 g as a once-daily dosage for the next 3 months and patient was advised to take the drug empty stomach (1 hour before food or 2 hours after meals). MTX was prescribed as a 7.5 mg weekly dosage for 7–9 months and the dose was titrated depending upon the clinical response. The patient was advised to take the drug empty stomach and to take it with food if gastrointestinal disturbances occurred. The patient was advised to avoid milk-rich products along with the drug. Post-treatment biopsy was done for assessment of cdk4 and p16 expression. Assessment was made for unwanted side effects such as burning or taste malfunction and any abnormal alteration in the appearance of mucosa. Assessment for abnormal vital signs and systemic side effects was done. Serological evaluation of liver function and kidney function was done biweekly, along with weekly evaluation of platelet count and hemoglobin level.

Immunohistochemical Analysis (Cdk4 and p16 expression):

Tissue was formalin-fixed and embedded into paraffin blocks. Sections of 4-μm thickness were cut from the blocks and mounted on albumin-coated glass slides. Tissue was deparaffinized and rehydrated. Endogeneous peroxidize activity was blocked with incubation in 3% H2O2 for 30 minutes. Sections were heated in a microwave at 20°C for 30 minutes with intermittent cooling after every 5 minutes in 10-mM citrate buffer for antigen retrieval. Thorough washing of sections was done with phosphate-buffered saline. Sections were treated with primary antibody (Santa Cruz biotechnology; SC260) and kept overnight at 4°C. Thereafter, sections were washed in phosphate buffer saline (PBS; pH: 7.0–7.5) supplemented with 0.05% of Tween-20 (buffer A) for 3 × 5 minutes. Secondary antibody was applied and left for 15 minutes. Tertiary antibody was applied and left for 15 minutes. Thoroughly washed in PBS for 3 × 5 minutes and after cleaning the surrounding area of section HRP–Peroxide–DAB was applied. Chromogen (DAB) was applied and left for 10 minutes. Sections washed in water for 2 × 5 minutes. Counterstaining was done in hematoxylin for 5 minutes and followed by washing in distilled water for 3 × 2 minutes. Slide mounted for observation.

For cdk4 expression, cells showing golden brown staining in the cytoplasm or/and nucleus were considered as positive. The percentage of positive versus negative cells was counted. Presence of more than 5% positive cells was considered significant.

For p16 expression cells showing golden brown staining in cytoplasm or/and nucleus were considered positive. Percentage of positive versus negative cells was counted. Presence of more than 5% positive cells was considered significant. Comparison of expression of cdk4 and p16 markers pre and post treatment (treatment groups- pre- vs. post-treatment and among various treatment groups) has been done. Comparison of efficacy of each drug at the molecular level was done and compared with the other drugs.

Statistical Analysis Plan

Because some variables in the study groups were not normally distributed; nonparametric statistics were applied. Only two-sided probability values less than 0.05 were considered as significant. The data were entered in software (SPSS 13 for windows, SPSS Inc, Chicago, III). Continuous data were reported as mean ± SD. A Krusal–Wallis test was carried out for multiple comparisons, while Mann–Whitney tests were performed to detect a difference between each pair of groups in rank sign. Intragroup comparison of various parameters at different points of time (Pre and post treatment difference of various markers) was done by using Wilcoxon signed-rank test.

Results

This study demonstrated a significantly high expression of cdk4 in the cytoplasm (P < 0.05) and p16 in the cytoplasm (P = 0.011) as well as nuclei (P < 0.001) in OLP compared to normal mucosa, contrary to a previous study where cdk4 showed significantly higher expression in nuclei only in lesional tissue of OLP.[5] Similarly, cdk4 and p16 expression were significantly higher in OSCC in both cytoplasm and nuclei (P < 0.0001), a finding supported by the previous studies.[48]

A significant decrease in cdk4 cytoplasmic positivity was noted in all three treatment groups and was especially more significant with MTX group (P < 0.0001) than the other two groups (P < 0.001) [Wilcoxon signed-rank test]. While a significant decrease in the cdk4 nuclear staining was noted with only systemic treatment groups of MMF (P < 0.05) and MTX (P < 0.01), both supplemented with pimecrolimus. However, no significant difference was seen between groups with respect to a decrease in the percentage number of cells having cytoplasmic staining [Kruskal–Wallis test].

A significant decrease in cdk4 count post-treatment was seen in 65% of the 80% cases showing increased cdk4 expression and remained the same in 13% and increased in only 3.3% of the cases where a significant increase was seen in cdk4 while p16 decreased in one case.

No significant decrease in P16 levels was noted in the MTX group while a significant decrease in cytoplasmic p16 levels was noted in the other two groups, although no significant decrease in p16 nuclear staining was noted with any treatment.

Regarding safety profile, a significant decrease in platelet count was seen in 40% of the patients in the MTX group at 3–4th month period, requiring tapering of the drug for a period of about 15 days–1 month based upon the restoration of count although, there was no significant change in liver and kidney function profile and there was no gastrointestinal upset during use. Mycophenolate was well tolerated in all the cases and there was no complaint of nausea, vomiting, and diarrhea, and kidney and liver profiles were stable. Transient burning sensation was noted with pimecrolimus after application for 15–20 days after beginning treatment but it was not a hindrance to patient compliance and treatment.

Discussion

This study demonstrated a significantly high expression of cdk4 in the cytoplasm (P < 0.05) and p16 in the cytoplasm (P = 0.011) as well as nuclei (P < 0.001) in OLP compared to normal mucosa, contrary to a previous study where cdk4 showed significantly higher expression in nuclei only, in lesional tissue of OLP.[5] Similarly, cdk4 and p16 expression was significantly higher in OSCC in both cytoplasm and nuclei (P < .0001), a finding supported by the previous studies.[48] Similar findings were reported by our group in previous studies.[23]

It is accepted that the malignant transformation rate in OLP is lower than that of oral leukoplakia where overexpression of cdk4 occurs but no changes in p16 are observed.[824] In this context, it can be postulated that loss of p16 expression may be an initial sign of malignant transformation in OLP with high cdk4 expression. Thus, the combination of cdk4 and p16 may be a useful tool in predicting malignant transformation in OLP.

The percentage of cytoplasmic cdk4 positive cells decreased significantly after treatment in all three groups, and the change was especially more significant in the MTX group (P = 0.0001) than in the other two groups (P = 0.001), and the percentage of nuclear cdk4 positive cells decreased significantly in only MTX (P = 0.011) and MMF (P = 0.027) groups [Figure 1, Tables 1 and 2]. These findings may suggest targeting the cell cycle molecular pathways involved in OLP by pimecrolimus, MTX, and MMF; especially by MTX and that cdk4 may be a useful marker in assessing the therapeutic potential of a drug, disease status, and malignant potential in OLP. Although a significant decrease in cdk4 expression was noted in all three groups, while a significant decrease in cytoplasmic p16 expression was noted in the pimecrolimus (P = 0.003) and MMF (P = 0.024) group only, and in the MTX group, change in p16 expression was not significant (P = 0.307), pointing toward favorable efficacy outcome of MTX in terms of controlling the malignant potential [Table 2].

Figure 1

Comparison of pretreatment to post-treatment mean difference of cytoplasmic and nuclear cdk4 and p16 in various treatment groups. T1-Pimecrolimus, T2-Mycophenolate Mofetil with Pimecrolimus, T3-Methotrexate with Pimecrolimus

Table 1

Cdk4 and P16 Staining pre and post treatment

Pre/post cdk4 and p16	Pattern of staining	Treatment Group
		T1	T2	T3
Pre cyt.cdk4	No. of cases % cells stained	17 (85%)	15 (75%)	16 (80%)
		30.10±26.52	14.40±14.41	14.35±20.53
		20 (0-70)	11 (0-42)	7 (0-80)
Post cyt.cdk4	No. of cases % cells stained	6 (30%)	8 (40%)	6 (30%)
		4.80±10.49	1.15±1.78	0.65±1.04
		0.00 (0-40)	0.00 (0-6)	0.00 (0-3)
Pre Nuc.cdk4	No. of cases % cells stained	4 (20%)	6 (30%)	9 (45%)
		1.60±1.40	4.10±10.34	3.55±6.58
		0.00 (0-20)	0.00 (0-42)	0.00 (0-28)
Post Nuc.cdk4	No. of cases % cells stained	1 (5%)	1 (5%)	4 (20%)
		0.1±0.44	0.10±0.44	0.45±0.94
		0.00 (0-2)	0.00 (0-2)	0.00 (0-3)
Pre cyt.p16	No. of cases % cells stained	16 (80%)	14 (70%)	11 (55%)
		30.50±29.79	12.61±16.43	4.85±9.50
		21 (0-90)	5 (0-50)	2.00 (0-42)
Post cyt.p16	No. of cases % cells stained	13 (65%)	12 (60%)	5 (25%)
		12.25±18.17	5.40±5.47	1.95±3.62
		5 (0-80)	5 (0-16)	0.00 (0-10)
Pre Nuc.p16	No. of cases % cells stained	4 (20%)	3 (15%)	4 (20%)
		7.55±19.29	3.35±11.34	0.80±1.73
		0.00 (0-65)	0.00 (0-50)	0.00 (0-5)
Post Nuc.p16	No. of cases % cells stained	1 (5%)	0 (0%)	2 (10%)
		0.25±1.11	0.00±0.00	0.65±2.05
		0.00 (0-5)	0.00 (0)	0.00 (0-8)

T1 - topical Pimecrolimus, T2 - Patients on oral Mycophenolate mofetil therapy alongwith topical pimecrolimus, T3 - Patients on oral Methotrexate therapy alongwith topical pimecrolimus.

Table 2

Difference between pre and post-treatment CDK4 and p16

Treatment Group	cdk4 cytoplasmic % stained cells (Pre-post)	cdk4 Nuclear % stained cells (Pre-post)	p16 cytoplasmic % stained cells (Pre-post)	p16 Nuclear % stained cells (Pre-post)
T1	25.30±23.75112	1.5±4.54799	18.25±22.10233	7.30±19.42895
	20 (­10-68) (P=0.001)*	0.00 (0-20) (P=0.068)	12 (-14-60) (P=0.003)*	0.00(-5-65) (P=0.080)
T2	13.25±14.75011	4.00±10.34154	8.45±15.6994	3.35±11.34286
	11(-4-42) (P=0.001)*	0.00 (0-42) (P=0.027)*	3.50(-7-50) (P=0.024)*	41.75(-181-819.78) (P=0.109)
T3	13.70±20.7367	3.10±6.45552	2.90±10.1872	0.15±2.5808
	4.50 (0-80) (P=0.0001)*	0.00 (0-28) (P=0.011)*	0.00(-10-42) (P=0.307)	00(-6-5) (P=0.891)

*Statistically significant difference within group pre to post treatment (By Wilcoxon signed rank test).

MMF was well tolerated in all the cases and it was in accordance with previous studies.[1721222526] Thus, MMF can be substituted in place of corticosteroids as a safe immunosuppressant.

MTX was well tolerated by all patients, a finding supported by other studies[1819]. However, contrary findings were seen in other studies.[27] It can be used safely in hypertensive, diabetic, and old patients in comparison to corticosteroids[27], and the same was found in our study.

So, comparison of various therapeutic modalities for OLP and their effects on various pathogenic aspects of the disease (cdk4 and p16 markers) reveals that there is a marked possibility for these markers to predict the malignant potential of these lesions and in the management of recalcitrant chronic OLP cases based on the use of these markers in deciding the best possible therapy for a case.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.