Leukoplakia of oral cavity and urinary bladder are premalignant to squamous carcinoma

Abstract

A critical work published by Panchannavar et al on the close link between oral leukoplakia (OLK) and oral squamous cell carcinoma (OSCC), the expression of epithelial mesenchymal transition proteins ZEB1 and E-cadherin would predict the progression of OLK to OSCC. In connection with similar clinical status, there are cumulative data that the urinary bladder leukoplakia (Bladder-LK) progress to invasive bladder squamous cell carcinoma (SCC) which does not response to local therapy that indicates immediate cystectomy, these findings were established with multiple publications with clinical follow-up that showed the progression of leukoplakia to SCC in both oral cavity and urinary bladder. Oral leukoplakia, oral SCC, Bladder-LK, urothelial carcinoma (UC) of the bladder, squamous differentiation of UC, and bladder-SCC showed expression of programmed cell death-ligand 1 (PD-L1). In recent years, immune checkpoint inhibitors immunotherapy for malignant tumors became a promising cancer immunotherapy. However, the role of PD-L1 inhibitors in OLK OSCC is controversial. Objectives are to highlight the rationale for prospective treatment of OLK and OSCC with PD-L1 inhibitors immunotherapy.

Key Words: Leukoplakia; Squamous cell carcinoma; Programmed cell death-ligand 1; Immune checkpoint inhibitors; Oral cancer; Bladder cancer

Core Tip: Oral leukoplakia and urinary bladder leukoplakia are premalignant lesions that progress to squamous cell carcinoma (SCC), both lesions and their corresponding SCC express programmed cell death-ligand 1 (PD-L1) indicating response to PD-L1 inhibitors. A recent approach with consideration of recent finding of expression of PD-L1, genetic analysis, microenvironment landscape, recruited clinical trials, and animal model studies would advocate possible treatment of oral leukoplakia with PD-L1 inhibitors aiming at cure and prevent progression to SCC.

TO THE EDITOR

An important work published by Panchannavar et al[1] on the close link between oral leukoplakia (OLK) and oral squamous cell carcinoma (OSCC) entitled “immunohistochemical expression of epithelial mesenchymal transition (EMT) proteins ZEB1 and E-cadherin in OLK and oral squamous cell carcinoma”. They studied expression of EMT proteins ZEB1 and E-cadherin in 60 patients with OLK/epithelial dysplasia (OED) (n = 30), and OSCC (n = 30). Immunohistochemistry with anti-ZEB1 antibody, and anti-E-cadherin antibody was performed on tissue samples. Results showed reduction in the expression E-cadherin and altered localization were noted from OLK/OED to OSCC. Loss of E-cadherin and up-regulation of ZEB1 from OED to OSCC, predisposes to induction of EMT, the study concluded that the EMT phenomenon in oral premalignant and malignant lesions suggests that EMT is an early change and can predispose carcinogenesis. There is cumulative data that urinary bladder leukoplakia (Bladder-LK) progresses to invasive bladder squamous cell carcinoma (Bladder-SCC) which does not response to local therapy that indicates immediate cystectomy, these findings were established with multiple publications with clinical follow-up that showed the progression of leukoplakia to SCC in both oral cavity and urinary bladder.

Oral leukoplakia, OSCC, Bladder-LK, squamous differentiation of urothelial carcinoma (SqD-UC), and bladder SCC showed expression of programmed cell death-ligand 1 (PD-L1). In recent years, immune checkpoint inhibitors (ICIs) immunotherapy for malignant tumors became a promising cancer immunotherapy. Clinical studies showed favorable response with PD-L1 inhibitors in treatment of bladder SCC, and SqD-UC, with similar results to urothelial carcinoma (UC). Experimental studies showed good response of OLK to PD-L1 inhibitors. However, the role of PD-L1 inhibitors in OLK and OSCC is controversial. The results of treatment of SqD-UC squamous bladder lesions which have similarity in cellular level to oral squamous lesions could initiate the need for clinical trials on immunotherapy of OLK and OSCC. Objectives are to highlight the rationale for prospective treatment of OLK and OSCC with PD-L1.

Objectives of the study are to highlight the rationale for prospective treatment of OLK and OSCC with ICIs immunotherapy which is based on the confirmed positive expression of PD-L1 in these tumors. These findings are correlated with paired data with Bladder-LK, SqD-UC, and pure Bladder-SCC.

ORAL LEUKOPLAKIA AND SQUAMOUS CELL CARCINOMA

In recent years, PD-L1 inhibitors are implemented in cancer immunotherapy with promising results. However, the role of PD-L1 in OSCC and OLK is highly considered controversial. To reveal the expressions of PD-L1 and CD8 in OLK and OSCC an investigation was carried on 41 OSCC, and 21 OLK, and 25 normal mucosa samples, immunohistochemistry with performed using mono-clonal antibody. The expression density of PD-L1 was correlated with CD8 expression, additionally the PD-L1 protein was detected in 97.6% of cases OSCC, and in 61.9% of OLK. PD-L1 positivity was significantly associated with CD8 density. Cases of OSCC showed that PD-L1 expression was positively correlated with the tumor grade, lymph node metastases, and recurrence. Additionally PD-L1 expression was upregulation in OLK. The upregulation of PD-L1, and CD8+ tumor-infiltrating lymphocytes are linked to disease progress in OLK and OSCC[2].

CD8+ and CD163+ cells and PD-L1 immunoexpression were highly expressed in OLK and oral OSCC[3]. The immune microenvironment in OLK and OSCC is influenced by expression of programmed death 1 (PD-1)/PD-L1. PD-L1 expression within the tumor microenvironment of OSCC and OLK has a high clinical and biological significance regarding tumor progression[4,5].

Recently it was identified that immune cells would contribute to transformation of OLK to OSCC. The T regulatory cells, CD8+ effector T cells, and immune checkpoint molecules (PD-1 and PD-L1) were identified to be expressed across oral dysplastic, OLK, and OSCC, detection was carried out by immunohistochemistry in tissue sections. The staining intensity correlated with higher grade dysplasia, SCC, and disease progression. The presence of immune checkpoint markers PD-1 and PD-L1, in addition to CD8+ effector T cells were also associated with oral cancer progression. These findings indicate targeted interventions coupled with ICI[6].

With the introduction of artificial intelligence (AI) technology, it is becoming feasible for to visualize and identify OSCC in optical coherence tomography images from the extracted tumor tissues to differentiate the normal epithelium, dysplasia, OLK, and OSCC, these AI data are based on the features extracted from the oral computer tomography (OCT) images. The application of OCT imaging in combination with a deep learning model would be a helpful tool for identification of OSCC, OLK, and normal areas for proper diagnosis, local therapy, and during surgical excision[7].

The National Comprehensive Cancer Network guidelines on the treatment for OSCC are surgery, followed by chemoradiation. Despite advancements in multimodality treatments for OSCC, the clinical outcomes are poor. Clinical evidences pointed out that failure of treatment and cancer recurrence could be caused by the cancer cells involved in DNA damage repair, EMT, and tumor immune microenvironment[8].

PD-1/PD-L1 inhibitors nivolumab and pembrolizumab immune chick-point inhibitors are used for treating OSCC with promising results, this treatment strategy would prevent transformation of OLK cells into SCC[9].

Utilizing gene expression profiles as well as immunostaining and semi-quantitative evaluation of distinctive cell population, it was detailed that immunological changes are detected in early stages of oral tumorigenesis. Changes were distinguished with M2 macrophages. All immune cell populaces (T- and B cells, macrophages) are increasing early amid mucosal change. Stromal gene expression changes that were distributed across the diverse stages of tumorigenesis[10].

Xu et al[11] investigated the immune signature of oral dysplasia and explored the results of treatment with an immunotherapy with vidutolimod + immune checkpoint blockades to determine whether it can induce regression of oral dysplasia or prevent progression to SCC via activating the toll-like receptors 9, they investigated vidutolimod which activate dendretic cells. A murine model was used to induce oral dysplasia and their progressions aiming at investigate the response to in-situ injection of immunotherapeutic during the premalignant phase. The treatment showed that the sera of the mice excremental models there are elevated levels of interferon-gamma, interleukin-12, and tumour necrosis factor alpha, as well as the draining lymph nodes of the oral cavity exhibited an increased expression of proliferating T cells and plasmacytoid dendritic cells. The oral lesion revealed decreased expression of Ki-67. The findings indicated the immune-boosting effect of immunotherapy with vidutolimod combined with ICIs, implicating that immunotherapy with ICIs regime may prevent lesion progression.

Wang et al[12] used the 4-nitroquinoline-1-oxide mouse model of oral carcinogenesis to evaluate the hypothesis that PD-1 blockade may control the progression of oral premalignant lesions (OPL). Mice received blocking antibody for PD-1 inhibitors. They found that anti-PD-1 treatment altogether diminished the number of oral lesions that developed in these mice and stopped tumor progression. Low-grade dysplastic lesions reacted to PD-1 inhibitor with a noteworthy increment within the enrollment of CD8+ and CD4+ T cells in the microenvironment. The amassing of cytotoxic T-lymphocyte antigen 4 (CTLA-4) and T cells in their microenvironment. Strikingly, PD-1 inhibitors was associated with induction of type III interferon-λ, STAT1 activation and the generation of the T-cell effector granzyme B in infiltrating cells, and through the induction of apoptosis within the epithelial cells of the oral lesion.

Monteiro et al[13] proved that PD-1 inhibitors diminished the incidence of oral dysplasia in a carcinogen-induced murine model. They study to characterize the immunological landscape of oral dysplasia and OLK. The study revealed the impact of targeting the PD-1, CTLA-4, CD40, or OX40 pathways on the development of oral dysplasia in the 4-nitroquinoline 1-oxide mouse model. The immune pathways were revealed using PD-L1-knockout mice. After intervention. The effect of focusing on the PD-1, CTLA-4, CD40, or OX40 pathways on the advancement of OPLs and verbal carcinomas within the 4-nitroquinoline 1-oxide show. Targeting PD-L1 inhibitors, reduced progression of OPLs to OSCC. Distinct patterns of immune system modulation were observed for blockade of the PD-1/PD-L1.

The retrieved data recapitulated that discrete approaches may be valuable in the process of the development of OSCC. PD-1/PD-L1 blockades were preventing progression into OSCC in a murine model. A distinguishable pattern of immune modulation was proved when PD-1/PD-L1 pathways were targeted.

Ries et al[14] demonstrated that high levels of PD-1 and PD-L1 are related to judge the potential of transformation in OLK, to OSCC. The increased levels of PD-L1 levels establish an immunosuppressive microenvironment that could favor immune escapeand contribute to progression to malignant transformation. These findings indicate that ICI could abolish tumor development in OLK and may serve as efficient therapeutic strategy. The introduction of ICI to head and neck squamous cell carcinoma (HNSCC), offering improved outcomes dependent on the tumor and the immune cell proportion of PD-L1. Multiple trials have subsequently explored immunotherapies in combination with other modalities in oral premalignant lesions, OSCC, and HNSCC. Immunotherapy regimens may be personalized by tumor biomarkers, including PD-L1 content. Gaps remain for protocols using immunotherapy to reverse OLK. A phase II nonrandomized controlled trial, using the ICI nivolumab, showed a 2 years cancer-free survival of 73%, although larger sample size and longer follow-up trials are required[15-17].

A phase II nonrandomized controlled trial (NCT03692325) assessed the safety and efficacy of nivolumab in 33 patients with high-risk OLK, defined as multifocal, contiguous, or a single lesion ≥ 4 cm. The results showed that at follow-up, the 2 years cancer-free survival was 73%, these data is consistent with potential activity of anti-PD-1 inhibitors; however, follow-up was for a median of 21.1 (5.4 to 43.6) months[18].

Ongoing immunopreventive studies include the phase II IMPEDE (NCT04504552) using avelumab on patients with high-risk OPL, a phase I trial (NCT05327270) evaluating the response of OPLs to intralesional nivolumab, a phase II trial (NCT03603223) studying the response of leukoplakia to intravenous pembrolizumab, and a phase II trial (NCT02882282) studying response of oral intraepithelial neoplasms to intravenous pembrolizumab[15].

LEUKOPLAKIA AND SQUAMOUS CELL CARCINOMA OF THE URINARY BLADDER

Urothelial epithelium is the urinary bladder mucosa would be replacement with stratified squamous cells which is defined as squamous metaplasia which can be keratinizing or non-keratinizing. The keratinizing squamous metaplasia of the bladder is defined as Bladder-LK. Many authors consider Bladder-LK a premalignant lesion. Diagnosis of Bladder-LK is proven with cystoscopy. Biopsy and histopathology. The treatment of Bladder-LK is controversial, currently there is no effective medical therapy is available for its treatment[19].

Bladder-SCC has also been associated with chronic irritation of the urinary bladder due to recurrent infections, urinary bladder calculi, prolonged catheterization in patients with neurologic bladder dysfunction mostly in patients had spinal cord injuries. Urinary schistosomiasis is a risk factor in certain countries, however, its incidence is declining today. Bladder-LK is a rare entity. It is considered a premalignant condition associated with invasive Bladder-SCC.

It is reported that after cystoscopy with biopsy confirmed Bladder-LK, radical cystectomy with an ileal continent reservoir was performed. The pathology of the cystectomy specimen revealed focal erosion and diffuse keratinizing squamous metaplasia of the bladder. It is concluded that patients with spinal cord injury who are dependent on bladder indwelling catheters for bladder management are at higher risk of developing keratinizing squamous metaplasia (Bladder-LK). Surveillance for early detection of Bladder-LK in patients with prolonged indwelling catheter this is entirely recommended. Prophylactic cystectomy is recommended by many authors. An interdisciplinary approach is recommended for the management of bladder-LK[20].

Bladder-SCC, squamous metaplasia, and SqD-UC are infrequent findings in western countries. The prognostic and natural history of bladder-LK have been under investigation, revealing premalignant characteristics. DNA methylation is a common occurrence in bladder cancer, playing a role in the initiation and advancement of tumors. This type of cancer is a significant health concern for individuals with neurogenic bladder, where it is marked by aggressive histopathological features, advanced stages at the time of diagnosis, and elevated mortality rates. The methylation profiles of the promoters of five genes linked to bladder cancer in the bladder tissue of patients with neurogenic bladder revealed that the use of quantitative methylation-specific polymerase chain reaction to assess the methylation status of the promoters of RASSF1, RARβ, DAPK, hTERT, and APC genes in bladder tissue samples indicated high levels of DNA hypermethylation connected to Bladder-SCC in these patients. This suggests an epigenetic field effect likely resulting from chronic infection and squamous dysplasia[21].

Bladder-SCC, squamous metaplasia, and SqD-UC are infrequent findings in western countries. Prognostic, clinical significance and natural history of squamous cell lesions have undergone investigation that revealed individual premalignant characteristics.

Recently, investigating the molecular characterization of squamous alterations of the urinary bladder urothelium indicated pathogenetic similarities and interrelations, these data might lead to more precise tumor classification and risk stratification[22].

Evaluation of expression of different immunohistochemical markers in tissues of bladder carcinomas in patients with neurogenic bladder a study was conducted in UC and Bladder-SCC. Immunohistochemistry of tissue micro-array sections was performed to reveal the expression of CK7, CK14, CK20, FoxP3, GATA3, STAG2, CD3, CD8, Ki67, and P53. The study showed that the expression of CK7, CK14, and CK20 was significantly higher in both UC and Bladder-SCC compared to controls. A significant higher expression of STAG2 was found compared to SCC[23]. PD-1 and PD-L1 inhibitors immunotherapy for carcinoma of the bladder is implemented in SCC and metastatic muscle invasive UC[24].

Bladder cancer with divergent differentiation including SqD-UC and SCC, these subtypes comprise a differential expression of nectin-4 and PD-L1. Importantly, PD-L1 expressions are associated with responses to ICI[25].

Gulinac et al[26] investigated cohort included 105 cases of bladder cancer for expression of PD-L1, expression was performed using an anti-PD-L1. They found that PD-L1 was expressed in a significant percentage in UC with squamous differentiation (40%).

Morsch et al[27] analyzed the expression of PD-L1 in SqD-UC, Bladder- SCC, and patient with metastatic UC who received ICIs treatment. Tissue microarrays of 45 tissue samples of patients with SqD-UC, and 63 Bladder-SCC, the samples were immunohistochemically stained with anti-PD-L1 antibodies. Positive PD-L1 staining ranged between 4.8% and 61.9% for ICIs treated patients. There were no significant differences between SqD-UC and SCC patients. The FDA guidelines approved for first line therapy of UC with pembrolizumab, a subset of SCC patients up to 20% would be eligible for this treatment. The data reveals expression of PD-L1 in SqD-UC, these findings are comparable with shown current data of positive expression of PD-L1 in UC. In accordance with the encouraging clinical data, it is suggested ICIs treatment for SqD-UC, Bladder-LK, and pure Bladder-SCC.

DISCUSSION

Oral leukoplakia, is a common type of oral dysplasia, it is described as a white mucosal patch in the oropharingal cavity. Oral dysplasia is the commonest premalignant in the oral cavity. Pathologists distinguished two types of keranenised dysplasia of the oral cavity, OLK which is a simple plaque, and the papillary verrucous leucoplakia which in one of its variants is considered SCC. Bladder-LK is classified into keratinized squamous dysplasia and the aggressive variant verrucous leukoplakia which is considered SCC that indicated cystectomy in most of the reported cases.

Oral leukoplakia and Bladder-LK are premalignant tumors that would progress SCC, both tumors and their corresponding SCC are expressing PD-L1 indicating response to PD-L1 inhibitors[14]. Reports on ICI immunotherapy of variant histology of bladder cancer for SqD-UC and SCC showed good response and overall survival similar to pure UC. Experimental study on ICIs in animal model of OLK showed good response[11-13]. Nonrandomized phase II controlled trials (NCT03692325) evaluated the safety and efficacy of nivolumab in 33 patients with high-risk leukoplakias showed a 2 years cancer-free survival of almost 73% of patients[16-18]. Ongoing immunopreventive studies with ICIs on patients with high-risk OPL include the phase II IMPEDE (NCT04504552), a phase I trial (NCT05327270), and phase II trial (NCT03603223) studying the response of leukoplakia to intravenous ICI[15].

The question to be raised: Is leukoplakia of the oral cavity would be treated with ICI immunotherapy as they positively express PD-L1, treatment would be single agent or combined with other treatment options. Multidisciplinary approach and clinical trials are needed. Many treatment modalities were proposed for the treatment OLK that ranged from topical treatment, excision, LASER ablation, and all these modalities would not be successful on the long follow-up and would end with recurrence and progression to SCC. Recent studies on immunological and genetic investigations of OLK and OSCC showed a promising personalized treatment of these cases.

CONCLUSION

Oral leukoplakia and Bladder-LK are premalignant lesions that invariably progress to invasive SCC with worse prognosis. So far, ICI immunotherapy for PD-L1 positive is approved for advanced and metastatic UC of the bladder and other advanced tumors. Squamous differentiation of UC, pure SCC of the bladder, OLK, and oral SCC express PD-L1 positivity. However, there are no released clinical trials on the use of ICI in premalignant lesions. Immunotherapy approvals are primarily for advanced/metastatic diseases. Clinical evidence for treating premalignant lesions with ICI is lacking. Prospective clinical studies are needed to validate ICI in OLK and OSCC. Multidisciplinary approach with consideration of recent funding of expression of PD-I, genetic analysis would be of help for cure and prevent progression of OLK and SCC.