FOXP3 and CXCR4-positive regulatory T cells in the tumor stroma as indicators of tumor immunity in the conjunctival squamous cell carcinoma microenvironment.

Conjunctival squamous cell carcinoma (SCC) is the most common ocular surface neoplasia. The purpose of this retrospective study was to examine the role of regulatory T cell (Treg) activity in tumor immunity and investigate the tumor microenvironment as a new treatment focus in conjunctival SCC. Cancer progression gene array and immunohistochemical analyses of FOXP3 as a Treg marker, CD8 as a tumor-infiltrating lymphocyte marker, and CXCR4 expression on activated Tregs were conducted in a series of 31 conjunctival SCC cases. The objective was to investigate the immunoreactive response in tumor cells and stromal cells in the cancer microenvironment. The stroma ratio in tumor cells was investigated by monitoring α-smooth muscle actine (SMA) expression between carcinoma in situ (Tis) and advanced carcinoma (Tadv) (P<0.01). No significant change in PD-L1 expression was observed in this study (P = 0.15). Staining patterns of FOXP3, CD8, and CXCR4 were examined separately between tumor cells and stromal cells in SCC tumors. Differences in staining of FOXP3 in Tregs and CD8 in tumor-infiltrating lymphocytes in tumor stroma in the Tis group were observed compared with the Tadv group (each P<0.01). In addition, double immunostaining of CXCR4/FOXP3 was correlated with progression-free survival (P = 0.049). Double immunostaining of CXCR4/FOXP3 correlated with American Joint Committee on Cancer T-stage, independent of age or Ki67 index (P<0.01). Our results show that FOXP3 and the CXCR4/FOXP3 axis are important pathologic and prognostic factors of ocular surface neoplasia, including SCC. The tumor microenvironment of conjunctival SCC should be considered in the future development of treatment options.

New Therapeutic Approaches for Conjunctival Melanoma-What We Know So Far and Where Therapy Is Potentially Heading: Focus on Lymphatic Vessels and Dendritic Cells.

Conjunctival melanoma (CM) accounts for 5% of all ocular melanomas and arises from malignantly transformed melanocytes in the conjunctival epithelium. Current therapies using surgical excision in combination with chemo- or cryotherapy still have high rates for recurrences and metastatic disease. Lately, novel signal transduction-targeted and immune checkpoint inhibitors like cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibitors, programmed cell death protein-1 (PD-1) receptor inhibitors, BRAF- or MEK-inhibitors for systemic treatment of melanoma have improved the outcome even for unresectable cutaneous melanoma, improving patient survival dramatically. The use of these therapies is now also recommended for CM; however, the immunological background of CM is barely known, underlining the need for research to better understand the immunological basics when treating CM patients with immunomodulatory therapies. Immune checkpoint inhibitors activate tumor defense by interrupting inhibitory interactions between tumor cells and T lymphocytes at the so-called checkpoints. The tumor cells exploit these inhibitory targets on T-cells that are usually used by dendritic cells (DCs). DCs are antigen-presenting cells at the forefront of immune response induction. They contribute to immune tolerance and immune defense but in the case of tumor development, immune tolerance is often prevalent. Enhancing the immune response via DCs, interfering with the lymphatic pathways during immune cell migration and tumor development and specifically targeting tumor cells is a major therapeutic opportunity for many tumor entities including CM. This review summarizes the current knowledge on the function of lymphatic vessels in tumor growth and immune cell transport and continues to compare DC subsets in CM with related melanomas, such as cutaneous melanoma and mucosal melanoma.

Conjunctival cancer in people living with HIV.

PURPOSE OF REVIEW: Historically, conjunctival cancer has been associated with HIV particularly in sub-Saharan Africa. The human papilloma virus (HPV) has been implicated as a potential causative agent without conclusive evidence. This review covers recent evidence of the epidemiology, diagnosis and treatment of conjunctival cancer in people living with HIV (PLWH). RECENT FINDINGS: HIV infection has been attributed to 33% of squamous cell carcinoma of the conjunctiva in sub-Saharan Africa. Although clear evidence of the effect of immunodeficiency on conjunctival cancer risk has been demonstrated, the role of HPV on conjunctival cancer development is still unclear. Biomarkers such as the p16 protein are not always indicative of HPV infection. The Epstein-Barr virus (EBV) might potentially be another infectious agent of interest in the development of conjunctival cancer. There is some evidence of increased conjunctival cancer recurrence post treatment as well as increased probability of metastasis in PLWH. SUMMARY: Immunodeficiency increases the risk of conjunctival cancer in PLWH. Symptomatic screening of conjunctival cancer in PLWH should be encouraged. Research on HPV involvement should remain a priority and EBV considered as another etiologic agent of interest. More studies on treatment modalities in PLWH should be considered.

Successful Treatment of Conjunctival Kaposi Sarcoma in a Human Immunodeficiency Virus-Negative Kidney Transplant Recipient: A Case Report.

Kaposi sarcoma (KS) is the most common tumor in patients with human immunodeficiency virus (HIV), and its frequency is increasing after organ transplantation in HIV-negative patients. A 28-year-old woman had preemptive kidney transplantation from her 48-year-old mother. In the postoperative ninth month, an exophytic mass was found in the upper medial conjunctiva of the right eye. The lesion was excised under local anesthesia, and cryotherapy was applied to the surgical area. The biopsy result was reported as KS. Treatment with tacrolimus was switched to everolimus (EVO), but EVO was discontinued because of pneumonitis. The patient was followed without any recurrences after the excision of the conjunctival lesion. No local or systemic recurrence was observed in the 14th month after surgical excision and local cryotherapy. Local treatment and the reduction of immunosuppressive therapy may be effective in the treatment of conjunctival KS.

Conjunctival Melanoma: Current Treatments and Future Options.

Conjunctival melanoma is a rare tumor of the conjunctival epithelium with a heterogenous clinical presentation and a propensity for regional and distant metastatic spread. Guidelines for the treatment of local conjunctival melanoma are well-established, but there are no standard efficacious therapies for metastatic disease. Given that conjunctival melanoma is genetically similar to cutaneous melanoma and mucosal melanomas, targeted therapies effective in the treatment of these diseases, such as BRAF inhibitors and KIT inhibitors, may be effective in the treatment of patients with metastatic conjunctival melanoma. Other targeted small-molecule drugs in the drug development pipeline for the treatment of more prevalent melanomas could also be applicable to conjunctival melanoma. Furthermore, systemic immunotherapy treatments that are now a mainstay in the treatment of cutaneous melanoma, such as programmed cell death-1 and cytotoxic T lymphocyte-associated antigen-4 inhibitors, could also stand to benefit patients with metastatic conjunctival melanoma. Limited case reports provide clues about the effectiveness of both targeted small-molecule inhibitors and immunotherapy in patients with advanced local and metastatic conjunctival melanoma and give credence to the argument that conjunctival melanoma patients should be included in major trials studying new therapies in both cutaneous and mucosal melanomas where applicable.

Human papillomavirus (HPV)-associated trilateral squamous neoplasia in immunocompetent individual.

PURPOSE: To describe a case of bilateral conjunctival and cervical human papillomavirus (HPV)-related squamous neoplasia. CASE DESCRIPTION: A healthy immunocompetent 55-year-old lady came to our attention for prosecution of care of a right recurrent conjunctival squamous cell carcinoma. Upon examination, she was found disease-free in the right eye but displayed a left conjunctival intraepithelial neoplasia (CIN grade I) and low-grade cervical squamous dysplasia. HPV infection with genotypes 16 (right eye), 11 and 39 (left eye) and 39 (uterine cervix) was also detected. CONCLUSION: Simultaneous uterine and conjunctival HPV-related squamous neoplasia can occur in immunocompetent individuals. Auto-inoculation and repeated exposure to HPV could explain coexistence of different genotypes.

Immune Checkpoint Inhibitors for Treatment of Metastatic Melanoma of the Orbit and Ocular Adnexa.

Programmed cell death 1 (PD-1) inhibitors are members of a new class of drugs known as immune checkpoint inhibitors and have proven efficacy in the treatment of metastatic melanoma. Herein, the authors report the use of nivolumab and pembrolizumab, 2 recently Food and Drug Administration-approved PD-1 inhibitors, in 3 patients: 1 with metastatic conjunctival melanoma and 2 with metastatic cutaneous melanoma and orbital involvement. The patients' metastatic disease responded well to drug treatment. As of this writing, 2 patients have completed therapy and remain disease free at least 1 year after treatment completion; the other patient is still receiving treatment, and his orbital disease is responding. The authors herein describe the use of PD-1 inhibitors as a new alternative in the treatment of metastatic melanoma to the orbit or metastatic ocular adnexal melanomas in these clinical settings.

[Two cases of conjunctival intraepithelial neoplasia in immunosuppressed patients].

UNLABELLED: Background: Conjunctival intraepithelial neoplasia (CIN) is a precursor lesion of conjunctival squamous cell carcinoma (SCC). CIN recurs frequently but progresses less aggressively than SCC. We report 2 cases of recurrent CIN in immunosuppressed patients. CASE 1: A 60-year-old woman had been taking oral immunosuppressive drugs for 30 years for systemic lupus erythematosus. In 2005, both conjunctival tumor and high serum SCC levels were noted. Biopsy revealed right eye CIN and left eye SCC, and extended resection was performed. In 2008, right eye CIN recurred accompanied by high serum SCC levels and another extended resection was performed. The patient was free from recurrence for 1 year until her death. CASE 2: A 43-year-old woman who had been taking oral immunosuppressive drugs for 3 years for nephrotic syndrome. She had twice undergone right corneal transplantation for keratoconus. In 2011, right conjunctival tumor and high serum SCC levels were noted. Biopsy revealed right eye CIN, for which an extended resection was performed. At present she remains free from recurrence. CONCLUSION: CIN can recur in immunosuppressed patients. We suggest that serum SCC levels be monitored to help detect recurrence.

Isolated ocular surface squamous neoplasia in a renal transplant recipient.

Ocular surface squamous neoplasia is a rare complication after a kidney transplant, related with increased risk and poor prognosis. Generally, ocular surface squamous neoplasia in kidney transplant patients is associated with skin lesions. We report a case of ocular surface squamous neoplasia without skin lesions in a kidney transplant recipient. Systematic periodic ophthalmic evaluation of recipients may help ensure the early diagnosis of subtle ocular surface squamous neoplasia.

Clinical significance of tumor-infiltrating FOXP3+ T cells in patients with ocular adnexal mucosa-associated lymphoid tissue lymphoma.

We evaluated the association between tumor-infiltrating FOXP3+ T cells and clinical outcomes in patients with ocular adnexal lymphoma of mucosa-associated lymphoid tissue type (OAML). Pretreatment formalin-fixed paraffin-embedded tissues from 42 patients with OAML were stained with 236A/E7 anti-FOXP3 murine monoclonal antibody as well as CD3, CD4 and CD8 antibodies. The amount of FOXP3+ T cells was numerically quantified using an image analysis program. Front-line treatments were as follows: combination chemotherapy (n = 25); radiotherapy (n = 9); doxycycline (n = 6); and wait and see (n = 2). Complete response (CR) was observed in 20 (50%) of 40 evaluable patients. Median progression-free survival (PFS) was 50 months. A high number of FOXP3+ T cells (n = 21, ≥ 180/0.58 mm(2)) showed a higher CR rate (33%vs 71%, P = 0.013) and tendency towards prolonged PFS (48 vs 67 months, P = 0.110). In the combination chemotherapy group, a high number of FOXP3+ T cells was significantly associated with a higher CR rate (29%vs 82%, P = 0.008) and prolonged PFS (17 vs 79 months, P = 0.003). A high number of tumor-infiltrating FOXP3+ T cells correlates with a favorable clinical outcome in OAML patients.

Rituximab immunotherapy for ocular adnexal lymphoma: clinicopathologic correlation with 5-year follow-up.

Two patients with primary ocular adnexal mucosa-associated lymphoid tumor lymphoma were treated with rituximab immunotherapy. One patient had refused radiotherapy. The other had bilateral ischemic retinopathy, a relative contraindication to radiotherapy. Biopsies were performed 6 months after treatment. One patient had complete resolution of local disease, and the other had a partial response, remaining stable without signs of progression. During the 5 years of clinical follow-up, there was no evidence of systemic disease in either patient. Rituximab immunotherapy is an alternative to regional radiotherapy for ocular adnexal mucosa-associated lymphoid tumor lymphoma.

Use of similar immunoglobulin VH gene segments by MALT lymphomas of the ocular adnexa.

Extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue type (MALT lymphomas) develop from acquired reactive infiltrates directed against external or autoantigens. Although some European cases of ocular adnexal MALT lymphoma have been associated with Chlamydia psittaci infections, C. psittaci has not been detected in large studies of US-based cases. To evaluate whether the growth of US-based ocular adnexal MALT lymphomas may be promoted by a similar antigen, we identified and analyzed the expressed immunoglobulin VH genes in 10 cases. Interestingly, the VH genes in two cases used the same VH1 family V1-2 gene segment, and three cases used the same VH4 family V4-34 gene segment. The other five cases all used different gene segments V4-31, V5-51, V3-23, V3-30, and V3-7. All of the VH genes were mutated from germ line, with percent homologies ranging between 96.9 and 89.0%. The distribution of replacement and silent mutations within the VH genes was nonrandom consistent with the maintenance of immunoglobulin function and also strongly suggestive of antigen selection in the six VH genes with highest mutation loads. The CDR3 sequences in two of three VH-34 cases were the same size (15 amino acids) and had similar sizes in the two VH1-2 cases (18 and 16 amino acids). In conclusion, US-based MALT lymphomas of the ocular adnexa preferentially express a limited set of VH gene segments not frequently used by other MALT lymphomas and consistent with some recognizing similar antigens. Analysis of somatic mutations present within the VH genes is also consistent with antigen binding stimulating the growth of these lymphomas.

CD5-undetected by immunohistochemistry, t(11;14)(q13;q32)-positive conjunctival mantle cell lymphoma: a case report.

Most primary ocular adnexal lymphomas are extranodal marginal zone B-cell lymphomas of mucosa-associated lymphoid tissue (MALT). A few cases of ocular adnexal mantle cell lymphomas have been reported in the literature. We present a case of mantle cell lymphoma presenting as conjunctival mass. A 58-year-old man presented with a palpable mass in the left lower tarsal conjunctiva incidentally detected one month previously. Histopathologic examination showed proliferation of monomorphous small-to-medium sized lymphoid cells. On immunohistochemistry, tumor cells were positive for CD20, bcl-2, and cyclin D1, and negative for CD5. PCR analysis for immunoglobulin heavy chain gene rearrangement showed monoclonal B-cell proliferation. t(11;14)(q13;q32), involving the CCND1 and IGH genes, was detected in interphase fluorescent in situ hybridization using formalin-fixed, paraffin-embedded tissue; however, MALT1 gene translocation was not observed. The final diagnosis was mantle cell lymphoma. There was no lymphadenopathy; however, bone marrow involvement of the lymphoma was suspected. The patient has been receiving systemic chemotherapy. This case emphasizes the differential diagnosis of conjunctival mantle cell lymphoma from extranodal marginal zone B-cell lymphomas of MALT regarding the clinical and pathological aspects.

Efficient adenovector CD40 ligand immunotherapy of canine malignant melanoma.

Cutaneous canine melanomas are usually benign in contrast to human malignant melanoma. However, the canine oropharyngeal, uveal, and mucocutaneous neoplasms are aggressive and have metastatic potential. Surgery and to a lesser extent radiotherapy and chemotherapy are widely adopted treatments but are seldom curative in advanced stages. The similarities between human and canine melanoma make spontaneous canine melanoma an excellent disease model for exploring novel therapies. Herein, we report the first 2 adenovector CD40L immunogene (AdCD40L) treatments of aggressive canine malignant melanoma. Case no. 1 was an advanced stage III oral melanoma that was cured from malignant melanoma with 2 intratumor AdCD40L injections before cytoreductive surgery. After treatment, the tumor tissue was infiltrated with T lymphocytes and B lymphocytes suggesting immune activation. This dog survived 401 days after the first round of gene therapy and was free of melanoma at autopsy. Case no. 2 had a conjunctival malignant melanoma with a rapid progression. This case was treated with 6 AdCD40L injections over 60 days. One hundred and twenty days after start of gene therapy and 60 days after the last injection, the tumor had regressed dramatically, and the dog had a minimal tumor mass and no signs of progression or metastasis. Our results indicate that AdCD40L immunogene therapy is beneficial in canine malignant melanoma and could be considered for human malignant melanoma as well.

Assessment of the role of hepatitis C, Helicobacter pylori and autoimmunity in MALT lymphoma of the ocular adnexa in 45 Austrian patients.

INTRODUCTION: A recent series from Italy has suggested a pathogenic link between hepatitis C virus and MALT lymphoma of the ocular adnexa. The hypothesis of our study was to prove this concept in Austrian patients with MALT lymphoma of the ocular adnexa. MATERIALS AND METHODS: A total of 45 patients presenting with MALT lymphoma of the ocular adnexa were assessed for the presence of infection with hepatitis A, B and C. Furthermore, extensive staging to evaluate the extent of disease along with analysis of Helicobacter pylori-infection, the presence or absence of autoimmune diseases (AD) and assessment of MALT-lymphoma specific genetic changes was performed. RESULTS: Only 2/45 (4%) patients were tested positive for hepatitis C, while 10/45 (22%) had an underlying AD and 15/39 (38%) had HP infection. Chromosomal aberrations were detected in 19 (54%) of 35 patients analyzed. Disseminated disease was a significant risk factor for relapse (p=0.014). DISCUSSION: Our series suggests that infection with hepatitis C is not a significant contributor to the pathogenesis of ocular adnexal MALT lymphoma in the Austrian population, while a substantial proportion of these patients suffer from autoimmune conditions.

Ocular adnexal MALT lymphoma: an intriguing model for antigen-driven lymphomagenesis and microbial-targeted therapy.

Non-Hodgkin's lymphomas constitute one half of malignancies arising in the orbit and the ocular adnexae. Mucosa-associated lymphoid tissue (MALT)-type lymphoma is the most common histological category in this anatomic region. The incidence of ocular adnexal lymphoma of mucosa-associated lymphoid tissue-type (OAML) is increasing and recent studies offered new relevant insights in molecular, pathogenetic and therapeutic issues on these neoplasms. A pathogenetic model of antigen-driven lymphoproliferation similar to that reported for Helicobacter pylori-related gastric MALT lymphomas has been hypothesized for OAML. This notion is supported by the association between OAML and Chlamydophila psittaci infection, an association that is of likely pathogenetic relevance and may influence both the biological behavior and the therapeutic management of these neoplasms. However, this association displays evident geographical variability indicating that other etiopathogenic agents could be involved. These recent acquisitions coupled with the occurrence of chromosomal translocations and other genetic alterations, as well as additional risk factors like autoimmune disorders have contributed to render OAML an exciting challenge for a broad group of physicians and scientists. OAML is an indolent and rarely lethal malignancy that, in selected patients, can be managed with observation alone. Lymphomatous lesions are frequently responsible for symptoms affecting patient's quality of life, requiring, therefore, immediate treatment. Several therapeutic strategies are available, often associated with relevant side-effects. However, the therapeutic choice in OAML is not supported by consolidated evidence due to the lack of prospective trials. In this review, we analyze the most relevant biological, molecular, pathological and clinical features of OAML and propose some therapeutic guidelines for patients affected by this malignancy.

Monoclonal antibodies in the diagnosis of lymphoproliferative diseases of the orbit and orbital adnexae.

AIMS: To describe the value of monoclonal antibodies in the differential diagnosis of orbital lymphoproliferative disorders. METHODS: A total of 200 sequential cases of malignant lymphoma diagnosed at St Bartholomew's Hospital as part of the ocular lymphoma service at Moorfields Eye Hospital and the Institute of Ophthalmology have been examined. Cases were acquired between January 1998 and June 2005. Each case had detailed immunophenotypic analysis using a panel of monoclonal antibodies and was classified using the WHO classification of lymphoma. These cases are discussed in relation to earlier studies of orbital lymphoma reported by our group. RESULTS: Lymphomas fell into three main categories. Extranodal marginal zone lymphoma was the largest group with 151 cases, arising within the orbital soft tissue, conjunctiva and lacrimal gland. Cases arising in the conjunctiva and lacrimal gland showed a higher female predominance than those arising within the deeper soft tissue. A small number of cases were associated with organ specific autoimmunity, including thyroid eye disease complicating Graves' disease. Follicular lymphoma and diffuse large B-cell lymphoma formed the next two groups, occurring with equal frequency. Many of the follicular lymphomas had evidence of disseminated disease on completion of staging. A miscellaneous group of T-cell and B-cell lymphomas formed a minority of cases during the study period. CONCLUSION: Extranodal marginal zone lymphoma is the most frequent type of primary orbital and orbital adnexal lymphoma. Its major differential diagnosis is with orbital lymphoid hyperplasia, chronic dacryoadenitis, and follicular conjunctivitis. Systemic types of lymphoma may present within the orbit or involve the orbit secondarily.

Conjunctival T-cell lymphoma: a clinicopathologic case report.

OBJECTIVE: To report a patient with conjunctival T-cell lymphoma, an extremely rare entity. DESIGN: Single case report. METHODS: Based on clinical examination, an excisional biopsy and immunostaining were performed on the conjunctival lesion. For management, we excised and performed triple freeze-thaw cryotherapy to the involved area, and we consulted the oncology service. MAIN OUTCOME MEASURES: T-cell and B-cell markers, and clinical examination of the lesion. RESULTS: Both examination and laboratory assessment revealed no evidence of systemic involvement. Conjunctival biopsy showed expansion of the substantia propria with an infiltrate of chronic inflammatory cells (including lymphocytes, plasma cells, and eosinophils), and prominent lymphocyte exocytosis with reactive epithelial changes. The CD-45 RO (T-cell marker) was strongly positive, whereas the CD-20 (B-cell marker) was negative. The T-cell receptor gene rearrangement was positive with beta clonality, confirming the diagnosis of T-cell lymphoma. CONCLUSIONS: T-cell lymphoma is a rare but possible diagnosis of gelatinous conjunctival lesions. The oncology consultants were reluctant to treat the patient with systemic chemotherapy or radiation because extraconjunctival extension could never be documented. The answer to the question of what is the most appropriate treatment for conjunctival T-cell lymphoma remains unknown.