Regression of diffuse B-cell lymphoma of the leg with intralesional gentian violet

Abstract

In this case report, a patient of primary cutaneous diffuse B-cell lymphoma, leg type was treated with intralesional gentian violet as she was judged to be too medically fragile for conventional chemotherapy due to advanced age and multiple serious comorbidities. Gentian violet (crystal violet/hexamethyl pararosaniline) is a triphenylmethane dye. It has been shown to have an inhibitory effect on NADPH oxidase, an enzyme family which is found in abundance in reactive oxygen-driven tumors such as melanoma and lymphoma. We hypothesize that intralesional gentian violet treatment caused signalling changes in the lymphoma which allowed for immune clearance of the lymphoma. Complete resolution of the patient’s lesion was noted on a follow-up visit.

We present a case of an 84-year-old woman who presented in January 2015 with three nodules on her right leg. Biopsy of these lesions revealed primary cutaneous diffuse B-cell lymphoma, leg type (PCLBCL of the leg). The overlying epidermis was unremarkable. Provided immunohistochemical stains demonstrated the atypical lymphoid cells to be positive for CD20, BCL2 and BCL6 and negative for CD3 and CD30.

The patient was treated with radiation therapy due to multiple comorbidities, including coronary artery disease, diastolic heart failure, sleep apnoea and diabetes mellitus. She underwent radiation therapy of 30 Gray in 10 sessions, resulting in remission of the initial nodules. However, three new nodules appeared outside the radiation field, which resolved with a single intravenous dose of rituximab. She was lost to follow-up until December 2015, when she presented with a 3-cm nodule on her right anterior leg (Figure 1A).

FIGURE 1.

Primary cutaneous diffuse B-cell lymphoma, leg type: (A) Clinical appearance of the patient’s lesion at presentation. (B) Clinical appearance of the patient’s lesion at four-month follow-up. (C) Histology of representative tumor removed during biopsy at a low powered view. (D) Histology of representative tumor removed during biopsy at a high powered view (400 ×). Note the large atypical lymphoid cells with pleomorphic nuclei. (E) Interspersed CD3 positive T cells present with the atypical B cells seen before the injection of gentian violet in the punch biopsy cavity. (F) Generation of reactive oxygen species results in activation of NFκB, Akt and suppression of wild-type p53. In addition, SOX-2 may increase the production of immunosuppressive factors

A punch biopsy of the nodule was performed, and gentian violet was introduced into the cavity and allowed to heal by secondary intention. The biopsy was consistent with recurrent PCLBCL of the leg (Figure 1C,D), but did contain infiltrating T lymphocytes (Figure 1E). The tumor appeared negative for p53 (wild type) and also had very little staining for PD-L1. No systemic involvement was found on staging of tumor. Patient was referred for surgical excision of the solitary lesion. However, she was hospitalized for coronary artery disease and did not have her nodule excised. She followed up 4 months later to the dermatology clinic, where a complete resolution of the lesion was noted (Figure 1B). The patient refused further biopsy and will be monitored for recurrence. Patient has been in remission for over a year now.

Gentian violet (crystal violet/hexamethyl pararosaniline) is a triphenylmethane dye.^[1]

It is an over the counter drug with over a century of use in humans.^[2] Our laboratory has discovered that gentian violet is an inhibitor of NADPH oxidase, an enzyme family that converts molecular oxygen to superoxide and hydrogen peroxide.^[3] Reactive oxygen-driven tumors, which include some melanoma, glioblastoma and likely many lymphomas, have abundance of this enzyme and promote tumor growth by oxidizing the tumor suppressor genes like PTEN, Ik-B among others (Figure 1F).^[4]

We hypothesize that gentian violet treatment causes signalling changes in the lymphoma cells which allow immune clearance of the lymphoma. To study this phenomenon in greater depth, we would investigate the effects of intra-tumoral gentian violet in a syngeneic mouse lymphoma model. The tumors in the control and gentian violet group will be compared for the growth and for the presence and subtype of infiltrating immune cells, including CD4 and CD8 T lymphocytes, tumor-associated macrophages and Treg cells. Interestingly, a majority of diffuse B-cell lymphomas of the leg have the driver MyD88 L265P mutation, initially discovered in Waldenstrom’s microglobulinemia.^{[5, 6]} This could serve as a tumor antigen, and neoplastic B lymphocytes might be defective in presenting this tumor antigen to the host. We have found deficiencies in antigen presentation in warts,^[7] and lymphomas might be deficient in presentation of mutant proteins.

We also propose the following biomarkers to assess the sequence of events in gentian violet-mediated tumor regression. Sox2 is one of the Yamanaka factors in tumor stem cells, and we have recently demonstrated that gentian violet downregulates Sox2.^[8] Immunohistochemistry for Sox2 could be performed in vivo in lymphomas treated with gentian violet. Similarly, immunohistochemistry for antigen-presenting molecules such as TAP1 could be performed. We predict the following, intralesional gentian violet downregulates NADPH oxidase, leading to downregulation of NF-kB and Sox2 (Figure 1F). As a result, antigen presentation is enhanced, leading to presentation of tumor antigens on the cell surface. This might be associated in addition with increased expression of MHC class 1 and 2 molecules on the tumor surface. We would predict an increase in CD8 effector T cells, decreased tumor-associated macrophages and Treg cells. This would lead to selective tumor cytolysis. Current immunotherapies are associated with long term and sometimes fatal autoimmunity. NADPH oxidase inhibition, especially when locally delivered, offers the promise of less toxic immunotherapy.

What can be learned from this patient? It is likely that the regression of this tumor was immune mediated, as the gentian violet was applied only once and immunohistochemistry of the lesion before application of GV revealed a sparse T-cell infiltrate (Figure 1E). In this particular case, we cannot predict whether the patient has a long-lasting response. However, gentian violet deserves further evaluation in human lymphoma, as well as the novel gentian violet-like NADPH oxidase inhibitors^[9] agents which have been given systemically in preclinical studies.