ABSTRACT
BACKGROUND: The gut microbiome plays an important role in immune modulation. Specifically, presence or absence of certain gut bacterial taxa has been associated with better antitumor immune responses. Furthermore, in trials using fecal microbiota transplantation (FMT) to treat melanoma patients unresponsive to immune checkpoint inhibitors (ICI), complete responses (CR), partial responses (PR), and durable stable disease (SD) have been observed. However, the underlying mechanism determining which patients will or will not respond and what the optimal FMT composition is, has not been fully elucidated, and a discrepancy in microbial taxa associated with clinical response has been observed between studies. Furthermore, it is unknown whether a change in the microbiome itself, irrespective of its origin, or FMT from ICI responding donors, is required for reversion of ICI-unresponsiveness. To address this, we will transfer microbiota of either ICI responder or nonresponder metastatic melanoma patients via FMT. METHODS: In this randomized, double-blinded phase Ib/IIa trial, 24 anti-PD1-refractory patients with advanced stage cutaneous melanoma will receive an FMT from either an ICI responding or nonresponding donor, while continuing anti-PD-1 treatment. Donors will be selected from patients with metastatic melanoma treated with anti-PD-1 therapy. Two patients with a good response (≥ 30% decrease according to RECIST 1.1 within the past 24 months) and two patients with progression (≥ 20% increase according to RECIST 1.1 within the past 3 months) will be selected as ICI responding or nonresponding donors, respectively. The primary endpoint is clinical benefit (SD, PR or CR) at 12 weeks, confirmed on a CT scan at 16 weeks. The secondary endpoint is safety, defined as the occurrence of grade ≥ 3 toxicity. Exploratory endpoints are progression-free survival and changes in the gut microbiome, metabolome, and immune cells. DISCUSSION: Transplanting fecal microbiota to restore the patients' perturbed microbiome has proven successful in several indications. However, less is known about the potential role of FMT to improve antitumor immune response. In this trial, we aim to investigate whether administration of FMT can reverse resistance to anti-PD-1 treatment in patients with advanced stage melanoma, and whether the ICI-responsiveness of the feces donor is associated with its effectiveness. TRIAL REGISTRATION: ClinicalTrials.gov: NCT05251389 (registered 22-Feb-2022). Protocol V4.0 (08-02-2022).
Subject(s)
Melanoma , Neoplasms, Second Primary , Skin Neoplasms , Humans , Clinical Trials, Phase I as Topic , Fecal Microbiota Transplantation/adverse effects , Fecal Microbiota Transplantation/methods , Feces/microbiology , Immune Checkpoint Inhibitors/therapeutic use , Melanoma/therapy , Melanoma/etiology , Neoplasms, Second Primary/etiology , Randomized Controlled Trials as Topic , Skin Neoplasms/therapy , Skin Neoplasms/etiology , Treatment Outcome , Clinical Trials, Phase II as Topic , Melanoma, Cutaneous MalignantABSTRACT
We have isolated a hybridoma cell line (clone 1E10) producing a monoclonal antibody which specifically recognizes nucleoli. The antibody (IgM, k isotype) was found to react in a nucleolar pattern with a variety of cell types. Specific staining was only obtained on cryostat sections of unfixed tissues. Paraffin embedding destroyed the epitope. Tissue specificity or species specificity was not observed. Nucleoli in neoplastic cells were highly reactive, presumably due to the larger size of nucleoli in these cells. Immunoelectron-microscopy (using a pre-embedding as well as a post-embedding technique) confirmed the specific nucleolar localization of the immunoreactivity. Immunoreactivity was confined to the granular component of the nucleolus. The intensity of the immunoreactivity increased after cell or tissue pretreatment with DNase, pronase or trypsin, indicating that the target epitope is not DNA or a protein. On Western blots of immunoreactive cells no specific signal was obtained, which supports the non-protein nature of the epitope. Acid hydrolysis and RNase digestion abolished the immunoreactivity. Parallel staining experiments with methylgreen pyronin and acridin orange confirmed the RNA nature of the epitope. In spot blots, immunoreactivity was not found with tRNA or mRNA. These observations indicate that 1E10 recognizes a conformational RNA epitope which occurs only in the nucleolus.
Subject(s)
Antibodies, Monoclonal/immunology , Cell Nucleolus/immunology , RNA/immunology , Animals , Antibodies, Monoclonal/isolation & purification , Caco-2 Cells , Cell Nucleolus/chemistry , Colonic Neoplasms/immunology , Deoxyribonucleases/metabolism , Epitopes/immunology , Esophageal Neoplasms/immunology , Formaldehyde/pharmacology , Humans , Hybridomas , Immunoglobulin M/immunology , Immunohistochemistry , Mice , Mice, Inbred BALB C , Microscopy, Immunoelectron , Pronase/pharmacology , RNA/analysis , Ribonucleases/metabolism , Tumor Cells, CulturedABSTRACT
The proto-oncogene Bcl-2 encodes a protein that protects cells from programmed cell death (apoptosis). The protein is expressed in the proliferative compartment of several normal tissues, including normal colonic crypts. The aim of this study was to test Bcl-2 expression in colorectal neoplasms, assuming that, as a regulator of apoptosis, it might be involved in the progression from adenoma to carcinoma. To this end, Bcl-2 reactivity was tested by immunohistochemistry in hyperplastic polyps, colonic adenomas, and carcinomas and its expression was compared with staining for the proliferation-associated Ki-67 antigen, using the MIB-1 antibody. Bcl-2 expression occurred in 2 out of 10 hyperplastic polyps and in 31 out of 35 (tubular, villous, and tubulovillous) adenomas, irrespective of their degree of dysplasia. Of ten carcinomas, only three were focally Bcl-2-positive, all moderately to well differentiated. In two of four carcinomas in Bcl-2-positive adenomas, no Bcl-2 staining was observed. High numbers of MIB-1-positive cells were found in all hyperplastic and neoplastic lesions, without apparent correlation between proliferation and Bcl-2 expression. These findings suggest that in the pathogenesis of hyperplastic polyps, increased crypt cell proliferation is primarily involved, but in some lesions decreased apoptosis may play a role. Furthermore, the increased Bcl-2 expression in adenomas but not in the majority of the carcinomas suggests either that decreased apoptosis is not usually involved in the pathogenesis of these lesions or that the regulation of apoptosis in colorectal epithelia involves additional regulatory factors.