Durable remission of renal cell carcinoma in conjuncture with graft versus host disease following allogeneic stem cell transplantation and donor lymphocyte infusion: rule or exception?

Abstract

Allogeneic stem cell transplantation (alloSCT) followed by donor lymphocyte infusion (DLI) can be applied as immunotherapeutic intervention to treat malignant diseases. Here, we describe a patient with progressive metastatic clear cell renal cell carcinoma (RCC) who was treated with T cell depleted non-myeloablative alloSCT and DLI resulting in disease regression accompanied by extensive graft versus host disease (GVHD). We characterized the specificity of this immune response, and detected a dominant T cell population recognizing a novel minor histocompatibility antigen (MiHA) designated LB-FUCA2-1V. T cells specific for LB-FUCA2-1V were shown to recognize RCC cell lines, supporting a dominant role in the graft versus tumor (GVT) reaction. However, coinciding with the gradual disappearance of chronic GVHD, the anti-tumor effect declined and 3 years after alloSCT the metastases became progressive again. To re-initiate the GVT reaction, escalating doses of DLI were given, but no immune response could be induced and the patient died of progressive disease 8.5 years after alloSCT. Gene expression studies illustrated that only a minimal number of genes shared expression between RCC and professional antigen presenting cells but were not expressed by non-malignant healthy tissues, indicating that in patients suffering from RCC, GVT reactivity after alloSCT may be unavoidably linked to GVHD.

Figure 1. Clinical course.

DLI doses, donor chimerism and the clinical course following DLI are depicted during time after allo-SCT (months, x-axis). The infused dose of T cells (filled triangles) and chimerism status (% of donor cells as measured by XY-FISH in PBMC, open circles) are shown in the upper part of the graph. Rectangles in the lower part of the graph indicate tumor status, GVHD state and GVHD treatment.

Figure 2. FUCA2 encodes a novel MiHA presented by HLA-B*07∶02.

(A) HLA-restricted reactivity of a T cell clone with unknown specificity was determined by testing recognition of patient EBV-LCL pre-incubated with monoclonal antibodies against HLA class-I, HLA class-II and HLA-B*07 prior to addition of T cells. In addition, mock transduced and pLZRS-NGFR-HLA-B*07∶02-transduced third party EBV-LCL were used as test cells. Reactivity was measured by Elisa and is depicted as the concentration of IFN-γ (ng/ml) in the supernatant after 24 h of co-cultivation. (B) WGAs identified a region on chromosome 6 associated with T cell recognition. Each dot represents a SNP relative to its position on chromosome 6 and the significance of association is expressed by P value. Double-headed arrows locate the genes ADAT2, PEX3 and FUCA2. (C) The FUCA2 gene contains 2 associating non-synonymous SNP. The amino acid sequence containing these SNP was investigated for potential peptide binding to HLA-B*07∶02, resulting in 1 candidate peptide sequence spanning rs3762002. (D) Synthetic peptides containing the patient specific valine residue (closed circles) and donor specific methionine residue (open circles) were loaded on donor EBV-LCL and tested for recognition by the T cell clone. (E) LB-FUCA2-1V tetramers were used to stain a patient sample collected at the onset of GVHD.

Figure 3. LRH-1 and LB-FUCA2-1V recognition and gene expression of P2RX5 and FUCA2.

(A) LB-FUCA2-1V and LRH-1 specific T cells were tested against patient-derived cells (EBV-LCL and fibroblasts) and a panel of 3^rd party cells expressing HLA-B*07∶02 and the LB-FUCA2-1V and/or LRH-1 MiHA. Cell lines RCC 90.03 and RCC Mz1774 were retrovirally transduced to express B*07∶02. Fibroblasts, keratinocytes and RCC cell lines were tested after 24 h pre-incubation in the absence (open bars) or presence (hatched bars) of 100 IU/ml of IFN-γ. Reactivity was measured by Elisa and is depicted as the concentration of IFN-γ (ng/ml) in the supernatant after 24 h of co-cultivation. (B) Expression patterns of the MiHA encoding genes P2RX5 (LRH-1) and FUCA2 (LB-FUCA2-1V) were determined by quantifying mRNA levels using microarray analysis. Expression, depicted as mean fluorescence intensity (MFI), is shown in hematopoietic cells (PBMC, B cells, T cells, monocytes, immature and mature DC and EBV-LCL), non-hematopoietic cells (fibroblasts, keratinocytes and PTEC pretreated with and without IFN-γ) and RCC cell lines. Numbers between brackets indicate the number of analyzed individual samples.

Figure 4. Genes over-expressed in both malignant cells and monoDC as compared to healthy tissue cells.

Mature monocyte derived DC (monoDC), RCC cell lines, melanoma (MEL) cell lines, ALL and AML were investigated for gene expression using microarray techniques. For each cell type, 2 different samples were analyzed. Indicated are the numbers of genes showing more than 10, 30 or 100-fold over-expression in both the malignant cells and monoDC as compared to fibroblasts and keratinocytes. Fold over-expression was calculated from the difference in mean log-transformed values, and only genes with a significant difference (p<0.05) in mean log-transformed values as measured by a standard Student's t-test were selected.