The Chimeric Antigen Receptor T Cell Target Claudin 6 Is a Marker for Early Organ-Specific Epithelial Progenitors and Is Expressed in Some Pediatric Solid Tumor Entities

Abstract

Background/Objectives: The oncofetal membrane protein Claudin 6 (CLDN6) is an attractive target for T cell-based therapies. There is a lack of detailed analyses on the age-dependent expression of CLDN6 in normal tissues is lacking, which limits the expansion of CLDN6 CAR-T cell clinical trials to pediatric populations. Methods: We analyzed CLDN6 expression in extracranial solid tumors and normal tissues of children using RNA-sequencing data from over 500 pediatric solid tumor samples, qRT-PCR and immunohistochemistry (IHC) in more than 100 fresh-frozen tumor samples and, approximately, 250 formalin-fixed paraffin-embedded (FFPE) samples. We examined normal tissue expression via qRT-PCR in 32 different infant tissues and via IHC in roughly 290 tissues from donors across four age groups, as well as in fetal autopsy samples. Results: In fetal tissues, we detected CLDN6 expression primarily in the epithelial cells of several organs, including the skin, lungs, kidneys, intestinal tract, and pancreas, but not in undifferentiated blastemal cells. Postnatally, we found CLDN6-positive epithelial progenitors only during the first few weeks of life. In older-age groups, isolated clusters of CLDN6-positive progenitors were present, but in scarce quantities. In tumor tissues, we found strong and homogeneous CLDN6 expression in desmoplastic small round cell tumors and germ cell tumors. Wilms tumors demonstrated heterogeneous CLDN6 expression, notably absent in the blastemal component. Conclusions: These findings highlight an organ-specific presence of CLDN6-positive epithelial precursors that largely disappear in terminally differentiated epithelia within weeks after birth. Therefore, our data support CLDN6 as a viable therapeutic target in pediatric patients and justify their inclusion in basket studies for anti-CLDN6-based therapies.

Keywords: CAR-T cells; Claudin 6; Wilms tumors; desmoplastic small round cell tumors; germ cell tumors.

Figure 4

CLDN6 expression is lost after birth. IHC was performed with the indicated organs and in different age groups, including embryo. The picture frame in red indicates tissues isolated from an embryo at the fifth week of pregnancy, while blue indicates a newborn (1 week old) and green indicates 13- to 18-years-old donors. Expression of CLDN6 in sparse small clusters can be observed in pancreas, skin, kidney and lung after birth. Negative control (embryonic skin, right without frame). The arrows indicate the CLDN6 positive epithelial cells.

Figure 1

CLDN6 expression is predicted in solid and blood cancer in the pediatric population. Batch-corrected and transformed gene expression values of CLDN6 across blood cancer (A) and extracranial cancer types (B). The lower half represents a boxplot per tumor type, where the box range from the first to third quantiles, divided by a line indicating the median, and whiskers demonstrate the largest and lowest values no further than 1.5 × IQR from the hinge. The upper half shows the distribution of the samples, where each dot represents a sample. B-ALL = B-cell Acute Lymphoblastic Leukemia, T-ALL = T cell Acute Lymphoblastic Leukemia, AML = Acute Myeloid Leukemia, AMKL = Acute Megakaryoblastic Leukemia, AMML = Acute Myelomonocytic Leukemia, AL = Acute Leukemias, DLBCL = Diffuse Large B-cell Lymphoma, RMS = Rhabdomyosarcoma, DSRCT = Desmoplastic Small Round Cell Tumor.

Figure 2

CLDN6 mRNA is present in the normal tissues of infants and in pediatric tumors. (A) Distribution of the solid tumor samples analyzed by qRT–PCR. (B) Expression of CLDN6 analyzed by qRT–PCR in normal and tumor tissues. Relative expression is shown as a box and whisker plot; the box range is from minimum to maximum value. Each dot represents a sample. In the normal tissues boxplot, red dots indicate lung, purple dots indicate kidney, bright green dots indicate pancreas, blue dots indicate skin, dark green dots indicate renal pelvis and orange dots indicate colon samples. In the normal kidney boxplot, a purple triangle indicates the medulla and the star cortex samples. In the boxplot for “nephroblastoma,” the brown dots and triangles represent metastases from two different patients, while the yellow triangle indicates the nephroblastoma component of a tumor that also presented with nephroblastomatosis (depicted as a yellow dot in the “other tumor entities” boxplot). Further, in the boxplot “other tumor entities”, a pink dot represents a DSRCT sample. (C) Relative expression of CLDN6 in subtypes of GCT. (D) Relative expression of CLDN6 in subtypes of nephroblastoma.

Figure 3

Organ–specific presence of CLDN6–positive epithelial precursors (5th week of pregnancy). Tissues were stained with an anti–CLDN6 specific antibody. The evaluation of the staining can be found in Table 4. Embryonic epithelium of (A) kidney (score 2+/3+), (B) Intestine (score 3+), (C) liver ducts (score 2+/3+), (D) peritoneum (score 2+). (E) Yolk sac (score 2+/3+). (F) Chorionic epithelium of the placenta (score 2+/3+).

Figure 5

CLDN6 was analyzed by immunohistochemistry in DSRCT ((A), 100% of tumor cells are positive, with score 3+); Yolk sac tumor ((B), 95% of the tumor cells are positive, with score 2+/3+) and high-risk WTs after therapy (C,E,F). Blastema (F), tumor stroma and part of the tumor epithelium (E) were CLDN6-negative, individual tumor cells in the metastasis were positive (C), 5%. The persistent immature nephroblastomatosis foci were partially positive (D). In the graph, the intensity and heterogeneity of CLDN6 expression across pediatric tumor entities is reported. The y-axis indicates the percentage of positive tumor cells (with a score of 2+/3+). Each dot represents a sample. The median value is indicated. Within GCT, the yellow dot indicates a mature teratoma, the green dots yolk sac tumors, the pink dot a dysgerminoma and the black dot a mixed subtype. Within “WT”, the blue dots indicate high-risk patients.