Genomic and Immunologic Characterization of INI1-Deficient Pediatric Cancers

Abstract

Purpose: Several aggressive pediatric cancers harbor alterations in SMARCB1, including rhabdoid tumors, epithelioid sarcoma, and chordoma. As tumor profiling has become more routine in clinical care, we investigated the relationship between SMARCB1 genetic variants identified by next-generation sequencing (NGS) and INI1 protein expression. Therapeutic approaches for INI1-deficient tumors are limited. Early reports suggest a potential role for immune checkpoint inhibition in these patients. Thus, we also investigated PD-L1 and CD8 expression in INI1-negative pediatric brain and solid tumors.

Experimental design: We performed immunohistochemistry (IHC) for INI1 and immune markers (PD-L1, CD8, and CD163) and NGS on tumor samples from 43 pediatric patients who had tumors with INI1 loss on previous IHC or SMARCB1 genomic alterations on prior somatic sequencing.

Results: SMARCB1 two-copy deletions and inactivating mutations on NGS were associated with loss of INI1 protein expression. Single-copy deletion of SMARCB1 was not predictive of INI1 loss in tumor histologies not known to be INI1-deficient. In the 27 cases with INI1 loss and successful tumor sequencing, 24 (89%) had a SMARCB1 alteration detected. In addition, 47% (14/30) of the patients with INI1-negative tumors had a tumor specimen that was PD-L1 positive and 60% (18/30) had positive or rare CD8 staining. We report on 3 patients with INI1-negative tumors with evidence of disease control on immune checkpoint inhibitors.

Conclusions: A significant proportion of the INI1-negative tumors express PD-L1, and PD-L1 positivity was associated with extracranial tumor site. These results suggest that clinical trials of immune checkpoint inhibitors are warranted in INI1-negative pediatric cancers.

Figure 1:. Confirmatory SMARCB1 Sequencing and INI1 Expression in Sequencing Cohort.

A. Two cases with 2-copy gene deletion and one case with a nonsense mutation were identified. Repeat sequencing was successful and identified SMARCB1 alterations for 2 of the 3 cases and INI1 IHC was negative in all 3 cases. B. Fourteen cases with 1-copy gene deletion were identified. Repeat sequencing was successful for 13 cases and SMARCB1 alterations were confirmed in 10 cases. INI1 IHC was positive for 13 cases and negative for 1 case.

Figure 2.. Clinical and Genomic Features of INI1-Negative Tumors.

OncoPrint of SMARCB1 alterations, tumor mutational burden (TMB), PD-L1 and CD8 staining and clinical features including diagnosis, sample timing and lesion treatment in 30 INI1-negative tumors.

Figure 3.. Expression of SMARCB1 is Inversely Correlated with CD8A and PD-L1 mRNA levels in The Cancer Genome Atlas (TCGA) data.

Volcano plot showing the Spearman’s correlation and estimated significance of SMARCB1 with CD8A (left) and PD-L1 (right) mRNA levels from RNA sequencing data across TCGA cancer types calculated by TIMER. Each dot indicates a cancer type in TCGA and red dots denote significant correlations (p < 0.05).

Figure 4.. Patients Treated with Immune Checkpoint Inhibition.

A-D Epithelioid sarcoma case. A. INI1 immunohistochemistry (IHC) from initial diagnosis showing negative nuclear INI1 in tumor cells at 40x (400 x magnification). B. PD-L1 IHC from resection after neoadjuvant pazopanib and radiation therapy showing PD-L1 staining in tumor cells and peritumor lymphocytes. C. Cutaneous lesions prior to talimogene laherparepvec (T-VEC) injections. D. Cutaneous lesions decreased in size after 3 cycles of T-VEC and continued pembrolizumab treatment. E-H Chordoma case. E. INI1 IHC from initial diagnosis showing negative INI1 staining in tumor cells. F. PD-L1 IHC from resection after relapse showing PD-L1 staining in 5% of tumor cells. G. Computerized Tomography (CT) scan showing pulmonary nodule (arrow) measuring 1.2cm before treatment with nivolumab. H. CT scan showing persistent response of the same pulmonary nodule after 28 cycles of treatment with nivolumab. I-L Malignant Rhabdoid tumor. I. IHC from initial diagnosis showing negative INI1 staining in tumor cells. J. Magnetic resonance imaging (MRI) of soft tissue lesion (arrow) measuring 14.3mm by 8.9mm at the time of diagnosis. K. MRI of same lesion (arrow) measuring 12.2mm by 8.0mm after 6 weeks of treatment with pembrolizumab. L. MRI of same lesion (arrow) measuring 9.4mm by 6.9mm after 14 weeks of pembrolizumab therapy.