BACKGROUND. Germline mutations in telomerase and other telomere maintenance genes manifest in the premature aging short telomere syndromes. Myelodysplastic syndromes and acute myeloid leukemia (MDS/AML) account for 75% of associated malignancies, but how these cancers overcome the inherited telomere defect is unknown. METHODS. We used ultra-deep targeted sequencing to detect somatic reversion mutations in 17 candidate telomere lengthening genes among controls and patients with short telomere syndromes with and without MDS/AML, and we tested the functional significance of these mutations. RESULTS. While no controls carried somatic mutations in telomere maintenance genes, 29% (16 of 56) of adults with germline telomere maintenance defects carried at least 1 (P < 0.001), and 13% (7 of 56) had 2 or more. In addition to TERT promoter mutations, which were present in 19%, another 13% of patients carried a mutation in POT1 or TERF2IP. POT1 mutations impaired telomere binding in vitro and some mutations were identical to ones seen in familial melanoma associated with longer telomere length. Exclusively in patients with germline defects in telomerase RNA (TR), we identified somatic mutations in nuclear RNA exosome genes RBM7, SKIV2L2, and DIS3, where loss-of-function upregulates mature TR levels. Somatic reversion events in 6 telomere-related genes were more prevalent in patients who were MDS/AML-free (P = 0.02, RR 4.4, 95% CI 1.2–16.7), and no patient with MDS/AML had more than 1 reversion mutation. CONCLUSION. Our data indicate that diverse adaptive somatic mutations arise in the short telomere syndromes. Their presence may alleviate the telomere crisis that promotes transformation to MDS/AML. FUNDING. This work was supported by the NIH, the Commonwealth Foundation, the S&R Foundation Kuno Award, the Williams Foundation, the Vera and Joseph Dresner Foundation, the MacMillan Pathway to Independence Award, the American Society of Hematology Scholar Award, the Johns Hopkins Research Program for Medical Students, and the Turock Scholars Fund.

Purpose: Although immunotherapy regimens have significantly improved treatment options for patients with advanced hepatocellular carcinoma (HCC), the optimal use of these regimens in earlier disease stages remains poorly defined. Patients and Methods: We conducted a single-institution, single-arm pilot study (NCT04857684) of neoadjuvant stereotactic body radiotherapy (SBRT) followed by 2 cycles of atezolizumab plus bevacizumab and subsequent surgical resection in patients with initially resectable HCC (n = 8). The primary endpoint was safety as defined by the proportion of patients with grade 3 to 4 treatment-related adverse events (trAE) by Common Terminology Criteria for Adverse Events v5.0. Furthermore, we dissected the detailed remodeling of the tumor immune microenvironment following treatment using single-cell resolution spatial transcriptomic method. Results: Only 1 patient experienced a grade 3 trAE. Seven of 8 patients proceeded to surgery, and all achieved margin-negative (R0) resection; 1 patient did not proceed because of subsequent disagreement of resectability. One patient achieved a pathologic complete response, and all resected patients were relapse-free at data cutoff (median follow-up, 16.3 months; range, 2.1–19.9). Compared with unmatched treatment-naïve HCC specimens, posttreatment specimens showed significantly higher anticancer immune infiltration, including organized peritumoral aggregates. Immune infiltration and its proximity to tumor cells correlated with preoperative radiographic response. Conclusions: This study provides proof of concept that neoadjuvant SBRT and immunotherapy is safe and provides a clear rationale for additional prospective clinical studies utilizing this strategy.

Supplementary Table S5 from Neoadjuvant Stereotactic Body Radiotherapy plus Immune Therapy Favorably Remodels the Hepatocellular Carcinoma Tumor Microenvironment

Figure S1. (A) Cross-sectional imaging showing baseline and pre-surgery CT scans of remaining patients that underwent both sets of imaging. Patients a-c had partial response per RECIST 1.1 criteria. Patients d-f had stable disease per RECIST 1.1 criteria. (B) Alpha fetoprotein (AFP), absolute lymphocyte count (ALC), white blood cell count (WBC), hemoglobin (Hgb), and platelet (PLT) count trends with time over the trial treatment duration. (C) Trends in individual trial participant tumor sizes before/after neoadjuvant therapy, assessed radiographically. (D) Duration of follow-up for each individual participant in the trial.

Fig. S4. Spatial localization of immune aggregates. (A) Spatial distribution of immune aggregates in TMA1, TMA2, HCC1, HCC2, HCC3, HCC4, and HCC5 slides. Red dots indicate cells within immune aggregates; gray dots represent other cells. Red boxes indicate tumor FOVs, green boxes indicate normal liver FOVs, purple boxes represent both FOVs (containing both normal liver regions and malignant cell regions), and gray boxes indicate other FOVs. (B) Comparison of immune aggregate size between the TxN and postTx groups, with size defined by the number of constituent cells. (C) Comparison of cellular composition between FOVs containing immune aggregates (IA FOVs) and FOVs without immune aggregates (Non-IA FOVs) in the TxN and postTx groups. (D) Cellular composition of tumor margin immune aggregates (IA-margin) and intratumoral immune aggregates (IA-tumor) in the postTx group.