In ultraviolet (UV) radiation–exposed skin, mutations fuel clonal cell growth. The relationship between UV exposure and the accumulation of clonal mutations (CMs) and the correlation between CMs and skin cancer risk are largely unexplored. We characterized 450 individual-matched sun-exposed (SE) and non-SE (NE) normal human skin samples. The number and relative contribution of CMs were significantly different between SE and NE areas. Furthermore, we identified hotspots in TP53, NOTCH1, and GRM3 where mutations were significantly associated with UV exposure. In the normal skin from patients with cutaneous squamous cell carcinoma, we found that the cancer burden was associated with the UV-induced mutations, with the difference mostly conferred by the low-frequency CMs. These findings provide previously unknown information on UV’s carcinogenic effect and pave the road for future development of quantitative assessment of subclinical UV damage and skin cancer risk.

Osteolytic lesions (OL) characterize symptomatic multiple myeloma. The mechanisms of how malignant plasma cells (PC) cause OL in one region while others show no signs of bone destruction despite subtotal infiltration remain unknown. We report on a single-cell RNA sequencing (scRNA-seq) study of PC obtained prospectively from random bone marrow aspirates (BM) and paired imaging-guided biopsies of OL. We analyze 148,630 PC from 24 different locations in 10 patients and observe vast inter- and intra-patient heterogeneity based on scRNA-seq analyses. Beyond the limited evidence for spatial heterogeneity from whole-exome sequencing, we find an additional layer of complexity by integrated analysis of anchored scRNA-seq datasets from the BM and OL. PC from OL are characterized by differentially expressed genes compared to PC from BM, including upregulation of genes associated with myeloma bone disease like DKK1, HGF and TIMP-1 as well as recurrent downregulation of JUN/FOS, DUSP1 and HBB. Assessment of PC from longitudinally collected samples reveals transcriptional changes after induction therapy. Our study contributes to the understanding of destructive myeloma bone disease.

BackgroundMicrosatellite instability (MSI) occurs in 3% of urothelial carcinomas as a result of germline or somatic loss of function mutation in mismatch repair (MMR) proteins.1 Although MSH4 is a member of the DNA MMR mutS family, the association of MSH4 mutation with MSI has not been described. We report a complete responder to PD-L1 blockade who had MSH4 mutated metastatic bladder cancer with mixed histology and MSI. The genomics of urothelial, plasmacytoid and squamous histology was characterized individually through microdissection.Case presentationAn 81-year-old man was diagnosed with metastatic urothelial carcinoma 8 months after a cystectomy for muscle invasive bladder cancer. His disease was primary refractory to first-line platinum-based chemotherapy but attained complete response to second-line atezolizumab. PCR-based assay revealed MSI high. The tumor mutational burden was elevated to 36.7 mut/Mb. However, immunohistochemistry of MLH1, MSH2, MSH6 and PMS2 was intact. Whole exome sequencing confirmed that the above mentioned four classic MMR genes were wild type but revealed a deleterious MSH4 L359I mutation with variant allele fraction of 30% and Polyphen2 score of 0.873. The association of MSH4 alterations and MSI-H was independently verified in two publicly available MSI-H colorectal cancer datasets.ConclusionsThe novel MSH4 L359I mutation is associated with MSI and high mutational burden leading to remarkable response to PD-L1 blockade. More studies are warranted to establish the causality relationship between MSH4 and MSI.

Osteolytic lesions (OL) characterize symptomatic multiple myeloma. The mechanisms of how malignant plasma cells (PC) cause OL in one region while others show no signs of bone destruction despite subtotal infiltration remain unknown. We report the first single-cell RNA sequencing (scRNA-seq) study of PC obtained prospectively from random bone marrow aspirates (BM) and paired imaging-guided biopsies of OL. We analyzed 148,630 PC from 24 different locations in 10 patients and observed vast inter- and intra-patient heterogeneity based on scRNA-seq analyses. Beyond the limited evidence for spatial heterogeneity from whole-exome sequencing, we found an additional layer of complexity by integrated analysis of anchored scRNA-seq datasets from the BM and OL. PC from OL were characterized by differentially expressed genes compared to PC from BM, including upregulation of genes associated with myeloma bone disease like DKK1, HGF and TIMP-1 as well as recurrent downregulation of JUN/FOS, DUSP1 and HBB. Assessment of PC from longitudinally collected samples revealed transcriptional changes after induction therapy. Our study, based on the largest number of PC analyzed by scRNA-seq, contributes to the understanding of destructive myeloma bone disease.

Spatial heterogeneity is a common phenomenon in metastatic solid tumors and an evolving concept in multiple myeloma (MM). The interplay between malignant plasma cells (PC) and the microenvironment has not yet been analysed in MM. For this purpose, we performed bone marrow aspirates and imaging-guided biopsies of corresponding lesions in newly diagnosed (NDMM) and relapsed (RRMM) patients. PC were isolated and subjected to whole-exome sequencing (WES). Non-PC were studied with next-generation flow (NGF) and T-cell receptor sequencing (TCRseq) to analyze the connection between malignant and non-malignant cells in the bone marrow and in lesions. While we observed a strong overlap from WES, NGF and TCRseq in patients with intramedullary disease, WES revealed significant spatial heterogeneity in patients with extramedullary disease. NGF showed significant immunosuppression in RRMM compared to NDMM as indicated by less myeloid dendritic cells (mDC), unswitched memory B-cells, Th9 cells and CD8 effector memory T-cells but more NK and regulatory T-cells. Additionally, less TCR sequences were detected in RRMM compared to NDMM and healthy individuals. After induction therapy, TCR repertoire richness increased to levels of healthy individuals and NGF showed more regulatory T-cells and myeloid derived suppressor cells, regardless of depth of response. Clinical significance of imaging-guided biopsies of lesions was demonstrated by detection of monoclonal PC in patients without measurable residual disease (MRD) in aspirates from the iliac crest as well as identification of secondary primary malignancies in MRD-negative patients. Furthermore, site-specific clones with different drug susceptibilities and genetically defined high-risk features were detected by our workflow.