Spatial profiling of ovarian clear cell carcinoma reveals immune-hot features.

Ovarian clear cell carcinoma (OCCC) has a high incidence in Asia, with frequent occurrence at an early stage, but without sufficient data on molecular stratification for high-risk patients. Recently, immune-hot features have been proposed as indicators of poor prognosis in early stage OCCC. Specific patterns of intratumoral heterogeneity associated with immune-hot features must be defined. NanoString Digital Spatial Profiling technology was used to decipher the spatial distribution of the 18-plex protein panel. ROIs were collected based on the reference hematoxylin and eosin (H&E)-stained morphology. Areas of illumination (AOIs) were defined according to the ROI segmentation using the fluorescence signals of the visualization markers pan-cytokeratin (PanCK), CD45, or DNA. Unsupervised hierarchical clustering of 595 AOIs from 407 ROIs showed that the PanCK segments expressed different combinations of immune markers, suggestive of immune mimicry. Three immune-hot clusters were identified: granzyme B high (GZMB), immune signal high (IH), and immune-like cells (IL); two immune-cold clusters were identified: fibronectin high (FN) and immune checkpoint high cells (IC). In tumor samples at FIGO stage IC1/2 experiencing recurrence, there was an increased occurrence of PanCK+ AOIs with IH and IL groups in the papillary morphology surrounded by macrophage lineage tumor-infiltrating immune cells (TIIs). In contrast, for tumor samples at FIGO stage IC3/II with recurrence, PanCK + AOIs were prevalent in the FN group, particularly those with tubulocystic morphology surrounded by lymphoid lineage non-TIIs. Our work on the spatial profiling of early stage OCCC tumors revealed that the immune mimicry of tumor cells, presence of TIIs, and morphological patterns were associated with recurrence, which switched during tumor progression.

Identification of Early Events in Serrated Pathway Colorectal Tumorigenesis by Using Digital Spatial Profiling.

INTRODUCTION: The colorectal serrated pathway involves precursor lesions known as sessile serrated lesions (SSL) and traditional serrated adenomas (TSA). Mutations in BRAF or KRAS are crucial early events in this pathway. Additional genetic and epigenetic changes contribute to the progression of these lesions into high-grade lesions and, eventually, invasive carcinoma. METHODS: We employed digital spatial profiling to investigate the transcriptional changes associated with SSL and TSA. The genes identified are confirmed by immunohistochemical (IHC) staining. Colorectal cancer (CRC) cell lines with CEACAM6 overexpression and knockdown were established to study the roles of CEACAM6 on tumorigenesis of CRC. RESULTS: Ten genes were upregulated in SSL and TSA, and seven were upregulated in both types of lesions. IHC staining confirmed overexpression of CEACAM6, LCN2, KRT19, and lysozyme in SSL and TSA. CEACAM6 expression is an early event in the serrated pathway but a late event in the conventional pathway. Using cell line models, we confirmed that CEACAM6 promotes CRC cells' proliferation, migration, and invasion abilities. CONCLUSION: These results highlight that the transcriptional changes in the early stages of tumorigenesis exhibit relative uniformity. Identifying these early events may hold significant promise in elucidating the mechanisms behind tumor initiation.

Immune-Hot tumor features associated with recurrence in early-stage ovarian clear cell carcinoma.

Ovarian clear cell carcinoma (OCCC) is a distinct histotype of ovarian cancer, which usually presages a worse prognosis upon recurrence. Identifying patients at risk for relapse is an unmet need to improve outcomes. A retrospective cohort analysis of 195 early-stage OCCC patients diagnosed between January 2011 and December 2019 at National Taiwan University Hospital was conducted to identify prognostic factors for recurrence, progression-free survival (PFS) and overall survival (OS). Molecular profiling of tumors was performed in a case-controlled cohort matched for adjuvant therapy for biomarker discovery. Multivariate Cox proportional hazard model revealed that paclitaxel-based chemotherapy was associated with better PFS than nonpaclitaxel chemotherapy (HR = 0.19, P = .006). The addition of bevacizumab was associated with better PFS, compared to no bevacizumab (HR = 0.09, P = .02). Neither showed significant improvement in OS. Recurrence is associated with an Immune-Hot tumor feature (P = .03), the CTLA-4-high subtype (P = .01) and increased infiltration of immune cells in general. The Immune-Hot feature (HR = 3.39, P = .005) and the CTLA-4-high subtype (HR = 2.13, P = .059) were associated with worse PFS. Immune-Hot tumor features could prognosticate recurrence in early-stage OCCC.

A CRYBB2 mutation in a Taiwanese family with autosomal dominant cataract.

BACKGROUND/PURPOSE: To identify the underlying genetic cause of a Taiwanese family with autosomal dominant cerulean cataract. METHODS: A three-generation cerulean cataract family with 13 affected and 13 normal was identified. Whole exome sequencing, whole genome single nucleotide polymorphism genotyping and haplotype analysis, and fine mapping using polymorphic short tandem repeat markers were used to identify the causative gene mutation. RESULTS: Whole genome single nucleotide polymorphism genotyping and haplotype analysis mapped the candidate disease loci to chromosome 18 and chromosome 22. Polymorphic short tandem repeat markers further narrowed down the disease interval to chromosome 22 between markers D22S1174 and D22S1163. Whole exome sequencing was performed on selected individuals. Polymorphisms detected were filtered based on their genomic positions, allele frequency (<1%), and segregation within the pedigree. Affected individuals were found to be heterozygous carrying a C to T mutation on exon 6 of the CRYBB2 gene (with SNP ID: rs74315489). The mutation was predicted to produce a premature stop mutation Q155X. The mutation is co-segregation across the pedigree and the disease "T" allele was not detected in healthy members of the family and in additional 50 normal controls (100 chromosomes). Phylogenic protein alignment was also performed for the CRYBB2 gene across 68 species ranging from fishes, Sauropsida, Placentalia, carnivores, rodents, and primates with total 56 orthologous genes. The Q155 residue is 100% conserved across the evolutionary tree, indicating its crucial function. CONCLUSION: Here we identify the first Taiwanese cerulean cataract family carrying a CRYBB2_Q155X mutation.

Nodal reactive proliferation of monocytoid B-cells may represent atypical memory B-cells.

BACKGROUND: Reactive lymphadenopathies such as toxoplasmosis and cytomegalovirus lymphadenitis are associated with monocytoid cell proliferation. Monocytoid cells are B-lymphocytes with an undetermined subset. METHODS: Using digital spatial profiling whole transcriptome analyses, this study compared monocytoid and control B-cells. The B-cell subset of monocytoid cells was assigned according to gene expression profiles. RESULTS: This study identified 466 differentially expressed genes between monocytoid and control B-cells. The cellular deconvolution algorithm identified monocytoid cells as memory B-cells instead of as naïve B-cells. A comparison of the upregulated genes revealed that atypical memory B-cells had the largest number of genes overlapping with monocytoid cells compared with other memory B-cell subsets. Atypical memory B-cell markers, namely TBX21 (T-bet), FCRL4 (IRTA1), and ITGAX (CD11c), were all upregulated in monocytoid cells. Similar to atypical memory B-cells, monocytoid cells exhibited (1) upregulated transcription factors (TBX21, TOX), (2) upregulated genes associated with B-cell inhibition (FCRL5, FCRL4) and downregulated genes associated with B-cell activation (PIK3CG, NFKB1A, CD40), (3) downregulated cell cycle-related genes (CDK6, MYC), and (4) downregulated cytokine receptors (IL4R). This study also analyzed the expression of monocytoid cell signature genes in various memory B-cell subsets. Atypical memory B-cells exhibited a gene expression pattern similar to that of monocytoid cells, but other memory B-cell subsets did not. Furthermore, monocytoid cells and marginal zone lymphomas differed in gene expression profiles. CONCLUSION: Spatial transcriptomic analyses indicated that monocytoid cells may be atypical memory B-cells.