Natural history of clonal haematopoiesis seen in real-world haematology settings.

Recursive partitioning of healthy consortia led to the development of the Clonal Hematopoiesis Risk Score (CHRS) for clonal haematopoiesis (CH); however, in the practical setting, most cases of CH are diagnosed after patients present with cytopenias or related symptoms. To address this real-world population, we characterize the clinical trajectories of 94 patients with CH and distinguish CH harbouring canonical DNMT3A/TET2/ASXL1 mutations alone ('sole DTA') versus all other groups ('non-sole DTA'). TET2, rather than DNMT3A, was the most prevalent mutation in the real-world setting. Sole DTA patients did not progress to myeloid neoplasm (MN) in the absence of acquisition of other mutations. Contrastingly, 14 (20.1%) of 67 non-sole DTA patients progressed to MN. CHRS assessment showed a higher frequency of high-risk CH in non-sole DTA (vs. sole DTA) patients and in progressors (vs. non-progressors). RUNX1 mutation conferred the strongest risk for progression to MN (odds ratio [OR] 10.27, 95% CI 2.00-52.69, p = 0.0053). The mean variant allele frequency across all genes was higher in progressors than in non-progressors (36.9% ± 4.62% vs. 24.1% ± 1.67%, p = 0.0064). This analysis in the post-CHRS era underscores the natural history of CH, providing insight into patterns of progression to MN.

The BRAF oncoprotein functions through the transcriptional repressor MAFG to mediate the CpG Island Methylator phenotype.

Most colorectal cancers (CRCs) containing activated BRAF (BRAF[V600E]) have a CpG island methylator phenotype (CIMP) characterized by aberrant hypermethylation of many genes, including the mismatch repair gene MLH1. MLH1 silencing results in microsatellite instability and a hypermutable phenotype. Through an RNAi screen, here we identify the transcriptional repressor MAFG as the pivotal factor required for MLH1 silencing and CIMP in CRCs containing BRAF(V600E). In BRAF-positive human CRC cell lines and tumors, MAFG is bound at the promoters of MLH1 and other CIMP genes, and recruits a corepressor complex that includes its heterodimeric partner BACH1, the chromatin remodeling factor CHD8, and the DNA methyltransferase DNMT3B, resulting in hypermethylation and transcriptional silencing. BRAF(V600E) increases BRAF/MEK/ERK signaling resulting in phosphorylation and elevated levels of MAFG, which drives DNA binding. Analysis of transcriptionally silenced CIMP genes in KRAS-positive CRCs indicates that different oncoproteins direct the assembly of distinct repressor complexes on common promoters.

Prosurvival kinase PIM2 is a therapeutic target for eradication of chronic myeloid leukemia stem cells.

A major obstacle to curing chronic myeloid leukemia (CML) is the intrinsic resistance of CML stem cells (CMLSCs) to the drug imatinib mesylate (IM). Prosurvival genes that are preferentially expressed in CMLSCs compared with normal hematopoietic stem cells (HSCs) represent potential therapeutic targets for selectively eradicating CMLSCs. However, the discovery of such preferentially expressed genes has been hampered by the inability to completely separate CMLSCs from HSCs, which display a very similar set of surface markers. To overcome this challenge, and to minimize confounding effects of individual differences in gene expression profiles, we performed single-cell RNA-seq on CMLSCs and HSCs that were isolated from the same patient and distinguished based on the presence or absence of BCR-ABL. Among genes preferentially expressed in CMLSCs is PIM2, which encodes a prosurvival serine-threonine kinase that phosphorylates and inhibits the proapoptotic protein BAD. We show that IM resistance of CMLSCs is due, at least in part, to maintenance of BAD phosphorylation by PIM2. We find that in CMLSCs, PIM2 expression is promoted by both a BCR-ABL-dependent (IM-sensitive) STAT5-mediated pathway and a BCR-ABL-independent (IM-resistant) STAT4-mediated pathway. Combined treatment with IM and a PIM inhibitor synergistically increases apoptosis of CMLSCs, suppresses colony formation, and significantly prolongs survival in a mouse CML model, with a negligible effect on HSCs. Our results reveal a therapeutically targetable mechanism of IM resistance in CMLSCs. The experimental approach that we describe can be generally applied to other malignancies that harbor oncogenic fusion proteins or other characteristic genetic markers.

Molecular and immunophenotypic characterization of anal squamous cell carcinoma reveals distinct clinicopathologic groups associated with HPV and TP53 mutation status.

Squamous cell carcinoma (SqCC) is the most common malignancy of the anal canal, where it is strongly associated with HPV infection. Characteristic genomic alterations have been identified in anal SqCC, but their clinical significance and correlation with HPV status, pathologic features, and immunohistochemical markers are not well established. We examined the molecular and clinicopathologic features of 96 HPV-positive and 20 HPV-negative anal SqCC. HPV types included 89 with HPV16, 2 combined HPV16/HPV18, and 5 HPV33. HPV-positive cases demonstrated frequent mutations or amplifications in PIK3CA (30%; p = 0.027) or FBXW7 mutations (10%). HPV-negativity was associated with frequent TP53 (53%; p = 0.00001) and CDKN2A (21%; p = 0.0045) mutations. P16 immunohistochemistry was positive in all HPV-positive cases and 3/20 HPV-negative cases (p < 0.0001; sensitivity: 100%; specificity: 85%) and was associated with basaloid morphology (p = 0.0031). Aberrant p53 immunohistochemical staining was 100% sensitive and specific for TP53 mutation (p < 0.0001). By the Kaplan-Meier method, HPV-negativity, aberrant p53 staining, and TP53 mutation were associated with inferior overall survival (OS) (p < 0.0001, p = 0.0103, p = 0.0103, respectively) and inferior recurrence-free survival (p = 0.133, p = 0.0064, and p = 0.0064, respectively). TP53/p53 status stratified survival probability by HPV status (p = 0.013), with HPV-negative/aberrant p53 staining associated with the worst OS, HPV-positive/wild-type p53 with best OS, and HPV-positive/aberrant p53 or HPV-negative/wild-type p53 with intermediate OS. On multivariate analysis HPV status (p = 0.0063), patient age (p = 0.0054), T stage (p = 0.039), and lymph node involvement (p = 0.044) were independently associated with OS. PD-L1 expression (CPS ≥ 1) was seen in 30% of HPV-positive and 40% of HPV-negative cases, and PD-L1 positivity was associated with a trend toward inferior OS within the HPV-negative group (p = 0.064). Our findings suggest that anal SqCC can be subclassified into clinically, pathologically, and molecularly distinct groups based on HPV and TP53 mutation status, and p16 and p53 immunohistochemistry represent a clinically useful method of predicting these prognostic groups.

CD56 expression in basaloid anal squamous cell carcinoma - A potential diagnostic pitfall.

Anal squamous cell carcinoma (SqCC) is a morphologically heterogeneous entity. Basaloid and non-keratinizing anal SqCC may be confused with other tumors including neuroendocrine carcinoma due to morphologic overlap, and expression of neuroendocrine markers is not well-studied in anal SqCC. Prompted by a case of anal SqCC that was initially misdiagnosed as neuroendocrine carcinoma on the basis of morphology and CD56 expression, we retrospectively examined the expression of neuroendocrine markers CD56, synaptophysin, and chromogranin in 48 cases of basaloid anal SqCC, with clinicopathologic correlation. HPV16 was identified in 46 cases, HPV33 in one case, and one case was HPV-negative. Three (6.3%) cases demonstrated CD56 expression, including two with diffuse and one with focal expression. Two CD56-positive cases demonstrated basaloid morphology with peripheral palisading and the other demonstrated adenoid cystic/cylindroma-like morphology. None of the cases showed significant synaptophysin or chromogranin expression. The three cases expressing CD56 were HPV16-positive, and one demonstrated a CTNNB1 mutation. There was no difference in clinicopathologic features including stage, outcome, or HPV status, between CD56-positive and negative groups. Our findings support that CD56 expression is infrequently expressed in anal SqCC and is not indicative of neuroendocrine differentiation in the absence of expression of more specific neuroendocrine markers such as synaptophysin and chromogranin. Pathologists should be aware that CD56 expression may occur in basaloid anal SqCC and is a diagnostic pitfall due to morphologic overlap with neuroendocrine carcinoma and other tumors including basal cell carcinoma.

Dedifferentiated Carcinoma of the Endometrium Associated With Low-grade Müllerian Adenosarcoma: A Clinicopathologic Case Report Including the Immunohistochemical and Molecular Profile.

Dedifferentiated carcinoma is defined as undifferentiated carcinoma coexisting with a second component of FIGO grade 1 or 2 endometrioid carcinoma. It is a rare entity with highly aggressive behavior. Dedifferentiated carcinoma combined with another primary uterine tumor is even rarer. We describe a case containing 3 different morphologies comprised of a dedifferentiated carcinoma associated with a low-grade endometrioid carcinoma coexisting with a low-grade Müllerian adenosarcoma. We also used targeted genomic analysis to show all 3 components arise from the same founding clone and identify novel mutations that drive tumor progression.

Next-Generation Sequencing to Detect Deletion of RB1 and ERBB4 Genes in Chromophobe Renal Cell Carcinoma: A Potential Role in Distinguishing Chromophobe Renal Cell Carcinoma from Renal Oncocytoma.

Overlapping morphologic, immunohistochemical, and ultrastructural features make it difficult to diagnose chromophobe renal cell carcinoma (ChRCC) and renal oncocytoma (RO). Because ChRCC is a malignant tumor, whereas RO is a tumor with benign behavior, it is important to distinguish these two entities. We aimed to identify genetic markers that distinguish ChRCC from RO by using next-generation sequencing (NGS). NGS for hotspot mutations or gene copy number changes was performed on 12 renal neoplasms, including seven ChRCC and five RO cases. Matched normal tissues from the same patients were used to exclude germline variants. Rare hotspot mutations were found in cancer-critical genes (TP53 and PIK3CA) in ChRCC but not RO. The NGS gene copy number analysis revealed multiple abnormalities. The two most common deletions were tumor-suppressor genes RB1 and ERBB4 in ChRCC but not RO. Fluorescence in situ hybridization was performed on 65 cases (ChRCC, n = 33; RO, n = 32) to verify hemizygous deletion of RB1 (17/33, 52%) or ERBB4 (11/33, 33%) in ChRCC, but not in RO (0/32, 0%). In total, ChRCCs (23/33, 70%) carry either a hemizygous deletion of RB1 or ERBB4. The combined use of RB1 and ERBB4 fluorescence in situ hybridization to detect deletion of these genes may offer a highly sensitive and specific assay to distinguish ChRCC from RO.

Common BRAF(V600E)-directed pathway mediates widespread epigenetic silencing in colorectal cancer and melanoma.

During cancer development, it is well established that many genes, including tumor suppressor genes, are hypermethylated and transcriptionally repressed, a phenomenon referred to as epigenetic silencing. In general, the factors involved in, and the mechanistic basis of, epigenetic silencing during cancer development are not well understood. We have recently described an epigenetic silencing pathway, directed by the oncogenic B-Raf proto-oncogene (BRAF) variant BRAF(V600E), that mediates widespread epigenetic silencing in colorectal cancer (CRC). Notably, the BRAF(V600E) mutation is also present in 50-70% of melanomas. Here, we show that the same pathway we identified in CRC also directs epigenetic silencing of a similar set of genes in BRAF-positive melanoma. In both CRC and melanoma, BRAF(V600E) promotes epigenetic silencing through up-regulation of v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog G (MAFG), a transcriptional repressor with sequence-specific DNA-binding activity. The elevated concentration of MAFG drives DNA binding on the promoter. Promoter-bound MAFG recruits a set of corepressors that includes its heterodimeric partner BTB and CNC homology 1, basic leucine zipper transcription factor 1 (BACH1), the chromatin remodeling factor chromodomain helicase DNA-binding protein 8 (CHD8), and the DNA methyltransferase DNMT3B, resulting in hypermethylation and transcriptional silencing. Our results reveal a common BRAF(V600E)-directed transcriptional regulatory pathway that mediates epigenetic silencing in unrelated solid tumors and provide strong support for an instructive model of oncoprotein-directed epigenetic silencing.