Cloning Analysis Research Articles

How I Diagnose EBV-Positive B- and T-Cell Lymphoproliferative Disorders.

Epstein-Barr virus (EBV)-associated lymphoproliferative disorders (LPDs) encompass a group of well-defined entities of B-, T-, and natural killer (NK)-cell derivation. The diagnosis of these disorders is challenging because of clinical and morphologic features that may overlap with other benign and malignant EBV+ lymphoproliferations. This review describes our approach to the diagnosis of EBV-associated LPDs. Two cases are presented that illustrate how we diagnose EBV-associated LPDs. The first case represents a systemic EBV+ T-cell lymphoma of childhood and the second case an EBV+ mucocutaneous ulcer. The clinicopathologic features that help distinguish these entities from biological and morphologic mimickers are emphasized. The accurate diagnosis of EBV-associated LPDs requires the incorporation of histologic and immunophenotypic features, the assessment of the EBV latency program, and, most important, complete clinical findings. Clonality analysis is not helpful in distinguishing benign from malignant EBV+ LPDs. The better understanding of EBV-associated LPDs has resulted in the recognition of well-defined entities of B-, T-, and NK-cell derivation and consequently improvement of their treatment with curative intent. It is critical to distinguish benign from malignant EBV+ LPDs to avoid overtreatment.

Clonal relationship analysis of 4 cases of multifocal primary lung cancer diagnosed histologically based on next-generation sequencing

目的： 基于二代测序法，探讨经组织学诊断为同步多灶原发性肺癌（SMPLC）病例的分子病理学特征及克隆起源。 方法： 对4例SMPLC的8个病灶进行全基因组外显子测序，并进行基因突变、基因组稳定性、克隆性关系及克隆演化的分析。 结果： 突变分析显示例1、2及3的2个病灶间突变的一致性均为0，例4的一致性为3/（50+44）。例1的2个病灶基因组稳定性差别较大。例2的2个病灶基因组稳定性差别不大。例3、4的2个病灶基因组均稳定。用单核苷酸变异测试对样本突变信息进行分析，结果显示例1、2、3的2个病灶间相互独立。例4的2个病灶之间存在克隆关系。对样本拷贝数变异进行分析，认为例1、2的2个病灶彼此独立。对例4进行克隆演化分析，提示2个病灶来源于同一个克隆，可能为肺内转移。 结论： 二代测序分析是对多灶肺癌组织学诊断的重要补充。不同组织学类型，或含原位癌病灶是判断SMPLC较可靠的依据。但不同组织学亚型诊断价值有限。为保证肿瘤分期和术后治疗的精准性，有必要对多灶肺癌的每一个病灶进行基因突变检测。.

Single cell clonal analysis identifies an AID‐dependent pathway of plasma cell differentiation

Germinal centers (GC) are microstructures where B cells that have been activated by antigen can improve the affinity of their B cell receptors and differentiate into memory B cells (MBCs) or antibody‐secreting plasma cells. Here, we have addressed the role of activation‐induced deaminase (AID), which initiates somatic hypermutation and class switch recombination, in the terminal differentiation of GC B cells. By combining single cell transcriptome and immunoglobulin clonal analysis in a mouse model that traces AID‐experienced cells, we have identified a novel subset of late‐prePB cells (L‐prePB), which shares the strongest clonal relationships with plasmablasts (PBs). Mice lacking AID have various alterations in the size and expression profiles of transcriptional clusters. We find that AID deficiency leads to a reduced proportion of L‐prePB cells and severely impairs transitions between the L‐prePB and the PB subsets. Thus, AID shapes the differentiation fate of GC B cells by enabling PB generation from a prePB state.

Polyclonal evolution of Fanconi anemia to MDS and AML revealed at single cell resolution

BackgroundFanconi anemia (FA) is a rare disease of bone marrow failure. FA patients are prone to develop myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). However, the molecular clonal evolution of the progression from FA to MDS/AML remains elusive.MethodsHerein, we performed a comprehensive genomic analysis using an FA patient (P1001) sample that transformed to MDS and subsequently AML, together with other three FA patient samples at the MDS stage.ResultsOur finding showed the existence of polyclonal pattern in these cases at MDS stage. The clonal evolution analysis of FA case (P1001) showed the mutations of UBASH3A, SF3B1, RUNX1 and ASXL1 gradually appeared at the later stage of MDS, while the IDH2 alteration become the dominant clone at the leukemia stage. Moreover, single-cell sequencing analyses further demonstrated a polyclonal pattern was present at either MDS or AML stages, whereas IDH2 mutated cell clones appeared only at the leukemia stage.ConclusionsWe thus propose a clonal evolution model from FA to MDS and AML for this patient. The results of our study on the clonal evolution and mutated genes of the progression of FA to AML are conducive to understanding the progression of the disease that still perplexes us.

279. CLONAL RELATIONSHIP AND ALCOHOL CONSUMPTION-ASSOCIATED MUTATIONAL SIGNATURE IN SYNCHRONOUS HYPOPHARYNGEAL TUMORS AND ESOPHAGEAL SQUAMOUS CELL CARCINOMA

Abstract The patients with dual esophageal squamous cell carcinoma (ESCC) and head and neck cancers have poor prognosis, but the underlying genetic pathogenesis is unclear. We hypothesize that independent origin rather than multifocality, which means local or lateral spreading involves the development of synchronous ESCC and hypopharyngeal cancer (HPC). We also hypothesize that multi-regional assessment provides better understanding of the evolution of tumor subclones and assessment of intra-tumor heterogeneity to guide personalized treatment with prognostic implications. Multiple region whole-exome sequencing (M-WES) and clonality analysis were used to assess clonal relationship and spatial inter- or intra-tumor heterogeneity (ITH) in 62 tumor regions, six normal tissues, and 15 blood samples from eight dual ESCC/HPC and ten ESCC patients. All synchronous ESCC/HPC patients had COSMIC 16 mutation signature compared to only 40% ESCC in the current study (p = 0.013) and public ESCC dataset (n = 165, p = 0.003). The mutational landscape and copy number variation (CNV) profiles were completely distinct between the ESCC/HPC; clonality analysis further suggested the two primary tumors shared no or only one clone accompanying independent subclone evolution. The M-WES strategy demonstrated higher sensitivity and accuracy for detection of mutational prevalence and the late branch mutations among different regions in the ESCC tumors compared to traditional single biopsy strategy. Patients with high ITH was significantly associated with both relapse and survival. Our hypothesis-generating M-WES ITH assessment data have implications for prognostication. We are the first to show a distinctive CNV and genetic mutation profile, the predominant alcohol-related COSMIC 16 mutational signature in synchronous ESCC/HPC, providing genetic evidence for a multi-centric independent origin for clonal evolution and the theory of field cancerization implicating an etiologic role of alcohol metabolism in dual ESCC/HPC carcinogenesis.

Malignant clonal evolution drives multiple myeloma cellular ecological diversity and microenvironment reprogramming

BackgroundMultiple myeloma (MM) is a heterogeneous disease with different patterns of clonal evolution and a complex tumor microenvironment, representing a challenge for clinicians and pathologists to understand and dissect the contribution and impact of polyclonality on tumor progression.MethodsIn this study, we established a global cell ecological landscape of the bone marrow (BM) from MM patients, combining single-cell RNA sequencing and single-molecule long-read genome sequencing data.ResultsThe malignant mutation event was localized to the tumor cell clusters with shared mutation of ANK1 and IFITM2 in all malignant subpopulations of all MM patients. Therefore, these two variants occur in the early stage of malignant clonal origin to mediate the malignant transformation of proplasmacytes or plasmacytes to MM cells. Tumor cell stemness index score and pseudo-sequential clonal evolution analysis can be used to divide the evolution model of MM into two clonal origins: types I and IX. Notably, clonal evolution and the tumor microenvironment showed an interactive relationship, in which the evolution process is not only selected by but also reacts to the microenvironment; thus, vesicle secretion enriches immune cells with malignant-labeled mRNA for depletion. Interestingly, microenvironmental modification exhibited significant heterogeneity among patients.ConclusionsThis characterization of the malignant clonal evolution pattern of MM at the single-cell level provides a theoretical basis and scientific evidence for a personalized precision therapy strategy and further development of a potential new adjuvant strategy combining epigenetic agent and immune checkpoint blockade.

Molecular Epidemiological Characterisation of ESBL- and Plasmid-Mediated AmpC-Producing Escherichia coli and Klebsiella pneumoniae at Kamuzu Central Hospital, Lilongwe, Malawi.

The global rise in infections caused by multidrug resistant (MDR) Enterobacterales poses a public health problem. We have performed a molecular epidemiological characterisation of representative plasmid-mediated AmpC (pAmpC) and ESBL-positive clinical isolates of Escherichia coli (n = 38) and Klebsiella pneumoniae (n = 17) from a tertiary hospital in Malawi collected in 2017. BlaCTX-M-15 was the most prevalent ESBL-determinant in E. coli (n = 30/38) and K. pneumoniae (n = 17/17), whereas blaCMY-2 was detected in nearly all AmpC-phenotype E. coli (n = 15/17). Whole genome sequencing revealed dominant globally disseminated E. coli sequence types (STs); ST410 (n = 16), ST131 (n = 7), and ST617 (n = 6). The ST distribution in K. pneumoniae was more diverse but included ST101 (n = 2), ST14 (n = 2), and ST340 (n = 2), all considered high-risk MDR clones. The isolates expressed an MDR profile, including resistance against commonly used antibiotics, such as fluoroquinolones, aminoglycosides, and/or trimethoprim-sulfamethoxazole, and harboured corresponding resistance determinants. Clonal analyses of the major STs of E. coli revealed closely related genetic clusters within ST410, ST131, and ST617 supporting within-hospital transmission between patients and/or via a common reservoir. The overall findings add to the limited knowledge on the molecular epidemiology of MDR E. coli and K. pneumoniae in Malawi and may help health policy makers to identify areas to target when addressing this major threat of antibiotic resistance.

Discovery and characterization of heterogeneous and multipotent fibroblast populations isolated from excised cleft lip tissue

BackgroundRegularly discarded lip tissue obtained from corrective surgeries to close the cleft lip represents an easily accessible and rich source for the isolation of primary fibroblasts. Primary fibroblasts have been described to show compelling similarities to mesenchymal stem cells (MSCs). Hence, cleft lip and palate (CLP) lip-derived fibroblasts could be thought as an intriguing cell source for personalized regenerative therapies in CLP-affected patients.MethodsInitially, we thoroughly characterized the fibroblastic nature of the lip-derived mesenchymal outgrowths by molecular and functional assays. Next, we compared their phenotype and genotype to that of bone marrow-mesenchymal stem cells (BM-MSCs) and of human lung-derived fibroblasts WI38, by assessing their morphology, surface marker expression, trilineage differentiation potential, colony-forming (CFU) capacity, and immunomodulation property. Finally, to better decipher the heterogeneity of our CLP cultures, we performed a single cell clonal analysis and tested expanded clones for surface marker expression, as well as osteogenic and CFU potential.ResultsWe identified intriguingly similar phenotypic and genotypic properties between CLP lip fibroblasts and BM-MSCs, which makes them distinct from WI38. Furthermore, our own data in combination with the complex anatomy of the lip tissue indicated heterogeneity in our CLP cultures. Using a clonal analysis, we discovered single cell-derived clones with increased levels of the MSC markers CD106 and CD146 and clones with variabilities in their commitment to differentiate into bone-forming cells and in their potential to form single cell-derived colonies. However, we were not able to gain clones possessing superior MSC-like capacities when compared to the heterogeneous parental CLP population. Additionally, all clones could still generate contractile forces and retained robust levels of the fibroblast specific marker FSP1, which was not detectable in BM-MSCs.ConclusionsOur results suggest that we isolate heterogeneous populations of fibroblasts from discarded CLP lip tissue, which show a prominently multipotent character in their entirety avoiding the need for elaborate subpopulation selections in vitro. These findings suggest that CLP lip fibroblasts might be a novel potential cell source for personalized regenerative medicine of clinical benefit for CLP patients.

Abstract A17: The immune checkpoints TIGIT and PD-1 are markedly upregulated in NLPHL compared to classical Hodgkin Lymphoma

Abstract Hodgkin lymphoma (HL) comprises two distinct disease entities based on clinical, morphologic and genotypic characteristics. Relative to classical Hodgkin Lymphoma (cHL), nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) is rare, and its tumor microenvironment (TME) is very poorly characterized. With the exception of rituximab, there are no targeted treatments nor advances in the treatment of NLPHL for decades. Unlike cHL, the utility of immune-checkpoint blockade (ICB) has not been evaluated in NLPHL. Diagnostic samples were collected from 49 NLPHL patients from 4 Australian centres and compared with stage-matched cHL patients (and with normal lymph nodes). An integrative transcriptomic, proteomic, T-cell clonal and functional analysis was performed to enable a comparison of the composition of the TME and its contribution to immune-evasion in NLPHL with cHL. 730 cancer-immune related genes were digitally quantified. Relative to cHL, gene set enrichment analysis identified T-cell receptor (TCR) and immune-checkpoint signaling pathway dysregulation in NLPHL. Most striking was prominent differential expression of the immune-transcriptome, particularly enrichment for programmed cell death-1 (PD-1) and T-cell Ig and ITIM domain (TIGIT) in NLPHL versus cHL. These were also over expressed compared to normal lymph nodes. Consistent with this, there was also upregulation of numerous T-cell markers (CD247, CD3D, GZMK, CD28, EOMES) in NLPHL. In contrast, immunosuppressive macrophage (CD163, CD36, CD68, COLEC12, MARCO) and regulatory T-cell genes (FOXP3) were higher in cHL. Importantly, PD-L1 and CD155 (respective ligands for PD-1 and TIGIT) were expressed at the surface of NLPHL and cHL malignant B-cells. Multispectral immunofluorescent microscopy showed intratumoral TIGIT+CD4+ and PD-1+CD4+ T-cells were markedly increased in NLPHL versus cHL and localised within NLPHL follicles. Expanded populations of intratumoral CD4+ T-cell clones were predominantly PD-1+ and frequently also TIGIT+. Multi-parameter flow cytometry and dimensionality reduction was used to establish the distribution of immune checkpoints within circulating T-cell subsets. PD-1+TIGIT+CD4+ T-cells were raised in circulating Treg, TH1 and TH2 subsets in NLPHL versus cHL, and PD-1+TIGIT+ TH2 T-cells displayed raised levels of the exhaustion marker EOMES, collectively indicating systemic T-cell dysfunction. To functionally demonstrate the utility of ICB to stimulate T-cells, an assay using cHL and/or NLPHL cell-lines co-cultured with a genetically engineered effector T-cell-line was developed. This showed that TIGIT/PD-1 dual-blockade was more effective than mono-blockade in inducing NLPHL and cHL tumor-directed CD4+ T-cell activation. Overall, our results indicate that immune-evasion mechanisms in NLPHL are distinct to those operative in cHL, with markedly greater T-cell, PD-1 and TIGIT dysregulation. PD-1 and/or TIGIT blockade warrants evaluation in NLPHL. Citation Format: Jay Gunawardana, Soi C. Law, Muhammed B. Sabdia, Karolina Bednarska, Sandra Brosda, Andreea Zaharia, Hennes Tsang, Lilia M. de Long, Melinda Burgess, Dipti Talaulikar, Justina N. Lee, Emily Jude, Eliza A. Hawkes, Sanjiv Jain, Karthik Nath, Clare Gould, Fiona Swain, Joshua W. D. Tobin, Colm Keane, Simone Birch, Mohamed Shanavas, Cameron Snell, Maher K. Gandhi. The immune checkpoints TIGIT and PD-1 are markedly upregulated in NLPHL compared to classical Hodgkin Lymphoma [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A17.

BioIns-PO-08 - Use of the T cell receptor B-chain constant domains 1 and 2 (TCRBC1 and TCRBC2) studies for the analysis of T-cell clonality

BioIns-PO-08 - Use of the T cell receptor B-chain constant domains 1 and 2 (TCRBC1 and TCRBC2) studies for the analysis of T-cell clonality

Une maladie de Rosai–Dorfman–Destombes de présentation laryngée trompeuse

Rosai-Dorfman-Destombes disease is a rare non-Langerhansian cell histiocytosis characterized by the accumulation of large activated histiocytes in the affected tissues with images of emperipolesis. The diagnosis is not really problematic in the classical forms, with a lymph node presentation, whose histology is very suggestive. However, it can be much more difficult in the extra-nodal forms, which are misleading in both their clinical and histological presentation. We report here a case illustrating this diagnostic difficulty. Firstly, clinically, the disease was revealed by an unusual laryngeal location, responsible for acute obstructive respiratory distress and requiring urgent surgical management. Secondly, histologically, the diagnosis was not evoked in the first instance by analysis of the laryngeal lesion. Indeed, there was a not specific appearing polymorphic infiltrate, associating small lymphocytes, plasma cells and numerous histiocytes, without evidence for a lymphoma after immunohistochemistry and lymphocyte clonality analysis. However, after re-examination of the slides, the histiocytes sometimes appeared large or xanthomised and have a PS100+, CD1a-, langerhine- phenotype, with rare images of emperipolesis. These aspects finally suggested the diagnosis of Rosai-Dorfman-Destombes disease, then confirmed by a cervical lymph node biopsy showing characteristic histological features. Simultaneously, NGS analysis of the laryngeal lesion showed a mutation in the MAP2K1 gene, in accordance with the diagnosis. The patient was treated with revlimid and dexamethasone for 6 months, with complete remission, and is currently undergoing maintenance treatment with revlimid.

In Vivo Clonal Analysis Reveals Random Monoallelic Expression in Lymphocytes That Traces Back to Hematopoietic Stem Cells.

Evaluating the epigenetic landscape in the stem cell compartment at the single-cell level is essential to assess the cells’ heterogeneity and predict their fate. Here, using a genome-wide transcriptomics approach in vivo, we evaluated the allelic expression imbalance in the progeny of single hematopoietic cells (HSCs) as a read-out of epigenetic marking. After 4 months of extensive proliferation and differentiation, we found that X-chromosome inactivation (XCI) is tightly maintained in all single-HSC derived hematopoietic cells. In contrast, the vast majority of the autosomal genes did not show clonal patterns of random monoallelic expression (RME). However, a persistent allele-specific autosomal transcription in HSCs and their progeny was found in a rare number of cases, none of which has been previously reported. These data show that: 1) XCI and RME in the autosomal chromosomes are driven by different mechanisms; 2) the previously reported high frequency of genes under RME in clones expanded in vitro (up to 15%) is not found in clones undergoing multiple differentiation steps in vivo; 3) prior to differentiation, HSCs have stable patterns of autosomal RME. We propose that most RME patterns in autosomal chromosomes are erased and established de novo during cell lineage differentiation.

Analyzing Starvation-Induced Autophagy in the <em>Drosophila melanogaster</em> Larval Fat Body

Autophagy is a cellular self-digestion process. It delivers cargo to the lysosomes for degradation in response to various stresses, including starvation. The malfunction of autophagy is associated with aging and multiple human diseases. The autophagy machinery is highly conserved-from yeast to humans.The larval fat body of Drosophila melanogaster, an analog for vertebrate liver and adipose tissue, provides a unique model for monitoring autophagy in vivo. Autophagy can be easily induced by nutrient starvation in the larval fat body. Most autophagy-related genes are conserved in Drosophila. Many transgenic fly strains expressing tagged autophagy markers have been developed, which facilitates the monitoring of different steps in the autophagy process. The clonal analysis enables a close comparison of autophagy markers in cells with different genotypes in the same piece of tissue. The current protocol details procedures for (1) generating somatic clones in the larval fat body, (2) inducing autophagy via amino acid starvation, and (3) dissecting the larval fat body, aiming to create a model for analyzing differences in autophagy using an autophagosome marker (GFP-Atg8a) and clonal analysis.

A clonality analysis based on T-cell receptor beta and delta loci for high-grade gastrointestinal lymphoma in dogs.

Clonality assays based on antigen receptors are used as adjunct examinations in the diagnosis of lymphoproliferative diseases. We investigated the usefulness of the T-cell receptor beta (TRB) and T-cell receptor delta (TRD) loci in clonality assays for high-grade gastrointestinal (GI) lymphoma in dogs. For TRB, we used primers reported previously; for TRD, we designed primers for each of the V and J genes based on genomic sequences. Genomic DNA was extracted from 39 formalin-fixed, paraffin-embedded sections of high-grade GI lymphoma diagnosed histologically. The sensitivity of TRB and TRD primers for GI lymphoma was 41.0% and 38.5%, respectively, which was lower than the 82.1% sensitivity of T-cell receptor gamma (TRG) primers However, some cases that could not be detected using TRG primers had clonality with either TRB or TRD primers. We found the TRG locus to be more suitable as a first choice for the assay of canine lymphoma clonality than the TRB and TRD loci. However, the detection rate of T-cell clonality may be enhanced using TRB and TRD primers for lymphoma cases not detected using TRG primers.

Antibiotic resistance, biofilm forming ability, and clonal profiling of clinical isolates of Staphylococcus aureus from southern and northeastern India.

Staphylococcus aureus is a pathogen endemic in India and sometimes deadly for patients in intensive care units. To determine the antibiotic-resistance pattern, biofilm forming ability, and clonal type of S. aureus from isolates collected in Tamil Nadu (south) and the Mizoram (northeast) regions of India. We collected S. aureus isolates from diagnostic laboratories in Tamil Nadu and Mizoram. An antibiotic susceptibility test was performed according to Clinical Laboratory and Standards Institute methods. Antibiotic-resistant determinants such as mecA, mecC, blaZ, vanA, vanB, and vanC were confirmed by polymerase chain reaction (PCR). All isolates were further studied for biofilm forming ability. Enterobacterial repetitive intergenic consensus (ERIC)-PCR was used for clonal analysis. A study of 206 clinical isolates showed 52.9% prevalence of methicillin-resistant S. aureus in Tamil Nadu and 49.4% in Mizoram. Minimum inhibitory concentration tests showed a high prevalence of 67% oxacillin resistance in isolates from Tamil Nadu and 49% in isolates from Mizoram. PCR showed 53% mecA in Tamil Nadu and 49% mecA in Mizoram. Vancomycin-intermediate resistance S. aureus (VISA) prevalence was lower in isolates from Tamil Nadu (4%) and Mizoram (5%). All methicillin-resistant S. aureus (MRSA) isolates formed biofilms. Clonal analysis revealed a genetic relatedness between the isolates. The prevalence of MRSA is high in the regions studied, with most of the clinical isolates being multidrug resistant. Adopting appropriate community-based preventive measures and establishing antimicrobial stewardship is highly recommended to minimize the dissemination in antibiotic resistance.

Enabling Clonal Analyses of Yeast in Outer Space by Encapsulation and Desiccation in Hollow Microparticles.

Studying microbes at the single-cell level in space can accelerate human space exploration both via the development of novel biotechnologies and via the understanding of cellular responses to space stressors and countermeasures. High-throughput technologies for screening natural and engineered cell populations can reveal cellular heterogeneity and identify high-performance cells. Here, we present a method to desiccate and preserve microbes in nanoliter-scale compartments, termed PicoShells, which are microparticles with a hollow inner cavity. In PicoShells, single cells are confined in an inner aqueous core by a porous hydrogel shell, allowing the diffusion of nutrients, wastes, and assay reagents for uninhibited cell growth and flexible assay protocols. Desiccated PicoShells offer analysis capabilities for single-cell derived colonies with a simple, low resource workflow, requiring only the addition of water to rehydrate hundreds of thousands of PicoShells and the single microbes encapsulated inside. Our desiccation method results in the recovery of desiccated microparticle morphology and porosity after a multi-week storage period and rehydration, with particle diameter and porosity metrics changing by less than 18% and 7%, respectively, compared to fresh microparticles. We also recorded the high viability of Saccharomyces cerevisiae yeast desiccated and rehydrated inside PicoShells, with only a 14% decrease in viability compared to non-desiccated yeast over 8.5 weeks, although we observed an 85% decrease in initial growth potential over the same duration. We show a proof-of-concept for a growth rate-based analysis of single-cell derived colonies in rehydrated PicoShells, where we identified 11% of the population that grows at an accelerated rate. Desiccated PicoShells thus provide a robust method for cell preservation before and during launch, promising a simple single-cell analysis method for studying heterogeneity in microbial populations in space.

Clonality analysis in lymphoproliferation using next-generation sequencing

Clonality analysis in lymphoproliferation using next-generation sequencing

Cutaneous metastases of internal malignancies: a single-institution experience.

AimsCutaneous metastases of internal malignancies occur in 1–10% of cancer patients. The diagnosis can sometimes be challenging, especially in cases with an unknown primary cancer.Materials and methodsA retrospective case review was performed including all cases of skin metastases from primary internal malignancies diagnosed at the Department of Pathology at the Maastricht University Medical Centre+ from 2007 to 2021. The clinicopathological data were collected and immunohistochemical and molecular diagnostic tests were performed to confirm the primary origin of the metastases.ResultsWe identified 152 cases (71 female; 31 male patients) of cutaneous metastases of internal malignancies. 28 patients (20 women and 8 men) were diagnosed with multiple cutaneous metastases. Among the female patients, the most common primary tumour was breast cancer (50% of the cases), followed by lung (13.6%), gynaecological (7.3%), and gastrointestinal origin (7.3%). Among the male patients, the most common primary sites were gastrointestinal and lung origin (altogether, 50% of the cases). In 19 patients, the cutaneous metastasis was the first presentation of a clinically silent internal malignancy (18.6%), of which most (78.9%) represented metastatic lung carcinomas. Finally, metastasizing patterns were different across tumour types and gender.ConclusionBreast, lung, gastrointestinal, and gynaecologic cancers are the most common primary tumours demonstrating skin metastases. Infrequently, cutaneous metastases can be the first clinically visual manifestation of an underlying not yet diagnosed internal malignancy; therefore, occasional broad immunohistochemical profiling, molecular clonal analysis, and a continuous high level of awareness are necessary for a precise diagnosis of cutaneous metastases of internal malignancies.

The combination of clinical parameters and immunophenotyping of intraepithelial lymphocytes allows to assess disease severity in refractory celiac disease

BackgroundFlow cytometry of intestinal lymphocytes is discussed to be a stronger predictor of enteropathy-associated T-cell lymphoma development in refractory celiac disease than T-cell clonality analysis. AimsTo investigate possible associations between clinical characteristics of refractory celiac disease patients and aberrant intraepithelial lymphocytes and to evaluate the accuracy of immunophenotyping for the identification of high-risk refractory celiac disease. MethodsFlow cytometry of isolated lymphocytes from duodenal biopsies of controls and celiac disease patients was performed and results were compared to clinical data. ResultsFlow cytometry analysis was performed on 42 controls, 37 non-complicated celiac disease and 30 refractory celiac disease cases with or without T-cell receptor clonality. Elevated aberrant intraepithelial lymphocyte counts were significantly associated with severe malabsorption. A 15% cut-off (aberrant lymphocytes among all lymphocytes) had the best discriminatory ability to identify high-risk patients. However, this technique failed to identify some high-risk cases (sensitivity 63%, specificity 100%). The severity of malabsorption was added to the criteria for high-risk refractory celiac disease, improving the correct patients’ allocation (sensitivity 100%, specificity 96%). ConclusionImmunophenotyping of aberrant intraepithelial lymphocytes is a good predictor for high-risk refractory celiac disease. Furthermore, adding the evaluation of malabsorption to the diagnostic assessment of refractory celiac disease optimizes accuracy.

Single-cell sequencing reveals CD133+CD44--originating evolution and novel stemness related variants in human colorectal cancer.

SummaryBackgroundTumor heterogeneity of human colorectal cancer (CRC)-initiating cells (CRCICs) in cancer tissues often represents aggressive features of cancer progression. For high-resolution examination of CRCICs, we performed single-cell whole-exome sequencing (scWES) and bulk cell targeted exome sequencing (TES) of CRCICs to investigate stemness-specific somatic alterations or clonal evolution.MethodsSingle cells of three subpopulations of CRCICs (CD133+CD44+, CD133−CD44+, and CD133+CD44− cells), CRC cells (CRCCs), and control cells from one CRC tissue were sorted for scWES. Then, we set up a mutation panel from scWES data and TES was used to validate mutation distribution and clonal evolution in additional 96 samples (20 patients) those were also sorted into the same three groups of CRCICs and CRCCs. The knock-down experiments were used to analyze stemness-related mutant genes. Neoantigens of these mutant genes and their MHC binding affinity were also analyzed.FindingsClonal evolution analysis of scWES and TES showed that the CD133+CD44− CRCICs were the likely origin of CRC before evolving into other groups of CRCICs/CRCCs. We revealed that AHNAK2, PLIN4, HLA-B, ALK, CCDC92 and ALMS1 genes were specifically mutated in CRCICs followed by the validation of their functions. Furthermore, four predicted neoantigens of AHNAK2 were identified and validated, which might have applications in immunotherapy for CRC patients.InterpretationAll the integrative analyses above revealed clonal evolution of CRC and new markers for CRCICs and demonstrate the important roles of CRCICs in tumorigenesis and progression of CRCs.FundingA full list of funding bodies that contributed to this study can be found in the Acknowledgements section.