Fresh Clinical Samples Research Articles

Single-cell profiling of surface glycosphingolipids opens a new dimension for deconvolution of breast cancer intratumoral heterogeneity and phenotypic plasticity

Glycosylated sphingolipids (GSLs) are a diverse group of cellular lipids typically reported as being rare in normal mammary tissue. In breast cancer (BCa), GSLs have emerged as noteworthy markers associated with breast cancer stem cells, mediators of phenotypic plasticity, and contributors to cancer cell chemoresistance. GSLs are potential surface markers that can uniquely characterize the heterogeneity of the tumor microenvironment, including cancer cell subpopulations and epithelial–mesenchymal plasticity (EMP). In this study, mass spectrometry analyses of the total sphingolipidome in breast epithelial cells and their mesenchymal counterparts revealed increased levels of Gb3 in epithelial cells and significantly elevated GD2 levels in the mesenchymal phenotype. To elucidate if GSL-related epitopes on BCa cell surfaces reflect EMP and cancer status, we developed and rigorously validated a 12-color spectral flow cytometry panel. This panel enables the simultaneous detection of native GSL epitopes (Gb3, SSEA1, SSEA3, SSEA4, and GD2), epithelial–mesenchymal transition markers (EpCAM, TROP2, and CD9), and lineage markers (CD45, CD31, and CD90) at the single-cell level. Next, the established panel was used for the analysis of BCa primary tumors and revealed surface heterogeneity in SSEA1, SSEA3, SSEA4, GD2, and Gb3, indicative of native epitope presence also on non-tumor cells. These findings further highlighted the phenotype-dependent alterations in GSL surface profiles, with differences between epithelial and stromal cells in the tumor. This study provides novel insights into BCa heterogeneity, shedding light on the potential of native GSL-related epitopes as markers for EMP and cancer status in fresh clinical samples. The developed single-cell approach offers promising avenues for further exploration.

Abstract 6081: DNA integrity and preanalytical handling of endocervical samples for use in liquid biopsy-based diagnostics and monitoring of gynecological cancer

Abstract Background: Liquid biopsies from the gynecological tract can be useful for analysis of cell free DNA (cfDNA) (1, 2, 3). Sampling procedure and optimal preanalytical handling are poorly investigated. In this study, we aimed to evaluate how the profile and integrity of DNA, sampled from the female genital tract, were affected by preanalytical handling. Methods: Biobanked/archival (stored at -80℃ after 48h at room temperature (RT)) and freshly obtained clinical samples were analyzed together with standardized in vitro samples. Archival and in vitro samples were preserved in methanol-based solution, imitating clinical handling of endocervical samples which are initially stored at RT for 48h before further handling. The fragmentation profile of archival and in vitro samples was analyzed. Time series of in vitro samples at RT and 4℃, with extraction at 6-hour intervals from 0 to 96h, were performed. The fresh clinical samples were collected from the endocervix in methanol-based solution and from the vagina in a solution designed to preserve DNA and placed at 4℃ resp RT. Extraction was performed within 4h and at 48h. Results: Accumulation of short DNA fragments were observed in the archival samples and in the in vitro samples, indicating that fragmentation was initiated prior to 48h. Time series showed that the quantity of short DNA fragments gradually increased with time at both RT and 4℃ but at a slower rate in 4℃. At RT the percentage of fragments between 100-350bp increased 12-fold (0.39% vs 4.75%, p=0.002). The freshly obtained endocervical samples showed a similar significant increase in short fragments in samples stored at RT compared with samples stored at 4℃ (p= 0.02 for 100-230bp; p= 0.007 for 230-350bp; p= 0.03 for 100-2500bp) at 4h-48h. Conclusion: Pre-analytical handling of endocervical samples can be optimized. Lowering temperature from RT to 4℃ immediately haltered DNA fragmentation. Citation Format: Jacob Malchau Lauesgaard, Therese Carlsson, Anna Linder, Sara Schumacher, Karin Sundfeldt. DNA integrity and preanalytical handling of endocervical samples for use in liquid biopsy-based diagnostics and monitoring of gynecological cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6081.

Abstract 5185: A multimodal, spatially defined, nanomechanical signature as predictive biomarker for breast cancer outcome: A 20 year survival study

Abstract Background: With the recent advancement in perioperative treatment protocols, the search for clinical biomarkers that can predict overall response and long-term cancer outcome independent from treatment acquires even more importance. Recently, it has become clear that mechanical properties of the tumor microenvironment in solid tumors play a key role in driving outcome, mediating immune infiltration and activation, and response to therapy. Moreover, a growing body of evidence supports the understanding that mechanical changes induced by a plethora of therapeutic regimens should be leveraged to elicit favorable outcome. Methods: In this work we propose a dual approach to reveal mechanical biomarkers predictive of long-term outcome in solid cancers. Our first approach is based on a newly developed, 60 channel, biomechanical imaging mass cytometry panel to elucidate biomechanical cues that drive tumor aggressiveness and immune infiltration. Our second approach utilizes the AFM-based Automated and Reliable Tissue Diagnostics (ARTIDIS) investigational device to measure mechanical properties of fresh clinical biopsy samples at a nanoscale resolution. Our analysis was performed on over 300 clinical samples, from routine baseline clinical biopsies collected from both the Breast Clinic, University Hospital Basel, and the University of Dundee, in the UK. Patients in these two cohorts were treated with either adjuvant or neoadjuvant approaches, including radiation, chemotherapy, hormonal, and surgical treatment regimens, with individual patient follow up as long as 20 years. Results: We demonstrated with two complimentary technologies that biomechanical spatial patterns drive outcome in breast cancer. Our results include a multi-platform elucidation of the biomechanical phenotypes of epithelial to mesenchymal transitions driven by hypoxic environments. Furthermore, we identified several biomechanical subpopulations of cancer associated fibroblasts, and their role in mediating cancer outcome, in relationship with survival time and several clinical variables (e.g. metastasis, nodes, grade). We mechanistically corroborated these results by evaluating the immune modulatory effect of the identified biomechanical microenvironments in breast cancer. Lastly, we correlated the identified biomechanical microenvironments with the ARTIDIS nanomechanical signature as a clinically integrated, rapid, single biomechanics signature at the nanoscale. Conclusion: Our results further validate the importance of biomechanics in driving tumor response and immune infiltration across a variety of clinical treatment setting. These result support the integration of ARTIDIS nanomechanical signature in clinical settings as a single predictive biomarker able to rapidly and accurately capture biomechanical alterations that drive cancer outcome. Citation Format: Sara Nizzero, Maria Pelaez Soni, Gregory Zaugg, Yitian Xu, Licheng Zhang, Junjun Zheng, Lee B. Jordan, Colin A. Purdie, Philip R. Quinlan, Chandandeep Nagi, Karla A. Sepulveda, Philipp Oertle, Tobias A. Appenzeller, Marko Loparic, Shu-Hsia Chen, Vittorio Cristini, Marija Plodinec, Alastair M. Thompson. A multimodal, spatially defined, nanomechanical signature as predictive biomarker for breast cancer outcome: A 20 year survival study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5185.

Abstract 6394: Tissue nanomechanical signature predicts response to neoadjuvant chemotherapy in patients with breast cancer

Abstract Background: Neoadjuvant chemotherapy (NACT) is increasingly used in patients with breast cancer. However, predicting response to NACT remains a challenge in everyday clinical practice. Increasing (pre)- and clinical evidence demonstrate the importance of cancer biomechanics in mediating therapy response. We and others have previously demonstrated that complex biomechanical alterations in cancer tissues, can be measured by the Atomic Force Microscopy (AFM) at the nanoscale level. Incorporating AFM measurements of clinical biopsies within standard of care is thus a promising avenue to leverage tissue mechanics as prognostic and predictive biomarker for solid cancers. Methods: Fresh clinical biopsy samples from 545 breast cancer patients were measured using the AFM-based Automated and Reliable Tissue Diagnostics (ARTIDIS) investigational device - ART-1. Measurements were performed on baseline biopsies within routine clinical workflow in the Breast Clinic, University Hospital Basel. This cohort comprises 30 patients who received NACT including: anthracycline and/or taxane based chemo (n=17), and anthracycline and/or taxane with platinum chemo (n=13). Study endpoints were radiological complete response (rCR) prior and pathological complete response (pCR) after surgery. Multiparametric nnanomechanical signature data has been analyzed in an automated manner using the proprietary ARTIDISNET software platform. Results: From the 30 patients who received NACT, 12 patients presented with rCR, 17 patients presented with rPR, and 1 patient presented with rNR. pCR has been achieved only in 9 patients. In this study, he ARTIDIS nanomechanical signature of response to NACT in breast cancer showed 100% negative predictive value (NPV) and 89% positive predictive value (PPV), with 93% of accuracy for patients receiving chemotherapy, independent of molecular subtype or treatment regimen. Sensitivity was 100% and specificity was 85% (AUC=0.91). Conclusion: ARTIDIS nanomechanical signature can be seamlessly integrated within clinical settings to predict response to standard of care NACT in patients with breast cancer in a prospective manner. These results are currently being validated in a global, multicentre prospective study in breast cancer (ANGEL, NCT06085833), enrolling in total 2706 patients, with the aim to support implementation of the ARTIDIS nanomechanical signature into routine clinical practice. Citation Format: Mariam Gachechiladze, Sara Nizzero, Tobias Appenzeller, Leonie Briner, Rosemarie Burian, Sabine Schädelin, Simone Muenst-Soysal, Tatjana Vlaijnic, Ellen Obermann, Sophie Dellas, Serafino Forte, Zlatko Marušić, Ahmed Jizawi, Papa Diogop Ndiaye, Philipp Oertle, Gregory Zaugg, Marko Loparic, Marija Plodinec. Tissue nanomechanical signature predicts response to neoadjuvant chemotherapy in patients with breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6394.

Commutability and Uncertainty of Blood Hemoglobin A1c Testing Materials.

Hemoglobin A1C (HbA1C) is used to evaluate glycemic control over a three-month period. Blood matrix-based HbA1C materials are needed for quality control and evaluation of HbA1C measurements. This study investigated the commutability of blood materials (BMs) and aimed to upgrade BMs for HbA1C testing for use as proficiency test (PT) material. We measured BMs from a DM blood donor (n = 1), an in vitro glycation procedure (n = 3), and from commercial sources (n = 2) for HbA1C in parallel with fresh unprocessed BMs (n = 3) and clinical blood samples (n = 25). Two NGSP-certified methods, including a turbidimetric and an enzymatic immunoassay, were used for HbA1C determinations. Commutability as investigated according to CLSI EP14-Ed4 guidelines. The commutable BMs exhibited a mean paired difference of 0% to 9% when compared to reference methods, whereas the non-commutable BMs represented a mean paired difference of 3% to 11%. Fresh, unprocessed BMs with a low HbA1C of 6.0% were more commutable than BMs with a high HbA1C. The values of HbA1C in BMs (mean and uncertainty following ISO Guide 35 for RM production) were applied to upgrade the PT material to be used as a reference material. The relative uncertainty of BM-Ndm-1 and BM-Gcb-3 were 1 and 0.4%, respectively. The commutability of hemoglobin in BMs is dependent on the preparation process. Blood materials with a high HbA1C content are usually less commutable versus materials with low HbA1C content when prepared by the same process. Our study showed BMs can be successfully used as quality control or PT materials.

Listeria monocytogenes, a silent foodborne pathogen in Ecuador.

Listeria monocytogenes is a foodborne pathogen that can produce serious, even fatal, infections. Among other foods, it can be found in unpasteurized dairy and ready-to-eat products. Surveillance of L. monocytogenes is of great interest since sources of infection are difficult to determine due to the long incubation period, and because the symptoms of listeriosis are similar to other diseases. We performed a genomic study of L. monocytogenes isolated from fresh cheeses and clinical samples from Ecuador. Sixty-five isolates were evaluated and sequenced, 14 isolates from cheese samples and 20 from clinical listeriosis cases from the National Institute of National Institute of Public Health Research, and 31 isolates from artisanal cheese samples from 8 provinces. All isolates exhibited heterogeneous patterns of the presence of pathogenicity islands. All isolates exhibited at least 4 genes from LIPI-1, but all references (26 L. monocytogenes closed genomes available in the NCBI database) showed the complete island, which encompasses 5 genes but is present in only two Ecuadorian isolates. Most isolates lacked gene actA. Genes from LIPI-2 were absent in all isolates. LIPI-3 and LIPI-4 were present in only a few references and isolates. With respect to the stress survival islets, our samples either presented SSI-1 or SSI-F2365, except for one isolate that presented SSI-F2365 and also one gene from SSI-1. None of the samples presented SSI-2. The predominant ST (sequence type) was ST2 (84.62% 55/65), and the only ST found in food (93.33% 42/45) and clinical samples (65% 13/20). Isolates were not grouped according to their sampling origin, date, or place in a phylogenetic tree obtained from the core alignment. The presence of ST2 in food and clinical samples, with high genomic similarity, suggests a foodborne infection risk linked to the consumption of fresh cheeses in Ecuador.

Widespread Multidrug Resistance of Arcobacter butzleri Isolated from Clinical and Food Sources in Central Italy.

The Arcobacter genus comprises a group of bacteria widely distributed in different habitats that can be spread throughout the food chain. Fluoroquinolones and aminoglycosides represent the most common antimicrobial agents used for the treatment of Arcobacter infections. However, the increasing trend of the antimicrobial resistance of this pathogen leads to treatment failures. Moreover, the test implementation and interpretation are hindered by the lack of reference protocols and standard interpretive criteria. The purpose of our study was to assess the antibiotic resistance pattern of 17 A. butzleri strains isolated in Central Italy from fresh vegetables, sushi, chicken breast, and clinical human samples to provide new and updated information about the antimicrobial resistance epidemiology of this species. Antimicrobial susceptibility testing was carried out by the European Committee on Antimicrobial Susceptibility Testing (EUCAST)'s disc diffusion method. All the strains were multidrug resistant, with 100% resistance to tetracyclines and cefotaxime (third generation cephalosporins). Some differences were noticed among the strains, according to the isolation source (clinical isolates, food of animal origin, or fresh vegetables), with a higher sensitivity to streptomycin detected only in the strains isolated from fresh vegetables. Our data, together with other epidemiological information at the national or European Union (EU) level, may contribute to developing homogeneous breakpoints. However, the high prevalence of resistance to a wide range of antimicrobial classes makes this microorganism a threat to human health and suggests that its monitoring should be considered by authorities designated for food safety.

Metaproteomic Analysis of Nasopharyngeal Swab Samples to Identify Microbial Peptides in COVID-19 Patients.

During the COVID-19 pandemic, impaired immunity and medical interventions resulted in cases of secondary infections. The clinical difficulties and dangers associated with secondary infections in patients necessitate the exploration of their microbiome. Metaproteomics is a powerful approach to study the taxonomic composition and functional status of the microbiome under study. In this study, the mass spectrometry (MS)-based data of nasopharyngeal swab samples from COVID-19 patients was used to investigate the metaproteome. We have established a robust bioinformatics workflow within the Galaxy platform, which includes (a) generation of a tailored database of the common respiratory tract pathogens, (b) database search using multiple search algorithms, and (c) verification of the detected microbial peptides. The microbial peptides detected in this study, belong to several opportunistic pathogens such as Streptococcus pneumoniae, Klebsiella pneumoniae, Rhizopus microsporus, and Syncephalastrum racemosum. Microbial proteins with a role in stress response, gene expression, and DNA repair were found to be upregulated in severe patients compared to negative patients. Using parallel reaction monitoring (PRM), we confirmed some of the microbial peptides in fresh clinical samples. MS-based clinical metaproteomics can serve as a powerful tool for detection and characterization of potential pathogens, which can significantly impact the diagnosis and treatment of patients.

Circ_0001589/miR-1248/HMGB1 axis enhances EMT-mediated metastasis and cisplatin resistance in cervical cancer.

Cervical cancer is the fourth most common malignant tumors in female worldwide. Cirular RNAs (circRNA) represent a new class of regulatory RNA and play a pivotal role in the carcinogenesis and development of tumors. However, their functions have not been fully elucidated in cervical cancer. In this study, we identified an upregulated circRNA, circ_0001589, both in fresh clinical samples and tissue microarray of cervical cancer. Transwell assay and cell apoptosis assay by flow cytometry demonstrated circ_0001589 promotes epithelial-mesenchymal transition (EMT)-mediated cell migration and invasion, and enhanced cisplatin resistance in vitro. In addition, in nude mice model, circ_0001589 increased the number of lung metastases and recovered xenograft growth from cisplatin treatment in vivo. Mechanistically, RNA pull-down assay, RNA immunoprecipitation, and dual-luciferase reporter assay disclosed that circ_0001589 function as an competing endogenous RNA to sponge miR-1248, which directly target the 3' untranslated region of high mobility group box-B1 (HMGB1). Thereby, circ_0001589 upregulated HMGB1 protein expression and accelerate cervical cancer progression. The rescue experiments also revealed that miR-1248 overexpression or HMGB1 knockdown partially reversed the regulatory functions of circ_0001589 on cell migration, invasion, and cisplatin resistance. In summary, our findings suggest the upregulation of circ_0001589 promoted EMT-mediated cell migration and invasion, and enhanced cisplatin resistance via regulating miR-1248/HMGB1 axis in cervical cancer. These results provided new evidence for understanding the carcinogenesis mechanism and finding new therapeutic target for cervical cancer.

Abstract 84: Single-cell transcriptional analysis of multiple myeloma bone marrow aspirates using particle-templated instant partition sequencing (PIPseq)

Abstract Single-cell RNA-seq technologies have dramatically evolved in recent years. Despite these advances, the field faces some significant limitations in the versatility of current single cell technologies. These limitations include speed, ease of use, and the ability to sample both vast and minimal numbers of cells in a single batch - particularly in human samples. The PIPseq approach is based on Particle-templated Instant Partitions (PIPs) that allow single-cell encapsulation and barcoding using uniform droplet emulsions with minimal lab hardware. PIPseq is particularly effective for routine, high-throughput analysis of multiple myeloma bone marrow biopsies. Patients with multiple myeloma undergo a series of treatments aimed at depleting their malignant plasma cells (PC). Nevertheless, a high rate of relapse is observed despite low PC levels detected in the bone marrow (BM). Deep molecular profiling of the cellular pathways of rare PC, as well as their unique microenvironment, may shed light on the mechanisms underlying resistance to conventional therapies. As part of an ongoing clinical research, a bone marrow sample from a patient with multiple myeloma was molecularly characterized using the PIPseq T2 3’ Single Cell RNA Kit. Two distinct cell populations from a frozen BM biopsy were sorted into the T2 PIPseq tube: CD45+ cells (tumor microenvironment; TME) and rare malignant PC. In spite of the limited number of cells in the biopsy, PIPseq analysis clearly resolved both the malignant PC and the TME, demonstrating its utility for extensive analyses of fresh or frozen clinical myeloma samples. Citation Format: Yonatan Katzenelenbogen, Mor Zada, Robert Meltzer, Kristina Fontanez, Ido Amit. Single-cell transcriptional analysis of multiple myeloma bone marrow aspirates using particle-templated instant partition sequencing (PIPseq) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 84.

Development of a Multiplex qPCR Assay for Fast Detection and Differentiation of Paracoccidioidomycosis Agents.

Classic paracoccidioidomycosis (PCM) is a potentially deadly neglected tropical systemic mycosis caused by members of the Paracoccidioides brasiliensis complex (P. brasiliensis s. str., P. americana, P. restrepiensis, and P. venezuelensis) and P. lutzii. The laboratorial diagnosis of PCM relies on observing pathognomonic structures such as the "steering wheel" or "Mickey Mouse" shape in the direct mycological examination, fresh biopsied tissue in 10% KOH, histopathological analysis, and/or the isolation of the fungus in culture. However, these procedures are time-consuming and do not allow for the speciation of Paracoccidioides due to overlapping morphologies. Here, we propose a new one-tube multiplex probe-based qPCR assay to detect and recognize agents of the P. brasiliensis complex and P. lutzii. Primers (Paracoco-F and Paracoco-R) and TaqMan probes (PbraCx-Fam, Plu-Ned, and Paracoco-Vic) were developed to target the rDNA (ITS2/28S) in the Paracoccidioides genome. A panel of 77 Paracoccidioides isolates revealed a 100% specificity (AUC = 1.0, 95% CI 0.964-1.000, p < 0.0001) without cross-reacting with other medically relevant fungi or human and murine DNA. The lower limit of detection was 10 fg of gDNA and three copies of the partial rDNA amplicon. Speciation using qPCR was in perfect agreement with AFLP and TUB1-RFLP markers (kappa = 1.0). As a proof of concept, we assessed a panel of 16 formalin-fixed and paraffin-embedded specimens from histopathologically confirmed PCM patients to reveal a significant sensitivity of 81.25% and specificity of 100% (AUC = 0.906 ± 0.05, 95% CI = 0.756-0.979, p < 0.0001, Youden index J = 0.8125). Our assay achieved maximum sensitivity (100%) and specificity (100%) using fresh clinical samples (n = 9) such as sputum, bronchoalveolar lavage, and tissue fragments from PCM patients (AUC = 1.0, 95% CI 0.872-1.000, p < 0.0001, Youden index J = 1.0). Overall, our qPCR assay simplifies the molecular diagnosis of PCM and can be easily implemented in any routine laboratory, decreasing a critical bottleneck for the early treatment of PCM patients across a vast area of the Americas.

Optimization of lateral flow assay for Canine morbillivirus detection and the application of the strip as sample substitute

Canine distemper is an emerging disease, caused by the Canine morbillivirus (CDV) of the Paramyxoviridae family. The virus has evolved as a multi-host pathogen as it affects many wildlife animal species. The development of specific and sensitive diagnostic tests is the need for a control program. Several diagnostic tests are available for the detection of CDV antigen and antibody. Lateral flow assay (LFA) is the most promising point of care diagnostic test because of its specificity, easy use, and instant result. This study was designed to develop a lateral flow assay using the in-house developed monoclonal antibody (mAb) against the nucleocapsid protein (N) of the ‘CDV/dog/bly/Ind/2018’ isolate, which represents the circulating strains of India. The two mAbs included in the study showed high binding affinity in indirect ELISA and dot blot assay. Out of two, one mAb was selected due to its comparatively higher binding affinity in LFA format, and less non-specific binding to the biological matrix and buffer components. The limit of detection was found to be 106.5 TCID50/ml with the assay run time of 5 min. The fresh clinical samples collected on the spot were distinctly detected by the LFA, whereas the stored samples with a reduced titre of the virus showed inconsistent results. Moreover, the blood samples showed a clear distinction of positive and negative than the swab and tissue homogenates. The RNA extraction from the strip was successful with the some modifications in the Trizol RNA extraction method and the N and H gene fragments were amplified. Therefore, the study concludes that the LFA is suitable for CDV antigen detection in the field conditions and the strips can be used as the sample substitute for molecular study.

P395 Clinical utility of semi-nested conventional PCR for diagnosis of mucormycosis in fresh clinical samples

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM ObjectivesWe aim to assess the clinical utility of the semi-nested conventional PCR in smear positive, culture-negative clinical samples for diagnosis of mucormycosis.MethodsThis prospective study was conducted for a period of 3 months (April-June 2021). A total of 218 clinical samples were included from patients in whom smear was positive for aseptate hyphae, but the culture failed to grow within 2 days of incubation or smear was negative but had high suspicion of mucormycosis. Molecular diagnosis was attempted using semi-nested PCR with Mucorales-specific primers targeting 18S region, described previously by Bialek et al.1. Phenol-chloroform based manual DNA extraction protocol was optimized in the mycology laboratory of the department of medical microbiology, PGIMER, Chandigarh. This method was applicable to different types of clinical samples to yield good quality DNA with minimal chances of extraneous contamination (Fig. 1). Amplified PCR products were further sequenced to identify the causative species (Fig. 2).ResultsAmong 218 patients with suspected mucormycosis included in this study, the majority were rhino-orbito-cerebral mucormycosis (ROCM), (77.7%, n = 169), followed by pulmonary mucormycosis (19.2%, n = 42), cutaneous (0.02%, n = 4), and gastro-intestinal (GI) mucormycosis (0.01%, n = 3). In 24 samples, the presence of both septate and aseptate hyphae was seen under microscopic examination raising the possibility of mixed infection. On microscopic examination, 90.3% samples (197/218) had aseptate hyphae while the remaining samples were smear-negative but had strong clinical suspicion of mucormycosis. The molecular technique was able to identify causative agent in 154 culture-negative samples (81.9%, 154/188) and 52.4% (11/21) in smear-negative cases. Among 218 patients, only 20 samples show delayed growth of Mucorales, and on comparison with molecular results 100% concordance was observed. However, 10 patients with strong clinical suspicion for mucormycosis were negative by both conventional and molecular methods. The low culture positivity necessities the molecular diagnosis based on in-house semi-nested PCR using above-mentioned primers followed by Sanger sequencing. In the case of 24 mixed infections with aseptate-septate hyphae, Mucorales-specific PCR correctly identified 23/24 (95.8%) Mucorales as a causative agent. The overall turn-around-time from the sample receiving to diagnosis was ˂48 h. Overall, R. arrhizus (85/143, 59.4%) was most commonly associated with ROCM, while R. microsporus (13/38, 34.2%), and R. homothallicus (5/38, 13.1%) were seen mainly with pulmonary mucormycosis. Apophysomyces and Saksenaea genus were associated with GI and cutaneous mucormycosis.ConclusionsThe molecular technique utilizing semi-nested PCR, followed by Sanger sequencing was able to identify Mucorales species 81.9% of culture-negative cases. Optimized manual DNA extraction protocol is suitable for different sample types, with minimum chances of extraneous contamination and offers low cost with a shorter turnaround time (TAT).

MiR-361-5p as a promising qRT-PCR internal control for tumor and normal breast tissues.

BackgroundOne of the most widely used evaluation methods in miRNA experiments is qRT-PCR. However, selecting suitable internal controls (IC) is crucial for qRT-PCR experiments. Currently, there is no consensus on the ICs for miRNA qRT-PCR experiments in breast cancer. To this end, we tried to identify the most stable (the least expression alteration) and promising miRNAs in normal and tumor breast tissues by employing TCGA miRNA-Seq data and then experimentally validated them on fresh clinical samples.MethodsA multi-component scoring system was used which takes into account multiple expression stability criteria as well as correlation with clinical characteristics. Furthermore, we extended the scoring system for more than two biological sub-groups. TCGA BRCA samples were analyzed based on two grouping criteria: Tumor & Normal samples and Tumor subtypes. The top 10 most stable miRNAs were further investigated by differential expression and survival analysis. Then, we examined the expression level of the top scored miRNA (hsa-miR-361-5p) along with two commonly used ICs hsa-miR-16-5p and U48 on 34 pairs of Primary breast tumor and their adjacent normal tissues using qRT-PCR.ResultsAccording to our multi-component scoring system, hsa-miR-361-5p had the highest stability score in both grouping criteria and hsa-miR-16-5p showed significantly lower scores. Based on our qRT-PCR assay, while U48 was the most abundant IC, hsa-miR-361-5p had lower standard deviation and also was the only IC capable of detecting a significant up-regulation of hsa-miR-21-5p in breast tumor tissue.ConclusionsmiRNA-Seq data is a great source to discover stable ICs. Our results demonstrated that hsa-miR-361-5p is a highly stable miRNA in tumor and non-tumor breast tissue and we recommend it as a suitable reference gene for miRNA expression studies in breast cancer. Additionally, although hsa-miR-16-5p is a commonly used IC, it’s not a suitable one for breast cancer studies.

High expression of PDGFA predicts poor prognosis of esophageal squamous cell carcinoma.

Platelet-derived growth factor A (PDGFA), the most known member of PDGF family, plays a crucial role in occurrence and progression of different tumors. However, PDGFA expression and its clinical significance in esophageal squamous cell carcinoma (ESCC) are not clear. The present study aimed to assess the expression and prognostic value of PDGFA in ESCC.The Gene Expression Omnibus databases (GSE53625, GSE23400, and GSE67269) and fresh clinical samples were employed for detecting PDGFA messenger RNA expression in ESCC. The associations of PDGFA expression with clinicopathological characteristics were evaluated by chi-square test. Kaplan–Meier analysis and Cox proportional hazard regression model were performed to determine the prognostic value of PDGFA in ESCC patients. PDGFA-related signaling pathways were defined by gene set enrichment analysis based on Gene Expression Omnibus databases.The PDGFA messenger RNA expression was upregulated in ESCC tissues compared with paired adjacent noncancerous tissues (P < .05) and was positively correlated with T stage (P < .05). Kaplan–Meier survival analysis suggested that ESCC patients with high PDGFA expression were associated with poorer overall survival compared to those with low PDGFA expression (P < .05), especially in advanced T stage (P < .05). Cox analyses showed that high expression of PDGFA was an independent predictor for poor prognosis in ESCC patients. Gene set enrichment analysis identified 3 signaling pathways (extracellular matrix receptor interaction, focal adhesion, and glycosaminoglycan biosynthesis chondroitin sulfate) that were enriched in PDGFA high expression phenotype (all P < .01).PDGFA may serve as an oncogene in ESCC and represent an independent molecular biomarker for prognosis of ESCC patients.

PGR and TUG1 overexpression: A putative diagnostic biomarker in breast cancer patients

BackgroundBreast cancer is highly prevalent globally and is one of the main causes of cancer-related deaths worldwide. Despite the advances, more insights are needed to decipher the regulatory mechanisms leading to breast cancer development and progression. Here, we examined the expression of PGR and TUG1 genes in breast cancer. Material and methodsqPCR was used to quantitatively measure the expression of PGR and TUG1 transcripts in normal and breast cancer fresh clinical samples. ResultsThe higher expression of both PGR and TUG1 was observed in the breast tumors as compared to the normal adjacent paired samples. The ROC curve analysis revealed that PGR and TUG1 expression patterns can discriminate breast tumor samples against normal tissues. Besides, there was a significant correlation between PGR and TUG1 expression in the studied samples. ConclusionPGR and TUG1 are significantly up-regulated in breast cancer, which both can distinct tumor from normal tissues. With respect to the positive correlation between PGR and TUG1, this gene signature might be used as a diagnostic biomarker for breast cancer detection.

MiR-195 down-regulation is a distinctive biomarker of HER2 positive state in breast cancer

BackgroundBreast cancer is the most common cancer among women worldwide. HER2-positive breast cancer accounts for nearly 20% of all cases. Upon targeted therapy resistance, these patients show fairly poor-prognosis. miRNAs have shown to be differentially expressed in HER2-positive cases that can decipher drug resistance. miR-195 down-regulation has been reported in breast cancer; however, very little is known about its expression particularly in HER2-positive breast cancers. Material and methodsqPCR was used to quantitatively measure the expression of miR-195 in normal, HER2-positive and HER2-negative fresh clinical samples. A computational approach was used to find unvalidated but potential targetome of miR-195. ResultsmiR-195 is down-regulated in breast cancer samples as compared to the normal controls. The level of down-regulation was significantly greater in HER2-positive tumors as compared to both normal and HER2-negative samples. Importantly, miR-195 down-regulation was demonstrated to be a potential diagnostic biomarker for distinguishing HER2-positive against both normal and HER2-negative samples. Besides, the in silico approach revealed that unvalidated miR-195 targetome play roles in oncogenic pathways related to breast tumorigenesis and progression. ConclusionmiR-195 is down-regulated in breast cancer, especially and significantly in HER2-positive cases. The expression of miR-195 could effectively be used as a distinctive diagnostic marker to discriminate HER2-positive from both normal and HER2-negative samples.

A single-cell and single-nucleus RNA-Seq toolbox for fresh and frozen human tumors

Single-cell genomics is essential to chart tumor ecosystems. Although single-cell RNA-Seq (scRNA-Seq) profiles RNA from cells dissociated from fresh tumors, single-nucleus RNA-Seq (snRNA-Seq) is needed to profile frozen or hard-to-dissociate tumors. Each requires customization to different tissue and tumor types, posing a barrier to adoption. Here, we have developed a systematic toolbox for profiling fresh and frozen clinical tumor samples using scRNA-Seq and snRNA-Seq, respectively. We analyzed 216,490 cells and nuclei from 40 samples across 23 specimens spanning eight tumor types of varying tissue and sample characteristics. We evaluated protocols by cell and nucleus quality, recovery rate and cellular composition. scRNA-Seq and snRNA-Seq from matched samples recovered the same cell types, but at different proportions. Our work provides guidance for studies in a broad range of tumors, including criteria for testing and selecting methods from the toolbox for other tumors, thus paving the way for charting tumor atlases.

Commutability of reference materials for alkaline phosphatase measurements

Commutability is a prerequisite when using a candidate reference materials (CanRMs) for calibration and trueness control of routine methods or for value transfer from the reference method to end-user calibrators of routine methods through a calibration hierarchy. The CanRMs include commercial general chemistry materials (GCs), enzyme trueness verification materials (ETVs), and secondary reference materials (SRMs). The commutability of CanRMs was evaluated based on the difference in bias and EP14-A3 to investigate their suitability for ALP measurement. A total of 44 fresh individual clinical samples (CSs) and the CanRMs were analyzed by six routine methods (validated methods) and by IFCC primary reference method (comparative method) for the determination of Alkaline Phosphatase (ALP). The commutability was performed based on IFCC working group recommendations for assessing commutability and analyzed according to EP14-A3 guide and difference in bias approach, respectively. The bias of the routine methods was assessed according to CLSI guidelines. The result of commutable materials obtained from the two evaluation approaches are not significantly different. It was observed that the routine methods with lower bias were associated with more commutable CanRMs. None of the CanRMs were suitable for use as commutable RMs. The findings of this study have a number of important implications for future practice.

Clinical Significance And Integrative Analysis Of Kinesin Family Member 18B In Lung Adenocarcinoma.

BackgroundKinesin family member 18B (KIF18B) is a member of the kinesin-8 superfamily, and functions as an oncogene in human cancers. However, its expression profile and role in lung adenocarcinoma (LUAD) remain unclear.Materials and methodsWe examined the expression profile of KIF18B using quantitative real-time reverse transcription polymerase chain reaction and immunohistochemistry in fresh clinical samples. Using data downloaded from the Cancer Genome Atlas database and Gene Expression Omnibus, we explored the clinical significance of KIF18B, potential mechanisms of its dysregulation and its underlying biological function in LUAD.ResultsKIF18B was significantly over-expressed in LUAD tissues relative to normal tissues. High KIF18B expression was associated with smoking history, positive nodal invasion, advanced clinical stage, death status and poorer prognosis. Cox regression analyses revealed that KIF18B overexpression was an independent prognostic biomarker for poor overall survival (OS) and recurrence-free survival in LUAD. In addition, KIF18B mutation was observed in 2.2% of LUAD cases. DNA copy number variation was correlated with upregulated expression of KIF18B in LUAD tissues and cell lines. The methylation level of some KIF18B DNA CpG sites was negatively associated with its mRNA expression. KIF18B was predictively targeted by miR-125a-5p, which was downregulated in LUAD tissues, inversely correlated with KIF18B mRNA expression and significantly associated with poor OS. Furthermore, gene set enrichment analysis revealed that genes positively co-expressed with KIF18B were mainly enriched in cell cycle signaling pathways.ConclusionOur results indicate that KIF18B is a promising prognostic biomarker for LUAD. DNA amplification, hypomethylation as well as miR-125a-5p downregulation may be involved in the mechanism of KIF18B dysregulation in LUAD. KIF18B might function as a novel oncogene through cell cycle regulation pathways in LUAD.