Paired Specimens Research Articles

B-355 Measurable Residual Disease Detection in Mantle Cell Lymphoma (MCL) Subjects Using NGS B-Cell MRD Assay

Abstract Background Mantle Cell Lymphoma (MCL) is an aggressive, rare form of non-Hodgkin B-cell lymphoma. Measurable Residual Disease (MRD) assessment can provide useful information when assessing and tracking response to therapy, refining treatment and predicting clinical outcome for subjects with B-cell lymphoproliferative diseases. One method of MRD detection is to identify and track specific clonal immunoglobulin heavy chain (IGH) gene rearrangement sequences on a next-generation sequencing (NGS) platform. Here, we present the NGS-based B-cell MRD Assay with 10−6 sensitivity for detecting MRD in malignancies in mantle cell lymphoma (MCL). Methods Residual DNA samples from different specimen types (44 BM and 77 PB) at different post-treatment time points were obtained from anonymized 47 MCL subjects that had been enrolled in a study approved by the Spanish Group of Lymphoma and Autologous Stem Cell Transplantation (clinical trial: NCT02682641, publication reference: 10.1200/JCO.21.02321). 121 of these follow up samples were tested by the B-cell MRD Assay by tracking the clonal sequences detected in corresponding baseline samples. Among them, 90 follow up samples were paired PB and BM samples. The B-cell MRD Assay workflow consists of DNA extraction and PCR based library preparation with proprietary multiplex master mixes (MMx) targeting either IGH Framework 1 (FR1) or 3 (FR3) regions, which both track the patient- and tumor-specific CDR3 sequence. Purified libraries are then equimolar pooled and sequenced on Illumina’s NextSeq Dx platform. The FASTQ output files from sequencing are analyzed using Invivoscribe’s B-Cell MRD Software-NextSeqDx. Results from Invivoscribe were compared to the results from Salamanca lab that were generated by qPCR. Results Based on the results from B-cell MRD Assay, 115/121 (95%) samples were valid, 42/115 (37%) valid samples had, “MRD Detected”, and 73/115 (63%) samples had, “MRD Not Detected”, with an “MRD Detection” rate of 28% in PB and 50% in BM specimen types. When comparing MRD detectable status between paired PB and BM samples from 36 MCL subjects, 30/44 (68%) paired samples had same MRD call and were concordant, whereas 14/44 (32%) calls were discordant. More specifically, 12/14 (86%) discordant samples had PB specimen type with a “MRD Not detected” call, whereas the paired BM specimen showed a “MRD Detected” call. Overall, in comparison with the MRD results (n = 84) provided by Salamanca lab using qPCR method, the B-cell MRD Assay demonstrated concordance of 80%, PPA of 79%, and NPA of 80% for MRD detection in MCL samples. Conclusion Good concordance was observed between the qPCR used at Salamanca and Invivoscribe’s B-cell MRD assay. It was important to note that the BM specimen type was determined to be more sensitive in detecting MRD when compared to PB in assessment of paired PB and BM samples. These results demonstrate that the B-cell MRD Assay can be utilized for MRD detection in follow-up sample types for MCL subjects.

B-072 Analysis of Anti-SARS-Cov-2 S1-RBD and N IgG in Saliva and Serum

Abstract Background Mucosal antibody testing may be a significant and more practical tool for measuring humoral immunity since it is non-invasive. The aim of this study was to evaluate the SARS-CoV-2-specific antibody prevalence in saliva compared to serum in 2022. Methods From February to December 2022, the paired serum and saliva specimens were collected twice over a period. The saliva specimens were collected using a Salivette Cortisol (Sarstedt AG & Co. KG, Germany) device according to manufacturers’ instructions. Antibody tests were performed using nucleocapsid protein-based Elecsys anti-SARS-CoV-2 (anti-N IgG) and spike protein targeted SARS-CoV-2 IgG II Quant (anti-S1-RBD IgG). Results 162 (97.6%) of 166 had anti-S1-RBD IgG in their serum. At the 1st visit and 2nd visit, 79 (47.5%) and 97 (58.4%) patients, respectively, had anti-N IgG in their serum. At 1st visit and 2nd visit, respectively, 14 (8.4%) and 13 (7.8%) patients had anti-S1-RBD IgG in their saliva. Four (2.4%) and five (3.0%) patients had anti-N IgG in their saliva during the 1st visit and 2nd visit, respectively. The correlation coefficient of the results of anti-S1-RBD IgG and anti-N IgG quantitative results between at 1st visit and 2nd visit were low (r = 0.1721 to 0.6448). All patients in the anti-S1-RBD IgG seronegative group (N = 4) had anti-N IgG in their serum, but neither anti-S1-RBD IgG nor anti-N IgG were detected in their saliva. All anti-N IgG seronegative patients (N = 68) had anti-S1-RBD IgG in serum, while 3 patients had anti-S1-RBD IgG and 7 patients had anti-N IgG in saliva. Conclusion Mucosal antibodies were detected in certain seronegative patients. The assessment of SARS-CoV-2-specific saliva antibodies should be regarded as a supplemental non-invasive diagnostic to serum to evaluate the prevalence and transmission of COVID-19.

Surface morphology of osteochondral grafts harvested from femoral condyles with free-hand technique: a cadaveric study.

The purpose of this cadaver study was to examine the surface morphology of the osteochondral grafts harvested from the femoral condyles using the free-hand graft harvesting technique. One hundred osteochondral grafts were harvested with 6.5mm chisels at ten different donor sites using the free-hand technique in five paired knee specimens (Mean age: 56.4years). The cartilage and subchondral bone surface angles were measured through multiplanar reconstruction computerized tomography examination. The cartilage thickness was measured with a MicroScribe G2X digitizer with an accuracy of 0.02mm. An acceptable congruity could be obtained when these plugs were transferred to a perpendicular socket (articular step-off of less than 1mm and 0.5mm) was evaluated. Four plugs were damaged or broken during harvesting due to technical difficulties; thus remaining 96 plugs were analyzed. The cartilage thickness varied between 1.36mm and 3.26mm across the donor sites. The cartilage was the thinnest in the medial intercondylar notch and thickest in the lateral supracondylar notch. Twenty of ninety-six plugs (20.8%) had unacceptable cartilage surface inclination according to the > 0.5mm protrusion criteria. Of these plugs, 14 were harvested from the lateral intercondylar notch, whereas five of 96 plugs (5.2%) had unacceptable cartilage surface inclination according to the > 1mm protrusion criteria. Of these plugs, all were harvested from the lateral intercondylar notch. High rates of unacceptable plugs (up to 100%) might be harvested from the lateral intercondylar notch. In large chondral lesions that require multiple plugs, lateral and medial supracondylar ridges were the best donor sites for perpendicular plug harvesting, whereas lateral intercondylar notch should be avoided.

Evaluation of enamel wear by 3 occlusal splint materials: An in vitro study

Statement of problemOcclusal devices used to manage bruxism have been commonly fabricated from polymethyl methacrylate with the powder-liquid technique. More recently, Vertex ThermoSens (VTS) and the biocompatible high-performance polymer (BioHPP), an optimized material having polyetheretherketone (PEEK) as its basis, have been used, but little is known about the wear of these materials on human enamel. PurposeThe purpose of this in vitro study was to assess via a mastication simulation test how 3 occlusal device materials affected the wear and roughness of enamel antagonists. Material and methodsA noncontact 3D optical profilometer was used to measure the enamel surface roughness (Ra) against 3 occlusal device materials: vertex regular, VTS, and PEEK high-performance polymer (BioHPP). A dual-axis mastication simulator was used to perform a 2-body wear test on specimens from each group. The test consisted of 10 000 cycles with a 70-N force and 5 to 55 °C thermocycling. Following simulated mastication, the weight of each specimen and the Ra change were compared with the Kolmogorov-Smirnov test, paired specimens t test, Wilcoxon signed-rank test, and 1-way analysis of variance (α=.05). ResultsThe polyamide group caused the lowest amount of enamel wear (P<.05), while the heat-polymerized acrylic resin group induced the largest amount of enamel wear (P<.05). For polyamide and PEEK, the change in enamel surface roughness exhibited a smooth texture, whereas it found a rougher surface for the heat-polymerized acrylic resin. ConclusionsAccording to this study, surface roughness and wear on human enamel were not correlated. PEEK is a promising material for the fabrication of occlusal devices.

Accuracy of Cytologic vs Histologic Specimens for Assessment of Programmed Cell Death Ligand-1 Expression in Non-Small Cell Lung Cancer: A Systematic Review and Meta-Analysis

Programmed cell death ligand-1 (PD-L1) expression on tumor cells, evaluated by immunohistochemistry, guides the use of immunotherapy in advanced non-small cell lung cancer (NSCLC). What is the sensitivity and specificity of PD-L1 testing performed in cytologic vspaired histologic specimens in patients with NSCLC? The MEDLINE, Embase, Web of Science, and Cochrane Library databases were searched through June 1, 2021. The primary outcome was pooled sensitivity and specificity of PD-L1 testing performed on cytologic specimens compared with the reference standard of histologic specimens, analyzed at the PD-L1 expression cutoffs (tumor proportion score)≥ 1%and≥ 50%. Pooled sensitivity and specificity, and associated 95%CIs, were estimated using bivariate generalized linear mixed models. Twenty-six articles were included, encompassing a total of 1,064 pairs of histology specimens and cytology cell blocks, and 267 pairs of histology specimens and direct smears. Among these, 946 paired specimens were acquired without interval treatment between the collection of histology and cytology samples. The pooled sensitivity and specificity of cytology specimens compared with paired histology specimens at the PD-L1 expression cutoff≥ 1%were 0.84 (95%CI, 0.77-0.89) and 0.88 (95%CI, 0.82-0.93), respectively, whereas the pooled sensitivity and specificity at cutoff≥ 50%were 0.78 (95%CI, 0.69-0.86) and 0.94 (95%CI, 0.91-0.96), respectively. When only paired specimens acquired without interval treatment were considered, the pooled sensitivity and specificity of cytology specimens at PD-L1 expression cutoff≥ 1%were 0.84 (95%CI, 0.76-0.90) and 0.89 (95%CI, 0.82-0.94), respectively, whereas the pooled sensitivity and specificity at cutoff≥ 50%were 0.80 (95%CI, 0.71-0.89) and 0.94 (95%CI, 0.91-0.96), respectively. Cytologic specimens provide an accurate assessment of PD-L1 expression in most patients with NSCLC, at both≥ 1%and≥ 50%cutoffs, when compared with histologic specimens. PROSPERO; No.: CRD42020153279; URL: https://www.crd.york.ac.uk/prospero/.

Therapeutic plasma exchange decreases plasma anti-SARS-CoV-2 spike IgG without increasing the proximate incidence of COVID-19.

Therapeutic plasma exchange (TPE) removes both pathologic and protective immunoglobulins (Ig). SARS-CoV-2 immunity is partially mediated by anti-SARS-CoV-2 spike antibodies (SAb), which impair viral host-cell invasion. Nonetheless, the systematic effect of TPE on SAb concentration and SARS-CoV-2 immunity is unknown. Paired plasma waste specimens from the first (first-TPE) and last (last-TPE) TPE treatment were collected from 9 patients between July 21, 2021 and March 1, 2022. The effects of TPE on Ig levels were assessed by quantitatively comparing the SAb, total IgG, and total IgM levels first-/last-TPE treatment. Complementary qualitative assessment for these changes was achieved via protein electrophoresis (PEP) and immunofixation (IFE). A retrospective review was performed to investigate the incidence of new SARS-CoV-2 infections following TPE v. other treatment at the same outpatient apheresis/infusion center during the same time frame. Median SAb levels between the first- and last-TPE waste specimens decreased significantly from 424.6 AU/mL to 17.0 AU/mL (P = 0.004). Concordantly, PEP and IFE analysis demonstrated broad Ig decreases. Cumulative incidence of subsequent COVID-19 diagnosis at 30, 90, and 180 days post-procedure did not differ between the TPE v. other treatment groups (n = 709 total patients). TPE significantly reduced SAb levels, a marker of SARS-CoV-2 immunity, but did not appear to provoke increased incidence of COVID-19 infections. Further investigation of the kinetics of TPE-mediated SAb decrease and post-TPE recovery are warranted.

Potential of miR-181a-5p and miR-630 as clinical biomarkers in NSCLC

BackgroundThe development of drug resistance and high mortality rates are the major problems observed in non-small cell lung cancer (NSCLC). Biomarkers indicating and predicting disease development towards these unfavorable directions are therefore on high demand. Many studies have demonstrated that changes in miRNAs expression may be associated with a response to treatment and disease prognosis, thus suggesting its potential biomarker value for a broad spectrum of clinical applications. The aim of the present study was to investigate the expression level of miR-181a-5p, miR-630, and its targets in NSCLC tumor tissue and plasma samples; and to analyze its association with NSCLC patient’s response to treatment and disease prognosis.MethodsThe study was performed in 89 paired tissue specimens and plasma samples obtained from NSCLC patients who underwent surgical treatment at the Department of Thoracic Surgery and Oncology of the National Cancer Institute. Analysis of miR-181a-5p and miR-630 expression was performed by qRT-PCR using TaqMan miRNA specific primers. Whereas BCL2, LMO3, PTEN, SNAI2, WIF1 expression levels were identified with KAPA SYBR FAST qPCR Kit. Each sample was examined in triplicate and calculated following the 2-ΔΔCt method. When the p-value was less than 0.05, the differences were considered statistically significant.ResultsIt was found that miR-181a-5p and miR-630 expression levels in NSCLC tissue and plasma samples were significantly decreased compared with control samples. Moreover, patients with low miR-181a-5p expression in tumor tissue and plasma had longer PFS rates than those with high miRNA expression. Decreased miR-630 expression in tumor was statistically significantly associated with better NSCLC patients’ OS. In addition, the expression of miR-181a-5p, as well as miR-630 in tumor tissue, are the statistically significant variables for NSCLC patients’ OS. Moreover, in NSCLC patient plasma samples circulating miR-181a-5p can be evaluated as significant independent prognostic factors for OS and PFS.ConclusionsOur findings indicate the miR-181a-5p and miR-630 expression levels have the potential to prognose and predict and therefore improve the treatment individualization and the outcome of NSCLC patients. Circulating miR-181a-5p has the potential clinical value as a non-invasive biomarker for NSCLC.

Clinical and Genomic Landscape of FGFR3-Altered Urothelial Carcinoma and Treatment Outcomes with Erdafitinib: A Real-World Experience.

Erdafitinib is the only FDA-approved targeted therapy for FGFR2/3-altered metastatic urothelial cancer. We characterized the genetic landscape of FGFR-altered urothelial carcinoma and real-world clinical outcomes with erdafitinib, including on-treatment genomic evolution. Prospectively collected clinical data were integrated with institutional genomic data to define the landscape of FGFR2/3-altered urothelial carcinoma. To identify mechanisms of erdafitinib resistance, a subset of patients underwent prospective cell-free (cf) DNA assessment. FGFR3 alterations predictive of erdafitinib sensitivity were identified in 39% (199/504) of patients with non-muscle invasive, 14% (75/526) with muscle-invasive, 43% (81/187) with localized upper tract, and 26% (59/228) with metastatic specimens. One patient had a potentially sensitizing FGFR2 fusion. Among 27 FGFR3-altered cases with a primary tumor and metachronous metastasis, 7 paired specimens (26%) displayed discordant FGFR3 status. Erdafitinib achieved a response rate of 40% but median progression-free and overall survival of only 2.8 and 6.6 months, respectively (n = 32). Dose reductions (38%, 12/32) and interruptions (50%, 16/32) were common. Putative resistance mutations detected in cfDNA involved TP53 (n = 5), AKT1 (n = 1), and second-site FGFR3 mutations (n = 2). FGFR3 mutations are common in urothelial carcinoma, whereas FGFR2 alterations are rare. Discordance of FGFR3 mutational status between primary and metastatic tumors occurs frequently and raises concern over sequencing archival primary tumors to guide patient selection for erdafitinib therapy. Erdafitinib responses were typically brief and dosing was limited by toxicity. FGFR3, AKT1, and TP53 mutations detected in cfDNA represent putative mechanisms of acquired erdafitinib resistance.

Integration of peripheral blood- and tissue-based biomarkers of response to immune checkpoint blockade in urothelial carcinoma.

As predictive biomarkers of response to immune checkpoint inhibitors (ICIs) remain a major unmet clinical need in patients with urothelial carcinoma (UC), we sought to identify tissue-based immune biomarkers of clinical benefit to ICIs using multiplex immunofluorescence and to integrate these findings with previously identified peripheral blood biomarkers of response. Fifty-five pretreatment and 12 paired on-treatment UC specimens were identified from patients treated with nivolumab with or without ipilimumab. Whole tissue sections were stained with a 12-plex mIF panel, including CD8, PD-1/CD279, PD-L1/CD274, CD68, CD3, CD4, FoxP3, TCF1/7, Ki67, LAG-3, MHC-II/HLA-DR, and pancytokeratin+SOX10 to identify over three million cells. Immune tissue densities were compared to progression-free survival (PFS) and best overall response (BOR) by RECIST version 1.1. Correlation coefficients were calculated between tissue-based and circulating immune populations. The frequency of intratumoral CD3+ LAG-3+ cells was higher in responders compared to nonresponders (p = 0.0001). LAG-3+ cellular aggregates were associated with response, including CD3+ LAG-3+ in proximity to CD3+ (p = 0.01). Exploratory multivariate modeling showed an association between intratumoral CD3+ LAG-3+ cells and improved PFS independent of prognostic clinical factors (log HR -7.0; 95% confidence interval [CI] -12.7 to -1.4), as well as established biomarkers predictive of ICI response (log HR -5.0; 95% CI -9.8 to -0.2). Intratumoral LAG-3+ immune cell populations warrant further study as a predictive biomarker of clinical benefit to ICIs. Differences in LAG-3+ lymphocyte populations across the intratumoral and peripheral compartments may provide complementary information that could inform the future development of multimodal composite biomarkers of ICI response. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Intestinal microbiota links to allograft stability after lung transplantation: a prospective cohort study

Whether the alternated microbiota in the gut contribute to the risk of allograft rejection (AR) and pulmonary infection (PI) in the setting of lung transplant recipients (LTRs) remains unexplored. A prospective multicenter cohort of LTRs was identified in the four lung transplant centers. Paired fecal and serum specimens were collected and divided into AR, PI, and event-free (EF) groups according to the diagnosis at sampling. Fecal samples were determined by metagenomic sequencing. And metabolites and cytokines were detected in the paired serum to analyze the potential effect of the altered microbiota community. In total, we analyzed 146 paired samples (AR = 25, PI = 43, and EF = 78). Notably, we found that the gut microbiome of AR followed a major depletion pattern with decreased 487 species and compositional diversity. Further multi-omics analysis showed depleted serum metabolites and increased inflammatory cytokines in AR and PI. Bacteroides uniformis, which declined in AR (2.4% vs 0.6%) and was negatively associated with serum IL-1β and IL-12, was identified as a driven specie in the network of gut microbiome of EF. Functionally, the EF specimens were abundant in probiotics related to mannose and cationic antimicrobial peptide metabolism. Furthermore, a support-vector machine classifier based on microbiome, metabolome, and clinical parameters highly predicted AR (AUPRC = 0.801) and PI (AUPRC = 0.855), whereby the microbiome dataset showed a particularly high diagnostic power. In conclusion, a disruptive gut microbiota showed a significant association with allograft rejection and infection and with systemic cytokines and metabolites in LTRs.

Comparative study of diagnostic efficacy of sputum and bronchoalveolar lavage fluid specimens in community-acquired pneumonia children treated with fiberoptic bronchoscopy

BackgroundCommunity-acquired pneumonia (CAP) is usually diagnosed in children, and the type of respiratory specimen is critical. Differences in pathogens detection between induced sputum (IS) and bronchoalveolar lavage fluid (BALF) have not been evaluated.MethodsIn 2018, paired sputum and BALF samples from CAP hospitalised children with indications for bronchoalveolar lavage (BAL) were subjected to multiplex PCR for the detection of 11 common respiratory pathogens.ResultsA total of 142 children with paired sputum and BALF were tested. The overall positivity rate was 85.9% (122/142) for sputum and 80.3% (114/142) for BALF. The two specimens presented almost perfect agreement between the detection on M. pneumoniae, influenza A, influenza B, bocavirus and RSV. In contrast, adenovirus had the lowest kappa value of 0.156, and a false negative rate (FNR) of 66.7%. Rhinovirus had the highest false positive rate (FPR) as 18.5%. The consistent rate was significantly higher in school-age children than those under 1 year old (p = .005). Bacterial co-infection in BALF specimens were observed in 14.8% (21/142). Of the 11 discordant pairs of specimens, 9 cases were sputum(+)/BALF(-) with adenovirus predominating.ConclusionOur findings suggest that the consistency of results between sputum and BALF is pathogen specific. Careful consideration needs to be given to whether sputum can be used as a substitute for BALF when children are young or co-infections with bacteria are suspected.

Genomic analysis and clinical correlations of non-small cell lung cancer brain metastasis

Up to 50% of patients with non-small cell lung cancer (NSCLC) develop brain metastasis (BM), yet the study of BM genomics has been limited by tissue access, incomplete clinical data, and a lack of comparison with paired extracranial specimens. Here we report a cohort of 233 patients with resected and sequenced (MSK-IMPACT) NSCLC BM and comprehensive clinical data. With matched samples (47 primary tumor, 42 extracranial metastatic), we show CDKN2A/B deletions and cell cycle pathway alterations to be enriched in the BM samples. Meaningful clinico-genomic correlations are noted, namely EGFR alterations in leptomeningeal disease (LMD) and MYC amplifications in multifocal regional brain progression. Patients who developed early LMD frequently have had uncommon, multiple, and persistently detectable EGFR driver mutations. The distinct mutational patterns identified in BM specimens compared to other tissue sites suggest specific biologic underpinnings of intracranial progression.

Subtype of SCLC Is an Intrinsic and Persistent Feature Through Systemic Treatment

IntroductionSCLC is an aggressive malignancy with poor outcome. Most patients have disease recurrence despite treatments with multiple modalities. Subtyping of SCLC has been proposed recently, and novel agents targeting specific subtypes are actively being investigated. In this study, we evaluated the plasticity of subtypes in paired pre- and post-treatment samples. The aim was to understand possible subtype evolution after chemotherapy resistance that could lead to alternate targeted therapy strategies. MethodsA total of 68 samples from 32 patients with sufficient paired specimens were identified from 1998 to 2022. ASCL1, NEUROD1, and POU2F3 immunohistochemistry studies were performed on all cases, and subtyping by predominant expression was determined. Subtype comparison in each patient was performed, and expression analysis was performed on the basis of subtypes. ResultsOf 32 cases, 28 (88%) had the same subtype in pre- and first post-treatment specimens. Protein expression level of subtype-specific transcription factor remained stable after chemotherapy. Two of five (40%) NEUROD1-predominant SCLC switched to ASCL1-predominant phenotype after treatment. One case had a pitfall of scoring ASCL1 on specimen with marked crushing artifacts. One case revealed the challenge of proper subtyping for samples with borderline POU2F3 expression. ConclusionsSubtype of SCLC generally remains the same after acquiring chemotherapy resistance. Plasticity was observed with rare cases switching from NEUROD1-predominant to ASC1-predominant SCLC. Resubtyping is unnecessary for the consideration of novel subtype-specific targeted agents, except cases with NEUROD1-predominant subtype.

Metastasis-related long non-coding RNAs AL359220.1, SH3BP5-AS1 and ZF-AS1 are significant for prognostic assessment of lung adenocarcinoma.

Metastasis of lung adenocarcinoma (LUAD) severely worsens prognosis. Genetic alteration in the tumor microenvironment (TME) is closely associated with metastasis and other malignant biological properties of LUAD. In this study, we establish a metastasis-related risk model to accurately predict LUAD prognosis. RNA-sequencing profiles and clinical data of LUAD patients including 503 tumor tissues and 54 adjacent normal tissues were collected in TCGA database. Additionally, the paired specimens from 156 LUAD patients were obtained in a single center. The metastatic relevance and clinical significance of metastasis-related long non-coding RNA (MRLNRs) was validated by series of in vitro experiments including western blotting, qPCR and transwell assays. Six MRLNRs were significantly correlated to prognoses of LUAD patients, of which AL359220.1, SH3BP5-AS1 and ZF-AS1 were further used to establish a metastasis-related risk scoring model (MRRS) due to the close associations with overall survival of LUAD patients. According to the MRRS, patients with higher scores in the high-risk group obtained poorer prognoses and survival outcomes. ZFAS1 expressed highly in tumor tissues and showed the inverse results compared to SH3BP5-AS1 and AL359220.1. In addition, the high expression of ZFAS1 was prominently correlated to the more advanced T-stage and distant metastasis. The reduction of ZFAS1 induced by siRNAs dramatically diminished the migration and invasion abilities of LUAD cells. In the present research, we elucidate the metastatic relevance and clinical significance of AL359220.1, SH3BP5-AS1 and ZF-AS1 in LUAD. Moreover, MRRS provide a promising assessing model for clinical decision making and prognosis of LUAD.

Does non-cavity distorting intramural fibroid affect endometrium around the time of embryo implantation?

The effect of the intramural fibroids not distorting the cavity remains controversial on implantation and pregnancy. The aim of this study was to examine the impact of non-cavity distorting intramural fibroids on endometrium. Fifty-six women with non-cavity distorting intramural fibroid were recruited in this study. Paired endometrial specimens, one from beneath the fibroid (ipsilateral endometrium) and the other from the opposite side of uterine cavity, away from the fibroid (contralateral endometrium) were obtained 7–9 days after the luteinizing hormone surge in a natural cycle. Histological dating, Mucin1 and Glycodelin expression and uterine natural killer (uNK) cell density were compared between the paired samples. The median (IQR) H-score of Mucin1 staining in the ipsilateral luminal epithelium was 210% (142–230%), which was significantly (p < 0.05) higher than that of the contralateral luminal endometrium (157%, IQR 114–176%). There was no significant difference in Mucin1 expression in the glandular epithelium. There was no significant difference in Glycodelin expression in luminal and glandular epithelium, uNK cells density or histological dating results between the paired endometrial samples. In conclusion, it is uncertain whether the altered expression of Mucin1 in luminal epithelium alone may have impact on implantation when other markers are not changed.

Low co-expression of PD-L1 and oncogenic receptor tyrosine kinases HER2 and cMET in urothelial carcinoma is associated with discordant expression between primary and metastatic sites

Novel regimens targeting immune checkpoints and the cMET or HER2 pathways are under investigation in metastatic urothelial carcinoma (mUC) though co-expression of these molecular targets has not been defined. We sought to characterize the protein co-expression rates of PD-L1, cMET and HER2 in primary and metastatic mUC lesions and agreement rates in paired biopsies.We assessed PD-L1, cMET and HER2 protein expression by immunohistochemistry (IHC) in archival mUC samples identified from an institutional database (n = 143). Correlation of expression between primary and metastatic biopsies was performed in patients with available paired biopsies (n = 79). Protein expression levels by predefined thresholds were measured, and Cohen's kappa statistics (κ) were utilized to assess the agreement in expression between paired primary and metastatic samples.In primary tumors (n = 85), high expression of PD-L1, cMET, and HER2 was observed in 14.1%, 34.1%, and 12.9%, respectively. In metastatic samples (n = 143), high expression of PD-L1, cMET and HER2 was detected in 9.8%, 41.3%, and 9.8%, respectively. Expression agreement rates between paired specimens (n = 79) were PD-L1: 79.7% (κ = 0.09), cMET: 69.6% (κ = 0.35), HER2: 84.8% (κ = 0.17). High PD-L1/cMET co-expression was observed in only 5.1% (n = 4) of primary and 4.9% (n = 7) of metastatic specimens. High co-expression of PD-L1/HER2 occurred in 3.8% (n = 3) of primary samples and no metastatic samples. The overall co-expression agreement between paired samples was 55.7% (κ = 0.22) for PD-L1/cMET and 67.1% (κ = 0.06) for PD-L1/HER2, but agreement for high co-expression between paired samples was very low (2.5% for PD-L1/cMET and 0% for PD-L1/HER2).Tumor co-expression of high cMET or HER2 and PD-L1 is low in this cohort. Agreement of high co-expression between primary and metastatic sites is rare. Biomarker-based strategies used in selection of patients for contemporary trials testing combinations of immune checkpoint inhibitors with either cMET or HER2-targeted agents should account for discordant biomarker expression between primary and metastatic sites.

Histologic Comparison of the Fascicular Area of Processed Nerve Allograft Versus Cabled Sural Nerve Autograft.

The use of multiple cables of sural nerve autograft is common for peripheral nerve reconstruction when injured nerve caliber exceeds the nerve graft caliber. Although the optimal matching of neural to nonneural elements and its association with functional outcomes are unknown, it is reasonable to consider maximizing the neural tissue structure available for nerve regeneration. No prior studies have compared directly the cross-sectional fascicular area between cabled nerve autografts and size-selected nerve allografts. This study evaluated the cross-sectional fascicular area between native nerve stumps and two reconstructive nerve grafting methods: cabled sural nerve autograft (CSNA) and processed nerve allograft (PNA). CSNA from matched cadaveric specimens and PNA were used to reconstruct nerve defects in the median and ulnar nerves of six pairs of cadaveric specimens. Nerve reconstructions were done by fellowship-trained hand surgeons. The total nerve area, fascicular area, and nonfascicular area were measured histologically. The CSNA grafts had significantly less fascicular area than PNA and caliber-matched native nerve. The PNA grafts had a significantly higher percent fascicular area compared with the intercalary CNSA graft. Fascicular area was significantly greater in PNA versus CSNA. The PNA consistently demonstrated a match in fascicular area closer to the native nerve stumps than CSNA, where CSNA had significantly smaller fascicular area compared with native nerve stumps.

MEKiAUTO: A multicenter phase 1 study of combination therapy with the MEK inhibitor, cobimetinib, immune checkpoint blockade, atezolizumab, and the autophagy inhibitor hydroxychloroquine (HCQ) in KRAS-mutated advanced malignancies.

45 Background: Autophagy, a catabolic cell survival process, is upregulated when cancer cells undergo MEK inhibition (MEKi) in preclinical models of pancreas adenocarcinoma (PDAC), and may function as a pathway of resistance. Simultaneous inhibition of the MEK and autophagy pathway in preclinical PDAC models has demonstrated anti-tumor and immune modulating effects. MEKiAUTO evaluated the tolerability and safety of combination cobimetinib, HCQ, and atezolizumab at three dose levels (DL), in advanced KRAS-mutated malignancies. Methods: This multicenter phase I study used the time-to-event continual reassessment method (TITE-CRM) with the primary objective of estimating the maximum tolerated dose (MTD) of the combination of cobimetinib (DL1 and DL2: 40mg; DL3: 60mg PO daily; on days 1-21); HCQ (DL1-DL3: 600mg PO BID), without (DL1) or with (DL2 and DL3) atezolizumab (840mg IV on days 1 and 15) every 28 days, in patients with advanced KRAS-mutated malignancies who had received at least one prior line of therapy (N=18). The secondary objective was to evaluate safety. Exploratory analysis included interrogating the MEK, autophagy, and key molecular pathways. Results: A total of 14 patients with PDAC were enrolled at Columbia University and Brown University between 02/24/20 through 03/15/22. The median age was 58, 57% were female. Of the 10 evaluable patients for dose limiting toxicity ((DLT); &gt;80% drug adherence and ≥4 week of follow-up), 4 experienced a DLT (3 out of 8 at DL1 and 1 out of 2 at DL2). The estimated MTD is dose level 1 with an estimated probability of DLT 0.37. Grade 3 or higher treatment-related adverse events occurred in 7 of 14 (50%) patients with elevation in AST and ALT (14%), acute kidney injury, hypokalemia, anemia, rash, and CPK increase (7% each). One grade 5 multi-organ failure and acute kidney injury occurred at DL1 possibly related to cobimetinib. Clinical benefit was observed in 5 of 14 (36%) patients, all of whom experienced stable disease as best response. The median PFS and OS for overall population was 7.7 weeks (95%CI: 6-NA) and 20.7 weeks (95%CI: 15.6-46.1). Patients with KRASG12R (N=7) had a trend toward longer OS (mOS: 20.9 vs 16.4 weeks) than patients with KRASnon-G12R mutations (N=7). Correlative analysis on paired tumor specimens using single nucleus RNAseq, multiplex immunofluorescence, and pathway analysis will discuss putative mechanisms of resistance. Conclusions: Combination cobimetinib, HCQ and atezolizumab had limited tolerability and efficacy in advanced KRAS-mutated PDAC. Patients with KRASG12R had a trend towards improved OS versus KRASnon-G12R mutations. Correlative analysis for pharmacodynamic and resistance mechanisms will be discussed. Funding: Roche-Genentech. Clinical trial information: NCT04214418 .

Diagnostic Performance of Urine-based HPV-DNA Test (CerviScan, Bio Farma) as Cervical Cancer Screening Tool in Adult Women

Objective: Detection of high-risk human papillomavirus (hr-HPV) in urine specimens has been introduced recently and a new local PCR kit has been developed in Indonesia (CerviScan, Bio Farma). The objective of this study was to obtain the accuracy of hr-HPV DNA testing using the new kit (CerviScan, Bio Farma) on urine specimens against the gold standard on cervical swabs.Method: Adult women (aged 20–50 years) underwent routine general check-up or Pap test were enrolled between July and September 2022. Pairs of urine and cervical swab specimens were obtained from all subjects. HPV-DNA tests were performed using the new local PCR kit (CerviScan, Bio Farma) and the standard procedure (COBAS® 6800 HPV, Roche Molecular System). Direct sequencing was added whenever there were dispute results between the two methods. Agreement between both methods was tested using Kappa statistics. Diagnostic performance test was done on CerviScan. Results: A total of 876 women completed the examination. Agreement between CerviScan and COBAS® 6800 was substantial (K=0.662; p&lt;0.001) and was almost perfect against COBAS® 6800 plus sequencing (K=0.828; p&lt;0.001). The accuracy of CerviScan on urine samples was 95.8% against COBAS® 6800 and increased to 97.8% after additional sequencing. The sensitivity and specificity of CerviScan on urine samples compared to cervical swabs are 73.1% and 97.3%, respectively.Conclusion: Urine-based HPV-DNA testing with CerviScan is a reliable tool to detect high-risk HPV subtypes. It could become an alternative method for HPV-DNA testing to improve the coverage of cervical cancer screening program.

Validation and benchmarking of targeted panel sequencing for cancer genomic profiling.

To validate a large next-generation sequencing (NGS) panel for comprehensive genomic profiling and improve patient access to more effective precision oncology treatment strategies. OncoPanScan was designed by targeting 825 cancer-related genes to detect a broad range of genomic alterations. A practical validation strategy was used to evaluate the assay's analytical performance, involving 97 tumor specimens with 25 paired blood specimens, 10 engineered cell lines, and 121 artificial reference DNA samples. Overall, 1107 libraries were prepared and the sequencing failure rate was 0.18%. Across alteration classes, sensitivity ranged from 0.938 to more than 0.999, specificity ranged from 0.889 to more than 0.999, positive predictive value ranged from 0.867 to more than 0.999, repeatability ranged from 0.908 to more than 0.999, and reproducibility ranged from 0.832 to more than 0.999. The limit of detection for variants was established based on variant frequency, while for tumor mutation burden and microsatellite instability, it was based on tumor content, resulting in a minimum requirement of 20% tumor content. Benchmarking variant calls against validated NGS assays revealed that variations in the dry-bench processes were the primary cause of discordances. This study presents a detailed validation framework and empirical recommendations for large panel validation and elucidates the sources of discordant alteration calls by comparing with "gold standard measures."