Diagnostic accuracy of autofluorescence-Raman spectroscopy for surgical margin assessment during Mohs micrographic surgery of basal cell carcinoma.

BACKGROUND: Autofluorescence (AF) - Raman spectroscopy is a technology that can detect residual basal cell carcinoma (BCC) on the resection margin of fresh surgically excised tissue specimens. The technology does not require tissue fixation, staining, labelling, or sectioning, and provides quantitative diagnosis maps of the surgical margins in 30 minutes. OBJECTIVES: To determine the accuracy of the AF-Raman instrument to detect incomplete excisions of BCC during Mohs micrographic surgery, using histology as reference standard. METHODS: Skin layers from 130 patients undergoing Mohs surgery at the Nottingham University Hospitals NHS Trust (September 2022 to July 2023) were investigated with the AF-Raman instrument. The layers were measured fresh, immediately after excision. The AF-Raman results and the intra-operative assessment by Mohs surgeons were compared to a post-operative consensus-derived reference produced by three dermatopathologists. The sensitivity, specificity, positive predictive value, and negative predictive value were calculated. RESULTS: The AF-Raman analysis was successfully completed for 125 out of the 130 layers. The AF-Raman analysis covered 91% of the specimen surface area on average, with the lowest being 87% for eyelid and the highest being 94% for forehead specimens. The AF-Raman instrument identified positive margins in 24 out of 36 BCC-positive cases, resulting in a 67% sensitivity (95% confidence intervals (CI): 49%-82%) and negative margins in 65 out of 89 BCC-negative cases, resulting in a 73% specificity (95% CI 63%-82%). Only one out of the 12 false negative cases was caused by misclassification by the AF-Raman algorithm. The other 11 false negatives cases were produced because no valid Raman signal was recorded at the location of the residual BCC due to either occlusion by blood or poor contact between tissue and cassette window. The intra-operative diagnosis by Mohs surgeons identified positive margins in 31 out of 36 BCC-positive cases, 86% sensitivity (95% CI: 70%-95%), and negative margins in 79 out of 89 BCC-negative cases, 89% specificity (95% CI: 81%-95%). CONCLUSIONS: This study shows that the AF-Raman instrument has potential for intra-operative microscopic assessment of surgical margins in surgery of BCC. Further improvements are required for tissue processing to ensure complete coverage of the surgical specimens. ClinicalTrials.gov ID NCT03482622.

Feasibility and safety of synchrotron-based X-ray phase contrast imaging as a technique complementary to histopathology analysis.

X-ray phase contrast imaging (X-PCI) is a powerful technique for high-resolution, three-dimensional imaging of soft tissue samples in a non-destructive manner. In this technical report, we assess the quality of standard histopathological techniques performed on formalin-fixed, paraffin-embedded (FFPE) human tissue samples that have been irradiated with different doses of X-rays in the context of an X-PCI experiment. The data from this study demonstrate that routine histochemical and immunohistochemical staining quality as well as DNA and RNA analyses are not affected by previous X-PCI on human FFPE samples. From these data we conclude it is feasible and acceptable to perform X-PCI on FFPE human biopsies.

NTRK Fusions in a Sarcomas Series: Pathology, Molecular and Clinical Aspects.

Targeting molecular alterations has been proven to be an inflecting point in tumor treatment. Especially in recent years, inhibitors that target the tyrosine receptor kinase show excellent response rates and durable effects in all kind of tumors that harbor fusions of one of the three neurotrophic tyrosine receptor kinase genes (NTRK1, NTRK2 and NTRK3). Today, the therapeutic options in most metastatic sarcomas are rather limited. Therefore, identifying which sarcoma types are more likely to harbor these targetable NTRK fusions is of paramount importance. At the moment, identification of these fusions is solely based on immunohistochemistry and confirmed by molecular techniques. However, a first attempt has been made to describe the histomorphology of NTRK-fusion positive sarcomas, in order to pinpoint which of these tumors are the best candidates for testing. In this study, we investigate the immunohistochemical expression of pan-TRK in 70 soft tissue and bone sarcomas. The pan-TRK positive cases were further investigated with molecular techniques for the presence of a NTRK fusion. Seven out of the 70 cases showed positivity for pan-TRK, whereas two of these seven cases presented an NTRK3 fusion. Further analysis of the fused sarcomas revealed some unique histological, molecular and clinical findings. The goal of this study is to expand the histomorphological spectrum of the NTRK-fused sarcomas, to identify their fusion partners and to correlate these parameters with the clinical outcome of the disease. In addition, we evaluated the immunohistochemical expression pattern of the pan-TRK and its correlation with the involved NTRK gene.

NTRK Gene Fusion Detection in a Pan-Cancer Setting Using the Idylla GeneFusion Assay.

Recently, approval of tyrosine receptor kinase (TRK) inhibitors by Food and Drug Administration and European Medicines Agency in NTRK fusion-positive cancer types has led to a variety of proposed testing algorithms. In this study, performance of the fully automated Idylla GeneFusion Assay was assessed in a set of clinically relevant cancer types, including glioblastoma, non-small-cell lung cancer, microsatellite instability-positive colorectal cancer, and thyroid carcinoma. Analysis with the Idylla GeneFusion Assay revealed significant differences in baseline RNA expression profile between the different cancer types, which corresponded to both literature and pan-TRK immunohistochemical staining. Compared with the RNA-based Oncomine Focus Assay, the Idylla GeneFusion Assay demonstrated an overall percentage agreement, positive percentage agreement, and negative percentage agreement of 92.7%, 81.8%, and 93.8%, respectively; and the pan-TRK immunohistochemistry demonstrated an overall percentage agreement, positive percentage agreement, and negative percentage agreement of 82.1%, 45.5%, and 85.7%, respectively. These findings highlighted the importance of tailoring NTRK testing algorithms per cancer type. In a small subset, data from the RNA-based Archer FusionPlex Assay were also available. NTRK fusion detection efficiency was compared between the four NTRK testing modalities, with a high concordance between the PCR-based methods. Last, RNA degradation was observed when using the Idylla GeneFusion Assay on snap frozen tissue samples as these are nonfixated. This might be countered by increasing the amount of sample input. To conclude, the Idylla GeneFusion Assay has shown a clear potential in identifying NTRK fusions.

Shallow whole-genome sequencing: a useful, easy to apply molecular technique for CNA detection on FFPE tumor tissue-a glioma-driven study.

Copy number alterations (CNAs) have increasingly become part of the diagnostic algorithm of glial tumors. Alterations such as homozygous deletion of CDKN2A/B, 7 +/ 10 - chromosome copy number changes or EGFR amplification are predictive of a poor prognosis. The codeletion of chromosome arms 1p and 19q, typically associated with oligodendroglioma, implies a more favorable prognosis. Detection of this codeletion by the current diagnostic standard, being fluorescence in situ hybridization (FISH), is sometimes however subject to technical and interpretation problems. In this study, we evaluated CNA detection by shallow whole-genome sequencing (sWGS) as an inexpensive, complementary molecular technique. A cohort of 36 glioma tissue samples, enriched with "difficult" and "ambiguous" cases, was analyzed by sWGS. sWGS results were compared with FISH assays of chromosomes 1p and 19q. In addition, CNAs relevant to glioblastoma diagnosis were explored. In 4/36 samples, EGFR (7p11.2) amplifications and homozygous loss of CDKN2A/B were identified by sWGS. Six out of 8 IDH-wild-type glioblastomas demonstrated a prognostic chromosome 7/chromosome 10 signature. In 11/36 samples, local interstitial and terminal 1p/19q alterations were detected by sWGS, implying that FISH's targeted nature might promote false arm-level extrapolations. In this cohort, differences in overall survival between patients with and without codeletion were better pronounced by the sequencing-based distinction (likelihood ratio of 7.48) in comparison to FISH groupings (likelihood ratio of 0.97 at diagnosis and 1.79 ± 0.62 at reobservation), suggesting sWGS is more accurate than FISH. We recognized adverse effects of tissue block age on FISH signals. In addition, we show how sWGS reveals relevant aberrations beyond the 1p/19q state, such as EGFR amplification, combined gain of chromosome 7 and loss of chromosome 10, and homozygous loss of CDKN2A/B. The findings presented by this study might stimulate implementation of sWGS as a complementary, easy to apply technique for copy number detection.

Evaluation of Cytologic Sample Preparations for Compatibility With Nucleic Acid Analysis.

OBJECTIVES: In this study, the influence of several key elements of the cytologic sample workflow on DNA and RNA content was evaluated. METHODS: The A549 cell line, patient-derived organoids, and pleural effusions were used to investigate the effect of (1) several collection media and delayed time to processing; (2) cytology specimens; (3) cytologic staining; and (4) formalin-fixed, paraffin-embedded (FFPE) cell block processing on nucleic acid quality and quantity as determined by fragment analyzer, Qubit analysis (Thermo Fisher Scientific), and quantitative polymerase chain reaction-based analysis on the Idylla platform (Biocartis). RESULTS: Alcohol-based collection media (CytoRich Red [Thermo Fisher Scientific] and EtOH95%) displayed high DNA and RNA preservation capacity, while phosphate-buffered saline and, to a lesser extent, formalin were associated with high RNA quality. Cytospin and smear cytology specimens were subject to DNA and RNA loss. Cytologic staining had no further impact on sample quality, hence destaining is not necessary. Both H&E-stained and unstained FFPE sections are compatible with nucleic acid analysis, despite a strong decrease in DNA and RNA quality. CONCLUSIONS: Although several key elements of the cytologic sample workflow have an influence on DNA and RNA quality and quantity, the selection of these elements is also dependent on the downstream (ancillary) testing methods.

Cusatuzumab for treatment of CD70-positive relapsed or refractory cutaneous T-cell lymphoma.

BACKGROUND: The clinical benefit of cusatuzumab, a CD70-directed monoclonal antibody with enhanced effector functions, was investigated in patients with relapsed/refractory (R/R) cutaneous T-cell lymphoma (CTCL). METHODS: In this cohort expansion of the ARGX-110-1201 study, 27 patients with R/R CTCL received cusatuzumab at 1 (n = 11) or 5 mg/kg (n = 16) once every 3 weeks to investigate its safety, dose, and exploratory efficacy. The pharmacokinetics, immunogenicity, CD70 expression, and CD70/CD27 biology were also assessed. RESULTS: The most common adverse events included infusion-related reactions, pyrexia, and asthenia. Eighteen serious adverse events (grade 1-3) were reported in 11 patients; 1 of these (vasculitis) was considered drug-related. For 8 of the 11 patients receiving 1 mg/kg, anti-drug antibodies (ADAs) affected the minimal concentration, and this resulted in undetectable cusatuzumab concentrations at the end of treatment and, in some cases, a loss of response. This effect was greatly reduced in the patients receiving 5 mg/kg. The overall response rate was 23%; this included 1 complete response and 5 partial responses (PRs) in 26 of the 27 evaluable patients. In addition, 9 patients achieved stable disease. The mean duration on cusatuzumab was 5.2 months, and the median duration was 2.5 months. Patients with Sézary syndrome (SS) achieved a 60% PR rate with a dosage of 5 mg/kg and a 33% PR rate with a dosage of 1 mg/kg; this resulted in an overall response rate of 50% for patients with SS at both doses. CONCLUSIONS: Cusatuzumab was well tolerated, and antitumor activity was observed at both 1 and 5 mg/kg in highly pretreated patients with R/R CTCL. The observed dose-dependent effect on exposure supports the use of 5 mg/kg for future development.

NTRK Fusions in Sarcomas: Diagnostic Challenges and Clinical Aspects.

Tropomyosin receptor kinase (TK) is encoded by the neurotrophic tyrosine receptor kinase genes (NTRK) 1, 2, and 3, whose activation plays an important role in cell cycle proliferation and survival. Fusions of one of these genes can lead to constitutive activation of TRK, which can potentially be oncogenic. NTRK fusions are commonly present in rare histologic tumor types. Among sarcomas, infantile fibrosarcoma shows NTRK fusion in more than 90% of the cases. Many other sarcoma types are also investigated for NTRK fusions. These fusions are druggable alteration of the agnostic type, meaning that all NTRK fused tumors can be treated with NTRK-inhibitors regardless of tumor type or tissue of origin. TRK-inhibitors have shown good response rates, with durable effects and limited side effects. Resistance to therapy will eventually occur in some cases, wherefore the next-generation TRK-inhibitors are introduced. The diagnosis of NTRK fused tumors, among them sarcomas, remains an issue, as many algorithms but no guidelines exist to date. Given the importance of this diagnosis, in this paper we aim to (1) analyze the histopathological features of sarcomas that correlate more often with NTRK fusions, (2) give an overview of the TRK-inhibitors and the problems that arise from resistance to the therapy, and (3) discuss the diagnostic algorithms of NTRK fused tumors with emphasis on sarcomas.

Immunohistochemistry as a screening tool for NTRK gene fusions: results of a first Belgian ring trial.

A Belgian ring trial for pan-TRK immunohistochemistry (IHC) staining was organised to harmonise pan-TRK IHC staining protocols and interpretation. As a reference method, the VENTANA pan-TRK Assay (clone EPR17341) on the Benchmark Ultra platform was selected. Six samples were selected: 2 negative, 2 fusion positive and 2 samples with wild-type endogenous TRK expression. Each participating laboratory stained the slides using their routine pan-TRK IHC and reported their results. In addition, they were asked to return one TRK-stained slide from each case. The coordinating lab evaluated these slides, compared them with the reference method and scored them. Two clones were used during the ring trial: A7H6R (Cell Signaling) and EPR17341 (Abcam/Ventana). Seven protocols achieved a sufficient performance mark, and three labs were advised to further optimise the protocol. Interpretation of pan-TRK IHC proved to be challenging in cases with physiological TRK expression. In addition, depending on the NTRK fusion partner, the staining can vary strongly in both intensity and staining pattern. Labs using the Ventana ready-to-use system based on the EPR17341 clone and using the recommended protocol settings scored best. However, given some small optimisation, all labs scored well on the technical staining and the succeeding evaluation.

Specialized Blood Collection Tubes for Liquid Biopsy: Improving the Pre-analytical Conditions.

INTRODUCTION: The potential of circulating cell-free DNA (cfDNA) analysis as a liquid biopsy has led to the development of several specialized measuring tools. Interest in the (pre-)analytical conditions of the liquid biopsy workflow has increased over the past few years. METHODS: In this study, we performed a systematic review of the cfDNA stabilizing efficacy in standard EDTA and specialized blood collection tubes (BCTs), namely CellSave, Norgen, PAXgene, Roche, and Streck tubes, and compared the efficacy of the latter three BCTs in a situation resembling the clinical setting. Blood samples were collected from ten KRAS-mutated metastatic cancer patients and stored for 72 h. During this time, samples were shaken and kept at either 6 °C or at room temperature for 24 h to mimic transport. RESULTS: We demonstrated that while cfDNA levels in EDTA tubes are only stable for a couple of (≤ 6) hours, they could be sustained for at least 48-72 h in all three specialized BCTs, irrespective of temperature. This timespan enables a fast turnaround time, which is one of the advantages of liquid biopsy. CONCLUSIONS: The choice between these specialized BCTs is less vital when they are processed correctly within a few days.

Clinical Performance of the Idylla MSI Test for a Rapid Assessment of the DNA Microsatellite Status in Human Colorectal Cancer.

In this study, the clinical performance of the Idylla MSI test (investigational use only) was evaluated in 330 colorectal carcinoma samples (all stages). This test is fully automated, from formalin-fixed, paraffin-embedded slide to result, and gives a result in <2.5 hours. Compared with the Promega MSI Analysis System version 1.2, an overall agreement, sensitivity, and specificity of 99.7%, 98.7%, and 100%, respectively, was reached. Whereas seven samples were invalid with the Promega MSI Analysis System, only two were invalid with the Idylla MSI test. Compared with the historical immunohistochemistry (IHC) data, overall agreement, sensitivity, and specificity of 98.7%, 94.4%, and 100%, respectively, were observed. Tumor mutation burden analysis of the discordant IHC cases was in favor of the Idylla MSI test result in three of the four samples. Furthermore, for those cases where the IHC data were invalid or hard to interpret because sole loss of one DNA mismatch repair deficiency marker was observed, Idylla MSI test results were always valid and accurate. Herein, the Idylla MSI test has been shown to be an accurate, fast screening assay for the detection of microsatellite status in colorectal cancer patients, with a low number of invalid results.

Circulating Cell-Free DNA and RNA Analysis as Liquid Biopsy: Optimal Centrifugation Protocol.

The combined analysis of circulating cell-free (tumor) DNA (cfDNA/ctDNA) and circulating cell-free (tumor) RNA (cfRNA/ctRNA) shows great promise in determining the molecular profile of cancer patients. Optimization of the workflow is necessary to achieve consistent and reproducible results. In this study, we compared five centrifugation protocols for the optimal yield of both cfDNA/ctDNA and cfRNA/ctRNA. These protocols varied in centrifugation speed, ambient temperature, time, and number of centrifugation steps. Samples from 33 participants were collected in either BD Vacutainer K2EDTA (EDTA) tubes or cell-free DNA BCT® (Streck) tubes. cfDNA concentration and fragment size, and cfRNA concentration were quantitated in all samples by digital droplet PCR (ddPCR) and quantitative PCR (qPCR). The KRAS-mutated ctDNA and ctRNA fraction was determined via ddPCR. In EDTA tubes, the protocol generating both plasma and platelets was found to produce high quality cfDNA and cfRNA concentrations. Two-step, high-speed centrifugation protocols were associated with high cfDNA but low cfRNA concentrations. High cfRNA concentrations were generated by a one-step, low-speed protocol. However, this coincided with a high amount of genomic DNA (gDNA) contamination. In Streck tubes, two-step, high-speed centrifugation protocols also generated good quality, high cfDNA concentration. However, these tubes are not compatible with cfRNA analysis.

A Multicenter Study to Assess EGFR Mutational Status in Plasma: Focus on an Optimized Workflow for Liquid Biopsy in a Clinical Setting.

A multicenter study was performed to determine an optimal workflow for liquid biopsy in a clinical setting. In total, 549 plasma samples from 234 non-small cell lung cancer (NSCLC) patients were collected. Epidermal Growth Factor Receptor (EGFR) circulating cell-free tumor DNA (ctDNA) mutational analysis was performed using digital droplet PCR (ddPCR). The influence of (pre-) analytical variables on ctDNA analysis was investigated. Sensitivity of ctDNA analysis was influenced by an interplay between increased plasma volume (p < 0.001) and short transit time (p = 0.018). Multistep, high-speed centrifugation both increased plasma generation (p < 0.001) and reduced genomic DNA (gDNA) contamination. Longer transit time increased the risk of hemolysis (p < 0.001) and low temperatures were shown to have a negative effect. Metastatic sites were found to be strongly associated with ctDNA detection (p < 0.001), as well as allele frequency (p = 0.034). Activating mutations were detected in a higher concentration and allele frequency compared to the T790M mutation (p = 0.003, and p = 0.002, respectively). Optimization of (pre-) analytical variables is key to successful ctDNA analysis. Sufficient plasma volumes without hemolysis or gDNA contamination can be achieved by using multistep, high-speed centrifugation, coupled with short transit time and temperature regulation. Metastatic site location influenced ctDNA detection. Finally, ctDNA levels might have further value in detecting resistance mechanisms.

The Trypanosoma cruzi satellite DNA OligoC-TesT and Trypanosoma cruzi kinetoplast DNA OligoC-TesT for diagnosis of Chagas disease: a multi-cohort comparative evaluation study.

BACKGROUND: The Trypanosoma cruzi satellite DNA (satDNA) OligoC-TesT is a standardised PCR format for diagnosis of Chagas disease. The sensitivity of the test is lower for discrete typing unit (DTU) TcI than for TcII-VI and the test has not been evaluated in chronic Chagas disease patients. METHODOLOGY/PRINCIPAL FINDINGS: We developed a new prototype of the OligoC-TesT based on kinetoplast DNA (kDNA) detection. We evaluated the satDNA and kDNA OligoC-TesTs in a multi-cohort study with 187 chronic Chagas patients and 88 healthy endemic controls recruited in Argentina, Chile and Spain and 26 diseased non-endemic controls from D.R. Congo and Sudan. All specimens were tested in duplicate. The overall specificity in the controls was 99.1% (95% CI 95.2%-99.8%) for the satDNA OligoC-TesT and 97.4% (95% CI 92.6%-99.1%) for the kDNA OligoC-TesT. The overall sensitivity in the patients was 67.9% (95% CI 60.9%-74.2%) for the satDNA OligoC-TesT and 79.1% (95% CI 72.8%-84.4%) for the kDNA OligoC-Test. CONCLUSIONS/SIGNIFICANCE: Specificities of the two T. cruzi OligoC-TesT prototypes are high on non-endemic and endemic controls. Sensitivities are moderate but significantly (p = 0.0004) higher for the kDNA OligoC-TesT compared to the satDNA OligoC-TesT.