Molecular Testing Research Articles

12279 Molecular Strategy And Genetic Characterization Of A Large Cohort Of Pediatric Patients With Hereditary Hypophosphatemic Rickets: Advantages Of Next Generation Sequencing

Abstract Disclosure: N. Perez Garrido: None. P. Ramirez: None. G.L. Viterbo: None. M. del Pino: None. S. Abbate: None. N.I. Saraco: None. F. Tesan: None. M. Ciaccio: None. V. Fano: None. A. Belgorosky: None. R. Marino: None. Background: Hereditary hypophosphatemic rickets (HHR) is a group of rare disorders characterized by renal phosphate wasting and impairment of vitamin D metabolism. Different genetic defects can cause HHR, although they share similar clinical, radiological and biochemical features. Dominant X-linked HR (XLHR) is the most frequent form (1/ 20,000) caused by inactivating variants in PHEX gene. As affected individuals present with a broad phenotypic spectrum next generation sequencing (NGS) represent a useful tool for molecular diagnosis characterization. Aim: To report a molecular strategy and genetic characterization of a large cohort of pediatric patients with HHR followed in a single tertiary institution. Patients and Methods: We analyzed 77 affected patients from 60 non related families (and 58 parents) sent to our laboratory for molecular genetic testing under suspicion of HHR based on clinical, radiological and laboratory studies.In patients without hypercalciuria, Sanger sequencing of PHEX gene was initially performed. In female cases in which no alteration was detected, MLPA analysis was used to detect copy number variations. A group of patients with no proven variations in PHEX gene and those with hypercalciuria were analyzed using a NGS custom panel including 14 related genes. Results: Of the total 60 non related cases a pathogenic variant was found in 52 (87%). Regarding PHEX gene analysis, a pathogenic variant was found in 48/60 (80%), 27 were sporadic and 21 familial cases. Forty-one different pathogenic variants were found (5 gross deletions, 13 nonsense, 9 frameshift, 5 splice-site and 9 missense) distributed throughout the gene with higher prevalence from exon 15 to 22. 18/41 (44%) of variants were novel. RNA analysis was performed in 2 novel splice variants (c.849G>A-p.Glu283Glu and c.1586+6T>A) in which an abnormal splicing was confirmed.We found a pathogenic variant in 5/9 cases analyzed by NGS. Three of them in SLC34A3 gene (2 homozygous, 1 compound heterozygous), 1 in ENPP1 gene (compound heterozygous) and 1 in PHEX gene non-detected by Sanger sequencing. Discussion: This molecular strategy, along with a careful clinical and biochemical selection of patients, allowed us to perform genetic diagnosis in 87 % of non-related families. This study reports 21 novel pathogenic variants and it shows the greatest genetic characterization in pediatric HHR patients in Argentine population. The results obtained confirmed that PHEX is the most responsible gene for HHR phenotype, as previously described. By NGS, less common forms of rickets could be detected. Among them, the most frequent was HHR with hypercalciuria due to recessive mutations in SLC34A3 gene. NGS studies represent a useful tool, especially in those complex cases, to reach an accurate diagnosis, and they contribute to improve long term follow up of patients and genetic counseling. Presentation: 6/2/2024

6688 Clinical Presentation and Management of Highly Differentiated Follicular Carcinoma Arising from Struma Ovarii with DICER1 Mutation: A Case Report

Abstract Disclosure: S.M. Seav: None. J. Shah: None. C. Dosiou: None. Background: Struma ovarii (SO) is a rare type of ovarian teratoma comprised of > 50% thyroid tissue. Extra-ovarian spread of SO, called peritoneal strumosis, was previously considered benign given the lack of histological malignant features. The 2020 WHO Classification of Female Genital Tumors has reclassified this as highly differentiated follicular carcinoma arising from struma ovarii (HDFCO), highlighting its low-grade malignant potential.[1] We report a case of HDFCO with extensive peritoneal disease and discuss its management. Clinical Case: A 38-year-old female presented to her OBGYN for irregular menses. Pelvic MRI showed an 8 cm right ovarian mass. Right salpingo-oopherectomy and pelvic peritoneal biopsies revealed well-differentiated thyroid tissue. Thyroid ultrasound demonstrated a multinodular goiter with ATA high risk nodules; 4 of 5 biopsied with benign results. Her thyroglobulin (Tg) was 170 ng/mL (normal 2 – 35 ng/ml). No further intervention was recommended. Two years later she was referred to our clinic. CT of abdomen/pelvis showed multifocal residual disease. We recommended more aggressive management, given the diagnosis of HDFCO. All visible disease was resected in a diagnostic laparoscopy with pelvic and omental biopsies. Pathology showed well-differentiated thyroid tissue. Molecular testing for the FGFR4 mutation associated with HDFCO was negative, but there was a DICER1 mutation c.5428 G>T of undetermined significance. DICER1 mutations are associated with thyroid cancers2. A total thyroidectomy and radioactive iodine ablation (RAI) were recommended. Thyroid pathology was benign. Tg was 9 ng/mL one month post-op. MRI six months post-laparoscopy revealed only one 6 mm omental lesion. Withdrawal I-123 whole body scan (WBS) showed multiple peritoneal implants. Tg was 214 ng/mL and TSH was 72 ulU/mL. The patient was treated with 157 mCi of oral I-131. Given the extensive peritoneal disease, she received prophylactic oral dexamethasone for 2 weeks after RAI to decrease the risk of peritoneal inflammation and bowel obstruction potentially mediated by local inflammation from radiation-related tissue injury. Her Tg three months post-RAI was 4 ng/mL. The surveillance plan is to alternate between RAI WBS and MRI abdomen/pelvis every 6 months. Conclusions: Peritoneal strumosis, previously considered benign, has recently been reclassified as HFDCO, an extremely rare low-grade malignancy with no standardized treatment. We report the first case of extensive HFDCO with a DICER1 mutation. Given the significant peritoneal involvement, our case was treated with total thyroidectomy, surgical excision of all implants, RAI, and prophylactic steroids post-RAI. Further research into the prognostic factors and optimal treatment of HFDCO is needed. Reference: [1]Giovanopoulou et al. Curr Oncol. 2022; 29(12): 9105-9116. 2Sauer et al. Head Neck Pathol. 2022; 16(1): 190–199. Presentation: 6/1/2024

12205 Challenging Case Of Anaplastic Thyroid Cancer With Unusual Histological Features: A Diagnostic Dilemma With High Grade Sarcoma

Abstract Disclosure: B. Gautam: None. B. Tiwari: None. M.S. Hossain: None. S. Hossain: None. S.C. Kumar: None. H. liao: None. Introduction: Anaplastic thyroid carcinoma (ATC) is a rare but highly aggressive thyroid cancer accounting for < 2% of cases, characterized by its rapid growth, extensive local invasion, and poor prognosis. Distinguishing ATC from other high-grade sarcomas, such as leiomyosarcoma, is challenging due to shared morphological features and overlapping immunoprofiles. We present a case involving a 73-year-old male presenting with rapidly growing neck mass, leading to diagnostic challenges differentiating between ATC and high-grade sarcomas. Case presentation: A 73-year-old male presented to ED with a rapidly growing painful neck mass for last 3 months, associated with dysphagia, dyspnea & dysphonia. He denied any fever, chills, weight loss and symptoms of hyperthyroidism or hypothyroidism. No personal or family history of thyroid cancer. On physical exam: vitals were normal, Neck exam revealed a left sided neck mass, mobile with deglutition, hard in consistency associated with hoarseness of voice. No stridor or wheeze noted on auscultation. Physical exam was otherwise normal. Laboratory tests showed normal CBC, CMP and TSH 1.45 mIU/L, T4 9.7 ug/dl. CT neck with contrast showed 5.4 x 5.6 x 8.2 cm large lobulated left thyroid mass with rightward airway deviation & substernal extension. Ultrasonogram of thyroid showed 9.6 x 5.3 x 3.3 cm left thyroid lobe with internal calcifications & extrathyroidal extension with several abnormal appearing cervical lymph node, largest measuring 4.9 x 2.4 x 3.3 cm. CT chest/abdomen/pelvis with contrast showed multiple bilateral lung nodules measuring up to 1.5 cm and 1.4 x 1.3 cm left gluteal soft tissue mass. IR-guided left neck mass biopsy was suggestive of primary spindle cell neoplasm with initial immunoprofiles consistent with a high-grade sarcoma, favoring leiomyosarcoma. Given the rapid growth of the neck mass with associated dyspnea, left thyroidectomy was performed and surgical pathology was suggestive of a high-grade sarcoma. However, subsequent pathology review made a final diagnosis of ATC, based on morphologic & immunohistochemical profile showing tumor cells strongly positive for p53 and Desmin with few cells positive for PAX-8, Cam 5.2 and CD68, and negative thyroglobulin, TTF-1, CK7, CK20, CK MNF116, pancytokeratin AE1/AE3 and CK5/6. Thyroseq revealed a distinctive molecular profile specifically positive for TERT and TP53 supporting diagnosis of ATC. Discussion: This case highlights the diagnostic dilemma posed by the resemblance between ATC and high-grade sarcomas due to overlapping clinical, morphological and immunohistology features. While both malignancies may exhibit similar morphological features, specific immunohistochemistry profiles along with molecular testing aid in distinguishing between them and guiding appropriate treatment strategies. Presentation: 6/1/2024

KRAS mutations detection methodology: from RFLP to CRISPR/Cas based methods.

In personalized cancer medicine, the identification of KRAS mutations is essential for making treatment decisions and improving patient outcomes. This work presents a comprehensive review of the current approaches for detection of KRAS mutations in different cancers. We highlight the value of fast and reliable KRAS mutations discovery and the effectiveness of molecular testing for selecting individuals who might benefit from targeted therapy. We provide an overview of various methods and tools available for detecting KRAS mutations, such as digital droplet PCR, next-generation sequencing (NGS), and polymerase chain reaction (PCR). We also address the difficulties and limitations in the identification of KRAS mutations, namely tumor heterogeneity and the emergence of resistance mechanisms. This article aims to guide clinicians in KRAS mutation identification.

8172 Retrospective Analysis of mRNA Expression Based Signatures of Thyroid Tumor Invasion and Metastases

Abstract Disclosure: S. Ahmadi: None. E. Namiranian: None. M. Alshalalfa: Employee; Self; Veracyte, Inc.. Stock Owner; Self; Veracyte, Inc. R. Jiang: Employee; Self; Veracyte, Inc.. Stock Owner; Self; Veracyte, Inc. Y. Hao: Employee; Self; Veracyte, Inc.. Stock Owner; Self; Veracyte, Inc. J.P. Klopper: Employee; Self; Veracyte, Inc.. Stock Owner; Self; Veracyte, Inc. R.T. kloos: Employee; Self; Veracyte, Inc.. Stock Owner; Self; Veracyte, Inc.. E. Marqusee: None. The American Thyroid Association thyroid cancer risk stratification system is driven primarily by the extent of vascular and extrathyroidal extension, as well as lymph node metastases. Minimizing surgical intervention for thyroid tumors from indeterminate thyroid nodules (ITN - Bethesda III and IV cytology) is often preferred to mitigate surgical complications and lessen the need for thyroid hormone supplementation post-operatively if clinical outcomes are not worsened. By leveraging the whole transcriptome derived Afirma Genomic Sequencing Classifier (GSC) thyroid nodule molecular testing platform, mRNA expression-based signatures were developed to predict thyroid tumors with a low risk of clinically significant invasion and metastases with a > 95% negative predictive value as part of the Afirma Genomics Resource for Intelligent Discovery (GRID). The objective of this study was to analyze the performance of these signatures on a retrospective cohort of patients with ITN who had Afirma GSC suspicious findings and thyroid surgery. Two hundred three subjects were evaluated, and pathology reports were scored for the extent of thyroid tumor invasion or metastases. Tumors with extensive vascular invasion or extrathyroidal extension are labeled as high risk (and otherwise are low risk). Tumors with lymph node metastases that either have documented > 2mm tumor deposits, > 40% of central nodes involved, or lateral lymph node metastases are labeled as high risk (and otherwise are low risk). The Afirma GRID invasion and metastases signatures were applied to correlate with the pathology scoring. The cohort was comprised of 144 (70%) female and 59 (30%) male patients with median age of 54 [IQR 40-65]. The cohort was mostly Bethesda III (n= 152, 75%). Fifty three percent (n=109) of the samples were malignant upon histology review. Two samples were scored as high risk for lymph node metastases (LNM) and 4 samples were scored as high risk for invasion (INV) based on final pathology. The LNM signature ruled out 55% of samples with 100% NPV and 55% specificity. No samples with high risk LNM scores were erroneously ruled out by the LNM GRID signature. The rule out % was similar in female (58%) and males (46%) (chi-squared test p=0.1). For the invasion model, 57% of samples were ruled out with 99% NPV and 57% specificity. One sample with extra-thyroidal invasion had a false negative GRID INV signature. The INV rule out % was similar in female (57%) and males (54%) (chi-squared test p=0.19). In summary, the Afirma GRID INV and LNM signatures ruled out clinically significant thyroid tumor features in > 50% of the studied cohort with > 95% NPV. Prospective studies should be performed to confirm the potential for Afirma GRID tumor behavior signatures to optimize and individualize surgical decision-making, decreasing the burden of unnecessary extent of thyroid surgery while maintaining outstanding clinical outcomes. Presentation: 6/3/2024

8109 Cancer-Associated Fibroblasts Correlate with Aggressive Thyroid Cancer Behavior: Insights from Four Large Patient Cohorts

Abstract Disclosure: M.A. Loberg: None. X. George: None. P.J. Courtney: None. H.C. Eric: None. A. Golding: None. B. David: None. M. Alshalalfa: Employee; Self; Veracyte, Inc.. Stock Owner; Self; Veracyte, Inc. Y. Hao: Employee; Self; Veracyte, Inc.. Stock Owner; Self; Veracyte, Inc. J.P. Klopper: Employee; Self; Veracyte, Inc.. Stock Owner; Self; Veracyte, Inc. R.T. Kloos: Employee; Self; Veracyte, Inc.. Stock Owner; Self; Veracyte, Inc.. V. Weiss: None. Thyroid tumor molecular analysis often utilizes fine-needle aspirate (FNA) samples to predict malignancy with high sensitivity. An outstanding question is if molecular sequencing can provide prognostic information to predict disease aggression, potentially informing management. Recently, the composition of the tumor microenvironment, in particular the presence of tumor-promoting fibroblasts, has been associated with aggressive thyroid cancer. However, fibroblast gene expression has not previously been analyzed in thyroid tumor FNA samples. In this work, we detect cancer-associated fibroblast (CAF) gene signatures across 4 distinct bulk RNA sequencing cohorts and confirm detection in FNA samples. Associations between CAF gene signatures and tumor characteristics were explored in: Afirma Genomic Sequencing Classifier (GSC) cohort from FNA (n=47,695), a Memorial Healthcare System (MHS) retrospective FNA cohort with pathology outcome data (n=318), TCGA papillary thyroid cancers (n=496), and a Vanderbilt University Medical Center-University of Washington (VUMC-UW) resection cohort (n=312). Published breast CAF gene sets were used to generate normal fibroblast and CAF subtype scores. Novel thyroid cancer specific CAF gene sets were generated from thyroid cancer single-cell RNA sequencing data. Scores for each gene set were calculated as the average Z-score. Across all cohorts, multiple CAF subsets were enriched in samples with BRAFV600E and papillary histology. The strongest enrichment was in an SFRP2+ CAF subset identified in thyroid cancer single-cell sequencing data. In Afirma GSC FNA samples, SFRP2+ CAFs were enriched in GSC-suspicious and Bethesda V/VI relative to GSC-benign nodules (Wilcoxon p value < 2e-[1]6 for all). In TCGA, SFRP2+ CAFs were associated with extrathyroidal extension, advanced disease stage, and aggressive histology. In the MHS cohort of 318 malignant FNA samples, SFRP2+ CAFs were significantly higher in patients with vascular invasion or lymph node metastasis (LNM) (OR:2.75 [95% CI 1.63-4.75], p=4e-5). In the VUMC-UW cohort, SFRP2+ CAFs were associated with shorter progression-free survival (PFS) (p=0.0033). In summary we created a novel thyroid cancer-specific CAF gene signature that is elevated in BRAFV600E+ and aggressive thyroid cancers across multiple large patient cohorts. We also demonstrated the ability to detect these CAF signatures in thyroid FNAs using the Afirma GSC molecular testing platform, with enrichment in suspicious and malignant samples. Amongst the CAF scores tested, thyroid-specific CAF subsets had the highest prognostic potential, and SFRP2+ CAFs, specifically, were associated with shorter PFS, tumor invasion and LNM. Together, these results highlight the potential of interrogating the thyroid tumor stroma in FNA using RNA-based assays to inform prognosis and possibly management. Presentation: 6/3/2024

Cartridge-Based Nucleic Acid Amplification Test Compared to Fine Needle Aspiration Cytology in Suspected Cases of Tubercular Lymphadenitis (the Indian Experience)

INTRODUCTION: Diagnosis of tubercular lymphadenitis is daunting as there are varied clinical presentations and no single confirmatory gold standard test. Cartridge-based nucleic acid amplification test (CBNAAT) of the lymph node is a rapid molecular diagnostic test for simultaneously detecting tuberculosis (TB) and rifampicin resistance. AIM: To evaluate the performance of the CBNAAT test for detecting M. tuberculosis in lymph node specimens compared to fine needle aspiration cytology (FNAC). MATERIALS AND METHODS: The study was conducted in a rural tertiary care hospital in central India. A total of 180 patients clinically suspected of tubercular lymphadenitis were included. The male-to-female ratio was 1:1.3. The average age was 33.3 years. The age group 21–40 years had the highest number of cases. The most common complaints among the patients were fever (29.4%), followed by loss of appetite (9.5%), weight loss (9.5%), and cough (6.6%). However, most patients presented to the hospital with only lymphadenopathy (44.4%). The most common site involved was the anterior cervical lymph node (78.8%), followed by the axillary group (10.5%), submandibular (2.8%), inguinal (2.8%), supraclavicular (2.2%), submental (1.7%) and infraclavicular (1.1%) group of lymph nodes. The patients were subjected to both FNAC and CBNAAT testing. Results were reported as positive or negative for M. tuberculosis as CBNAAT gives a semiquantitative estimate of the concentration of bacilli. Rifampicin resistance results were reported as detected or not detected. RESULTS: Cytological examination of the lymph node aspirates revealed that most were tubercular lymphadenitis cases. Cytomorphological analysis of the cases of tubercular lymphadenitis revealed Type 6 (tubercular abscess) as the predominant pattern. CBNAAT testing detected 26 cases of M. tuberculosis and three cases of rifampicin resistance. The study reported a specificity of 92.92% and low sensitivity of 26.86% of combined FNAC and CBNAAT is much higher compared to only CBNAAT. CONCLUSION: CBNAAT, along with FNAC, is a valuable addition in first-line investigations of tubercular lymphadenitis to make a timely diagnosis.

A potential conundrum in dermatopathology: molecularly confirmed superficial ossifying fibromyxoid tumors with unusual histomorphologic findings and a novel fusion.

Ossifying fibromyxoid tumor (OFMT) is a rare soft tissue neoplasm of uncertain histogenesis, primarily arising in subcutaneous tissues of the extremities, head and neck, or trunk. Most cases present as well-circumscribed masses with a characteristic morphologic appearance, comprising cytologically bland ovoid cells with fibromyxoid stroma, a peripheral rim of metaplastic bone, and lobulated architecture. Nevertheless, tumors displaying unusual morphologic characteristics pose significant diagnostic challenges, requiring the detection of a pathogenic fusion for a definitive diagnosis. The majority of OFMTs exhibits PHF1 gene rearrangements. Herein, we present six cases of molecularly confirmed OFMTs with uncommon histomorphologic features, including the absence of myxoid stroma, extensive chondroid differentiation, prominent clear cell morphology, collagen entrapment, interdigitating fibrocollagenous and fibromyxoid stromal elements, and conspicuous red blood cell extravasation. One case harbored a novel fusion (EPC1::SUZ12). This study emphasizes the broad range of morphologic manifestations that can be encountered in OFMT and the crucial role of molecular testing in establishing a conclusive diagnosis in such cases.

Molecular Testing in Gliomas: What is Necessary in Routine Clinical Practice?

A number of molecular characteristics are essential for accurate diagnosis and prognostication in glioma. The 2021 WHO classification of brain tumors and recent Food and Drug Administration (FDA) pathology agnostic drug approvals highlight the importance of molecular testing in the management of glioma. For diffuse gliomas, it is important to identify IDH mutations, given the favorable clinical behavior and potential for using FDA approved IDH inhibitors in the near future. MGMT promoter methylation testing is the most established molecular marker for response to temozolomide in IDH wild-type glioblastoma and in turn impacts overall survival. Moreover, identification of certain mutations and molecular markers, such as BRAF V600E, hypermutation or elevated tumor-mutational burden and NTRK fusions allow for the use of FDA approved agents that are tumor-agnostic. Finally, molecular testing opens options for clinical trials that are essential for diseases with limited treatment options like gliomas.

A-283 Serologic and molecular testing for the diagnosis of Rickettsia, Ehrlicha, and Anaplasma infections in patients in Maryland

Abstract Background Rickettsia, Anaplasma, and Ehrlichia are underrecognized tick-borne pathogens of increasing human importance. As tick habitats are expanding and populations are increasing, human exposure to these pathogens is also increased in recent years. Unfortunately, while treatment is usually simple, diagnosis can be difficult as it requires specialized testing for clinical diagnosis - generally performed only at reference laboratories. Methods To better understand the prevalence, diagnosis, and testing patterns for Rickettisal infections, Anaplasmosis, and Ehrlichiosis in Maryland, a retrospective analysis of all serologic and molecular testing for Rickettsia, Anaplasma, and Ehrlichia species performed for the University of Maryland Medical System from 2018-2022 was conducted. Eight hospitals within the University of Maryland Medical System (Central Maryland and Maryland’s Eastern Shore) diagnose Rickettsia, Anaplasma, and Ehrlichia infections using serologic and molecular testing. Results Between 2018-2022, an average of 413 individuals were tested for Rickettsia, Anaplasma, and Ehrlichia each year. On average 315 serologic tests for Rocky Mountain Spotted Fever (RMSF) group Rickettsia, 195 serologic tests for Ehrlichia chaffeensis (Anaplasma serology is not available), and 165 molecular tests for the detection of Anaplasma and Ehrlichia species were ordered each year. Clinical indications for testing included tick bite, fever, chills, severe headaches, muscle aches, and rash. Overall, 17% of individuals tested were positive for IgG antibodies to RMSF group Rickettsia, however only 1% were positive for IgM antibodies. Further, 20% of individuals tested were positive for IgG antibodies to Ehrlichia chaffeensis, while <1% were positive for IgM. Acute and convalescent samples were not tested for any individuals. Molecular evidence of infection was found in 8% of individuals tested. Nineteen precent were positive for Anaplasma phagocytophilum and the remaining 81% were positive for Ehrlichia chaffeensis. Interestingly, only two individuals had positive results from both molecular and serologic testing. Conclusions Results from serologic and molecular testing show that Rickettsia, Anaplasma, and Ehrlichia are important human pathogens with the potential to cause significant morbidity. However, lack of follow-up serology, and laboratory tests that confirm acute infection make definitive diagnosis difficult. Increased awareness of Rickettsial diseases and appropriate diagnostics is needed.

A-367 Development of a Multimodal Point-of-Care Diagnostic Platform to Perform Rapid, Accurate, and Cost-Effective Molecular, Immunochemistry, Clinical Chemistry, and Cytometry Tests

Abstract Background Access to point-of-care (POC) testing can make a significant impact on the quality of care. However, because POC diagnostic platforms available today are expensive and only perform one type of test, clinicians are unable to implement all the POC tests they wish to perform. Fluxergy is developing a multimodal POC diagnostic platform that consolidates diverse diagnostic modalities into a single, compact platform to minimize the cost of POC testing and standardize testing workflow for the most common clinical chemistry, hematology, immunochemistry and molecular tests. This study describes feasibility studies performed to benchmark these assay modalities on the Fluxergy Analyzer. Methods Fluxergy Analyzer: The Fluxergy Analyzer integrates conventional detection systems—colorimetric, fluorometric, and electrochemical—into a singular POC platform. This unique design expands -omics testing capabilities, including molecular, immunochemistry, clinical chemistry, and hematology within a single instrument. Benchmarking Feasibility Studies: Molecular benchmarking was performed using a multiplex respiratory panel on contrived samples in UTM/VTM. Immunochemistry benchmarking was performed using a procalcitonin assay on spiked sodium citrate plasma samples. Cytometry benchmarking was performed using a white blood cell count (WBC) assay and testing contrived samples whole blood samples. Results For molecular testing, the Fluxergy Analyzer was able to accurately detect flu A, flu B, SARS-CoV-2, and RSV with agreements with the Luminex ARIES® Flu A/B & RSV Assay and Cepheid Xpert® SARS-CoV-2 test results. For immunochemistry testing, the Fluxergy Analyzer accurate quantified PCT levels at 0.3, 0.6, 1.25 and 2.5 ng/mL. For hematology testing, the Fluxergy Analyzer demonstrated a strong correlation across the reportable range against the standard of care HemoCue analyzer. For clinical chemistry testing, the Fluxergy Analyzer demonstrated a strong correlation against the Siemen’s epoc® Blood Analysis System. Conclusions The Fluxergy Analyzer is uniquely suited to offer a comprehensive menu of the most common POC tests on a single, cost-effective platform.

Innovative COVID-19 Point-of-Care Diagnostics Suitable for Tuberculosis Diagnosis: A Scoping Review.

Rapid and accurate point-of-care (POC) tuberculosis (TB) diagnostics are crucial to bridge the TB diagnostic gap. Leveraging recent advancements in COVID-19 diagnostics, we explored adapting commercially available POC SARS-CoV-2 tests for TB diagnosis in line with the World Health Organization (WHO) target product profiles (TPPs). A scoping review was conducted following PRISMA-ScR guidelines to systematically map POC antigen and molecular SARS-CoV-2 diagnostic tests potentially meeting the TPPs for TB diagnostic tests for peripheral settings. Data were gathered from PubMed/MEDLINE, bioRxiv, medRxiv, publicly accessible in vitro diagnostic test databases, and developer websites up to 23 November 2022. Data on developer attributes, operational characteristics, pricing, clinical performance, and regulatory status were charted using standardized data extraction forms and evaluated with a standardized scorecard. A narrative synthesis of the data is presented. Our search yielded 2003 reports, with 408 meeting eligibility criteria. Among these, we identified 66 commercialized devices: 22 near-POC antigen tests, 1 POC molecular test, 31 near-POC molecular tests, and 12 low-complexity molecular tests potentially adaptable for TB. The highest-scoring SARS-CoV-2 diagnostic tests were the near-POC antigen platform LumiraDx (Roche, Basel, Switzerland), the POC molecular test Lucira Check-It (Pfizer, New York, NY, USA), the near-POC molecular test Visby (Visby, San Jose, CA, USA), and the low-complexity molecular platform Idylla (Biocartis, Lausanne, Switzerland). We highlight a diverse landscape of commercially available diagnostic tests suitable for potential adaptation to peripheral TB testing. This work aims to bolster global TB initiatives by fostering stakeholder collaboration, leveraging SARS-CoV-2 diagnostic technologies for TB, and uncovering new commercial avenues to tackle longstanding challenges in TB diagnosis.

A-236 Rapid multiplexed PCR (polymerase chain reaction) tests for respiratory disease improve process and time to diagnosis versus laboratory-based molecular testing

Abstract Background The Infectious Disease Society of America recommends molecular testing for symptomatic patients suspected of having COVID-19. Molecular tests can be performed at the point of care or in a centralized laboratory, using different methodologies, and can be singleplex or multiplex. Limited data are available examining the use of these tests in clinical practice and impact of different test types on patient outcomes; however, common antiviral treatments for both COVID-19 and influenza are more effective when taken earlier and are not indicated for use more than 5 and 2 days after symptom onset, respectively. The aim of this study was to describe the diagnostic journey for non-inpatients who received molecular testing for respiratory pathogens in the laboratory versus those who received testing using a rapid, multiplexed real-time RT-PCR test that can be performed at the point of care. Methods De-identified administrative claims were analyzed from July 1, 2020 through September 30, 2022 for commercially insured and Medicare Advantage enrollees in the Optum Labs Data Warehouse. Patients with ≥1 medical claim for a non-inpatient visit with a procedure code for testing for SARS-CoV-2 were identified; patients who received antigen testing at index were excluded. Patients were followed for up to 90 days. Two patient groups were selected for the final study sample: 1) Patients with ≥1 claim with CPT code 0240U or 0241U with a point-of-care or laboratory place of service on the index date (proprietary laboratory analyses codes for Xpert® Xpress CoV-2/Flu/RSV, or “Xpert Xpress”); and 2) Patients with other laboratory-based molecular testing on the index date. Standardized difference of >0.10 or <-0.10 was considered a significant imbalance in proportions or means. Results In total, 51,602 patients received testing with Xpert Xpress while 317,192 received laboratory testing. Patients who were tested with Xpert Xpress were significantly older (mean 49.82 vs 42.19, SD 0.3), had a higher Charlson Comorbidity Index (mean 0.7 vs 0.35, SD 0.29), and were more likely to have a respiratory risk factor (6.09% vs 2.38%, SD 0.19). On average, patients who were tested with Xpert Xpress had significantly fewer tests for SARS-CoV-2 on the index date (mean 1.2 vs 1.58, SD -0.55), and fewer tests for SARS-CoV-2, influenza, or RSV on the index date and through the end of the 90-day fixed follow-up period (mean 1.64 vs 2.57, SD -0.45). They were also significantly more likely to be diagnosed with COVID-19 on the index date (23.31% vs 16.53%, SD 0.17). Among patients with a diagnosis for COVID-19, mean time to diagnosis was significantly shorter among patients tested with Xpert Xpress (4.85 vs 8.86 days, SD -0.27; median 0 vs 4 days). This finding was also consistent for influenza (4.05 vs 20.28 days, SD -0.74; median 0 vs 5 days). Conclusions Testing with Xpert Xpress at index resulted in fewer total tests and faster diagnosis compared to testing in the laboratory. Rapid, multiplex PCR testing provides a faster time to diagnosis and may support more appropriate and effective treatment.

A-238 Lyophilized Bead Multiplex Assays for CT/NG/TV Detection

Abstract Background Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Trichomonas vaginalis (TV) cause the three most prevalent sexually transmitted infectious diseases in developed countries, contributing to approximately 300 million new infections worldwide, annually. Due to the challenges of culturing and using immunoassays to detect these pathogens, DNA-based molecular tests have been instrumental, and current trends in molecular testing for CT/NG/TV have moved toward point-of-care tests. Lyophilization provides benefits required for point-of-care tests, such as ambient storage, extended shelf life, simplified downstream workflow, and variable formats. A lyophilized-bead format enables an assay to be placed in sealed consumables and stored at room temperature for up to two years. An additional consideration is the need for endogenous and exogenous controls. Exogenous in-process controls can be used to validate the performance of workflows culminating in a qPCR reaction and provide flexibility to test developers. An ideal exogenous in-process control is nonhomologous to human or human pathogen genome sequences and does not align with primers or probes designed for pathogen detection. The objective of the current study was to develop a lyophilized-bead-format assay containing an ideal exogenous control and establish whether it is a valid option for the accurate detection of CT, NG, and TV. Methods A synthetic DNA control containing targets for CT/NG/TV/RNase P (RP)/exogenous control was used to establish an analytical LOD by qPCR. The 5-plex assay, lyophilized in beads, was hydrated and tested for linearity over an 8-log range of the synthetic DNA control. Further, the qPCR lyophilized bead with assay was tested with a proficiency sample by extracting DNA from ZeptoMetrix NATtrol CT/NG/TV Positive Control and spiking-in the exogenous DNA control. qPCR was performed on the extracted DNA and human cDNA. Results The analytical LOD for the control DNA was determined to be 5 copies/reaction. Additionally, linearity was observed by qPCR over an 8-log range of the synthetic DNA control for all five targets in multiplex with good efficiencies and R2 values. Finally, all targets were amplified and detected by qPCR using DNA extracted from ZeptoMetrix NATtrol CT/NG/TV Positive Control with spiked-in exogenous DNA control. The detection of the exogenous sequence in the qPCR multiplex reaction indicated the extraction was successful. Conclusions We have developed a multiplex qPCR assay in lyophilized-bead format that can identify infectious agents, such as CT/NG/TV, in a single reaction. We have included RP as a human host control, plus an exogenous in-process control, for a total of 5-plex in separate fluorescent channels. We have shown that our lyophilized beads with assay produce a positive qPCR result when combined with a proficiency sample.

A-268 Comparison of the Elecsys® HCV Duo Assay to Standard of Care Algorithmic Testing

Abstract Background Traditional hepatitis C virus (HCV) testing methods, though effective, may miss crucial diagnostic opportunities. The goal of this study was to assess the utility of Elecsys® HCV Duo antibody/antigen (Ab/Ag) assay as an alternative to traditional HCV algorithmic testing in the United States. Methods A prospective study was conducted at Barnes Jewish Hospital in St. Louis, Missouri. Patients with concurrent HCV Ab and molecular testing ordered within 48 h were enrolled. A total of 1,351 remnant serum and plasma-EDTA specimens were frozen following standard of care testing, which consisted of the Abbott Architect anti-HCV assay with reflex to cobas® HCV molecular assay. Patient demographics, risk factors, and results from both standard of care and HCV Duo testing were recorded. Results Among 1351 specimens, the HCV positivity rate as defined by algorithm was 19.9% (269/1351). Risk factors including homelessness and prior intravenous drug use were significantly associated with HCV diagnosis (P<0.0001). HCV Duo testing demonstrated a positivity rate of 19.6% (263/1345) based on Ab/Ag dual positivity. Comparing the HCV Duo Ab module to standard of care serology revealed a 95.1% PPA (95% CI, 93.1-96.5) and 98.8% NPA (95% CI, 97.7-99.4), while the HCV Duo Ag component exhibited a 72.3% PPA (95% CI, 66.7-77.3) and 99.6% NPA (95% CI, 99.1-99.9) when compared to molecular testing. In HCV-RNA+ patients, the Ag component was positively correlated to viral load with a Spearman r of 0.770 (95% CI, 0.715-0.815). The median viral load with for patients with Ag+ results was 6.27 log10 IU/mL (IQR, 5.90-6.69) and for patients with Ag- results was 5.08 log10 IU/mL (IQR, 4.47-5.61). Of HCV-RNA+ specimens , 266/271 (98.2%) were successfully detected on the Ab module. Among anti-HCV+/HCV-RNA+ cases, 99.2% (261/263) were detected as Ab+/Ag+ on the HCV Duo assay. These had a turnaround time (TAT) from serology to molecular results of 28.1 ± 23.5 h, which may have been significantly decreased if Ab/Ag dual positivity was solely used to establish a diagnosis (average TAT of 27 min). The remaining two discrepant specimens were from patients with a prior history of HCV infection, which may represent false negatives or seronegative chronic HCV infections. Notably, 5/8 (62.5%) anti-HCV-/HCV-RNA+ specimens were detected on the HCV Duo assay, which would have been missed by traditional algorithmic testing. These five patients had viral loads ranging from 323-41,400,000 IU/mL, compared to <15-5,400 IU/mL in the three undetected specimens. Of these five additional patient specimens captured on HCV Duo, two patients received an organ from an HCV+ donor, while the remaining three had no known history of HCV infection or transplant from an HCV+ donor. Conclusions The Elecsys® HCV Duo Ab/Ag assay, with its high sensitivity and specificity, drastically shortens the diagnostic window. Importantly, it successfully identified several anti-HCV-/HCV-RNA+ cases, a subgroup often undiagnosed by current algorithmic testing, demonstrating promise for improved diagnostic efficiency and accuracy in HCV detection. Further investigation is needed to determine whether improved diagnostic efficiency is due to enhanced antibody or antigen detection.

Standardization through education of molecular pathology: a spotlight on the European Masters in Molecular Pathology.

Despite advancements in precision medicine, many cancer patients globally, particularly those in resource-constrained environments, face significant challenges in accessing high-quality molecular testing and targeted therapies. The considerable heterogeneity in molecular testing highlights the urgent need to harmonize practices across Europe and beyond, establishing a more standardized and consistent approach in MP laboratories. Professionals, especially molecular pathologists, must move beyond traditional education to cope with this heterogeneity. This perspective addresses critical issues in molecular pathology (MP), such as limited access to high-quality molecular testing, leading to disparities in cancer treatment, and the consequences of inconsistent practices. Recognizing the necessity for a standardized framework for education to address these issues, educational programs play a pivotal role in updating professionals' skills to achieve standardization in MP. European experts from the Steering Committee, the Pathology Section of the European Union of Medical Specialists, and the European Society of Pathology have proposed creating a comprehensive Master's degree program called the "European Masters in Molecular Pathology" (EMMP). This program emerges as a strategic response to the demand for a specialized and standardized framework for education in MP, catering to professionals who concurrently work and study. The program's design aligns with evidence-based education methods, ensuring effective learning and engagement while integrating computational pathology to analyze complex molecular data, enhance diagnostic accuracy, and improve treatment outcomes. EMMP's structured curriculum, strategic partnerships, and regular updates underscore its significance in standardizing MP practices. Exploring future developments, this perspective delves into technology integration and interdisciplinary collaboration, anticipating ongoing advances and harmonization. Challenges and future directions in MP education are discussed, emphasizing the necessity for dynamic curriculum updates, seamless technology integration, and interdisciplinary cooperation. This perspective underscores EMMP's pivotal role in preparing pathologists for this dynamic field, advocating continuous advancements in education and training to uphold excellence in MP practices and maintain the highest patient care standards.

A-172 Feasibility of PT, aPTT, and Fibrinogen on a Multifunctional Digital Microfluidic Cartridge

Abstract Background Excessive bleeding is prevalent in severe trauma patients, and measurement of multiple coagulation parameters is vital to correcting coagulopathy and improving outcomes. There are no rapid, near-patient technologies that can perform multiple coagulation assays simultaneously on a single consumable. We are developing a point-of-care digital microfluidic (DMF) platform to test three critical coagulation markers: prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen. Methods Five proficiency testing plasma samples were obtained from the College of American Pathologists (CAP) with normal, intermediate, and prolonged response of PT and aPTT, and normal and elevated fibrinogen levels. After loading 10 μL of sample onto the cartridge, autonomous digital microfluidic operations divided the sample into three droplets, which were then combined with reagents for PT, aPTT or fibrinogen. The viscosity of these droplets, measured from the electrical impedance on a toaster-sized instrument, changes at a rate proportional to the amount of coagulation precursors present from which time/activity was calculated. Results Classification of samples into normal, intermediate or prolonged categories along with the respective times obtained from the DMF cartridges matched those obtained on the Stago Compact Max instrument (Figure). We also demonstrated separation of plasma from whole blood within the DMF cartridge using agglutinating reagents, enabling an easy-to-use workflow without the need for centrifugation. Conclusions Our preliminary results demonstrate that PT, aPTT and fibrinogen can be measured on a single cartridge with performance similar to comparator methods, with a time to result of <15 minutes. To our knowledge, this is the first time these 3 assays have been performed simultaneously on a single consumable and furthermore, this is the first time viscoelastic tests have been performed on a multifunctional instrument that also performs chemistry and molecular tests. Studies are underway to evaluate analytical and clinical performance.

A-170 Hypereosinophilic Syndrome and t(5;15): Case Report and Literature Review

Abstract Background Hypereosinophilic syndrome (HES) is a myeloproliferative disorder characterized by persistent eosinophilia (absolute eosinophil count >1,500/mm3), classified into primary/clonal, reactive and idiopathic. After ruling out secondary causes of eosinophilia (parasitic infections, allergic conditions, vasculitis and collagenosis, drugs, eosinophilic lung disease, allergic gastroenteritis and metabolic conditions such as adrenal insufficiency), the main hypotheses become oncohematological diseases. Main entities to consider are myeloid/lymphoid neoplasms associated with eosinophilia and rearrangement of PDGFRA, FDGFRB, FGFR1 or PCM1-JAK2 and chronic eosinophilic leukemia. Conventional and molecular cytogenetics are the basis for classification and diagnosis of various hematological malignancies. This report describes a rare case of HES with t(5;15) (q33;q22). Methods A 29-year-old male patient was admitted with leukocytosis with the following exams: hemoglobin 6.5g/dL, hematocrit 21.7%, platelets 71,000/mm3, leukocytes 56,190/mm3 (60% neutrophils with a left shift to pro-myelocyte; 6% monocytes and 24% eosinophils), DHL 277 U/L, B12 vitamin 932 pg/mL, folic acid 1.72ng/mL, ferritin 913ng/mL. Non-reactive results were detected for antinuclear antibody, rheumatoid Factor, HIV, hepatitis B and C, syphilis and chagas disease. Bone marrow was hypercellular for age with absolute granulocytic predominance (G:E ratio 64:1) and eosinophilia (38%) in all maturational stages without dysplasia. Bone marrow flow cytometry did not detect neither increases in blasts nor anomalous immunophenotype. Bone marrow biopsy demonstrated 99% cellularity with normal blasts. Cytogenetics with translocation between the long arms of chromosome 5 and 15 in 15 of 20 metaphases analyzed - 46,XY,t(5;15)(q33;q22). No other molecular tests were available for this patient. Results In the case described t(5;15)(q33;q22) was found, which may be related to the TP53BP1::PDGFRB gene rearrangement. This breakpoint generates a chimeric oncoprotein similar to other tyrosine kinase fusions responsive to Imatinib. 53BP1 is a cellular DNA damage response component that binds wild type (non-mutant) p53. Oncogenesis occurs since the coiled-coil domains of TP53BP1 can mediate the homodimerization of PDGFRB and the constitutive activation of its tyrosine kinase activity; on the other hand, the DNA damage response of TP53BP1 may be disrupted. Gene rearrangements involving PDGFRA and FDGFRB are associated with response to tyrosine kinase inhibitors. Elevated vitamin B12 is a common finding in myeloproliferations with eosinophilia due to the increased production of haptocorrins, which binds to vitamin B12 in serum and in other tissues. Conclusions To our knowledge, in the literature there is only one described case of Myeloproliferative Neoplasia with Eosinophilia related to t(5;15)(q33;q22).

A-368 Feasibility of a Five-Minute Sample-to-Answer Basic Metabolic Panel for Point-of-Care Testing on the Fluxergy Analyzer

Abstract Background Adoption of point-of-care (POC) platforms has been limited by their high cost and limited menu (e.g., molecular only). Fluxergy has developed a cost-effective multimodal POC platform capable of performing the most common clinical chemistry, immunochemistry, hematology, molecular tests in CLIA-waived settings. This method comparison study describes the performance of the Fluxergy’s feasibility stage basic metabolic panel (BMP) with contrived samples compared to the Siemen’s epoc® Blood Analysis System. Methods Fluxergy Analyzer: The Fluxergy Analyzer is a rapid and cost-effective multimodality platform that can perform lab-quality PCR, clinical chemistry, immunochemistry, and cytometry in a single instrument and if required, in a single cartridge. Fluxergy BMP: Fluxergy BMP rapidly quantifies key electrolytes (K+, Na+, Ca2+, Cl-) and metabolites (glucose, creatinine, and BUN) from whole blood in under five minutes on the Fluxergy Analyzer, utilizing the Fluxergy test cards with screen-printed electrochemical sensor. Feasibility Study: A method comparison study was conducted to assess the performance of the feasibility stage Fluxergy BMP using contrived samples generated by spiking known concentrations of K+, Na+, Ca2+, Cl-, and glucose into buffer solutions. Samples were tested in triplicate with both the Fluxergy BMP and Siemen’s epoc® Blood Analysis System at three different concentrations across the reportable range of the epoc® Blood Analysis System. Results The feasibility stage Fluxergy BMP demonstrated strong correlation across the reportable range for K+, Na+, Ca2+, Cl-, and glucose versus the epoc® Blood Analysis System (Table 1). The error percentage for all samples tested except one glucose sample (9.5 mM) was less than 10%. The coefficient of variance for all samples tested except one Ca2+ sample (0.44 mM) was less than ten percent. Conclusions This feasibility data demonstrates the Fluxergy Analyzer is well-suited to perform rapid and accurate metabolic testing from whole blood samples at the POC.

B-192 Covid on the PCR Pro: a consumer-friendly PCR test

Abstract Background The emergence of COVID-19 highlighted the need for rapid and accurate diagnostic solutions that can be deployed on a broad scale. The complexity and long turn-around-time of PCR testing typically performed in molecular laboratories is not compatible with community-wide screening. To overcome these challenges, we present the Co-Dx PCR Pro™, a rapid and simple test platform developed for self-collection and at-home use. The instrument is controlled from the user’s smartphone or tablet and performs sample treatment, RT-PCR, and DNA melting analysis. An automatic calling algorithm provides a simple, “Detected,” or, “Not detected,” result. We demonstrate the utility of this platform using a test for the detection of SARS-COV-2. Methods A self-collected anterior nasal swab sample and 2 mLs of water are added to the collection cup. Closing the lid of the cup initiates rehydrating a dried Proteinase K mixture to digest proteins in the sample. After a brief Proteinase K heat-kill step, the instrument mixes the sample with a dried PCR master mix. Primers target two SARS-COV-2 specific genes (RdRp and E gene) and the human RNase P gene as a control. The sample undergoes an RT step followed by 45 cycles of PCR and DNA melting analysis. Detection is by an automatic calling algorithm that identifies a SARS-COV-2 amplicon melting peak at 82°C and RNase P peak at 87°C. The total runtime is less than 30 minutes. The limit of detection for the assay was assessed by contriving intact virus in negative sample matrix, which was then applied to dry swabs. In addition, frozen and banked clinical samples were tested using the Logix Smart®™ Covid-19 test as a comparator assay. A third party performed a usability assessment by having (1) naïve users follow the instructions and self-administer the test (2) expert trained users perform an independent assessment of the assay. Participants were then asked to complete a usability assessment. Results The 95% limit of detection was 2,100 copies/swab using contrived nasal swabs. Clinical samples were blinded to the operator and run on the platform. All samples resulted in a diagnosis concordant with the comparator assay. The usability assessment was performed by six naïve users and resulted in a systems usability scale score of “Best” (84.2 ± 14.3, out of 100). The two expert users determined an 80% limit of detection of 700 genome equivalents and “ease of use,” and “satisfaction,” scores of 4.5/5.0 and 5.0/5.0, respectively. Conclusions Performance of the PCR Pro platform is on par with laboratory-based PCR testing. The benefits of our assay are its speed and the low complexity platform accessible to the lay. This platform could improve access to molecular diagnostic testing, including use at home. Future expansion to include common respiratory pathogens (Flu, RSV, Strep) will increase utility. Additionally, anonymized results can be aggregated and provide a wealth of epidemiological information at the community level to better inform public health initiatives and health outcomes.