Clinical application of targeted long read sequencing in prenatal beta-thalassemia testing and genetic counseling.

BACKGROUND: Beta thalassemia, related to HBB mutation and associated with elevated hemoglobin A2 (HbA2), is an important genetic hemoglobinopathy with high incidences of disease and carrier rates in Singapore. Carrier screening is essential to facilitate prenatal counseling and testing. However, when individuals with elevated HbA2 do not have an identifiable HBB disease-associated variant, there is ambiguity on risk to their offspring. METHODS: We describe a case report of a proband with elevated HbA2, no identifiable HBB disease-associated variant, whose partner was a beta thalassemia carrier. Through clinical HBB gene sequencing, multiplex ligation-dependent probe amplification (MLPA) analysis, as well as targeted Nanopore long read sequencing of selected genes, we performed a complete analysis of HBB including the promoter region, 5'UTR and coding gene sequence, as well as evaluation for potential modifier variants and other rare structural variants. RESULTS: This process identified that the proband was heterozygous for KLF1:c.544T>C (p.Phe182Leu), a potential functional polymorphism previously known to be associated with benign elevated HbA2 levels. The presence of disease variants in the HBB locus was excluded. CONCLUSION: This finding provided clarity and enabled family planning for the proband and her family.

The Prognostic Value of CD39 as a Marker of Tumor-Specific T Cells in Triple-Negative Breast Cancer in Asian Women.

Triple-negative breast cancer (TNBC) has a poor prognosis with limited therapeutic options available for affected patients. Efforts are ongoing to identify surrogate markers for tumor-specific CD8+ T cells that can predict the response to immune checkpoint inhibitor (ICI) therapies, such as programmed cell death protein 1 or programmed cell death ligand-1 blockade. We have previously identified tumor-specific CD39+CD8+ T cells in non-small cell lung cancer that might help predict patient responses to programmed cell death protein 1 or programmed cell death ligand-1 blockade. Based on this finding, we conducted a comparative interrogation of TNBC in an Asian cohort to evaluate the potential of CD39 as a surrogate marker of tumor-specific CD8+ T cells. Using ICI-treated TNBC mouse models (n = 24), flow cytometric analyses of peripheral blood mononuclear cells and tumor-infiltrating lymphocytes revealed that >99% of tumor-specific CD8+ T cells also expressed CD39. To investigate the relationship between CD39+CD8+ T-cell density and CD39 expression with disease prognosis, we performed multiplex immunohistochemistry staining on treatment-naive human TNBC tissues (n = 315). We saw that the proportion of CD39+CD8+ T cells in human TNBC tumors correlated with improved overall survival, as did the densities of other CD39+ immune cell infiltrates, such as CD39+CD68+ macrophages. Finally, increased CD39 expression on CD8+ T cells was also found to predict the response to ICI therapy (pembrolizumab) in a separate cohort of 11 TNBC patients. These findings support the potential of CD39+CD8+ T-cell density as a prognostic factor in Asian TNBC patients.

3D Printed Bioactive PLGA Dermal Scaffold for Burn Wound Treatment.

Burn injury represents a major global public healthcare problem and has a significant health-economics impact. In this study, we report on a 3D printed poly(lactic-co-glycolic acid) (PLGA) dermal scaffold containing bioactive PLGA for burn wound healing. Bioactive brush copolymers containing pendant side chains of PLGA and PEGylated Arg-Gly-Asp tripeptide (RGD) or hyaluronic acid (HA) were synthesized by ring-opening metathesis polymerization (ROMP). These copolymers exhibited good thermal stability for material processing using melt-extrusion-based methods. The copolymers were blended with commercial PLGA, extruded into filaments and 3D printed using fused filament fabrication (FFF) methods with incorporated porosities. The 3D printed scaffolds demonstrated good biocompatibility in in vitro cell assays and in vivo murine models. Porcine study based on partial thickness burn wound model showed that these PLGA scaffolds facilitated re-epithelization with reduced inflammation as compared to the clinical gold standard for second-degree burn wound treatment, Biobrane. The bioactive PLGA scaffolds presented herein are beneficial in wound healing and have therapeutic potential in burn wounds treatment.

Multimodal molecular landscape of response to Y90-resin microsphere radioembolization followed by nivolumab for advanced hepatocellular carcinoma.

BACKGROUND: Combination therapy with radioembolization (yttrium-90)-resin microspheres) followed by nivolumab has shown a promising response rate of 30.6% in a Phase II trial (CA209-678) for advanced hepatocellular carcinoma (HCC); however, the response mechanisms and relevant biomarkers remain unknown. METHODS: By collecting both pretreatment and on-treatment samples, we performed multimodal profiling of tissue and blood samples and investigated molecular changes associated with favorable responses in 33 patients from the trial. RESULTS: We found that higher tumor mutation burden, NCOR1 mutations and higher expression of interferon gamma pathways occurred more frequently in responders. Meanwhile, non-responders tended to be enriched for a novel Asian-specific transcriptomic subtype (Kaya_P2) with a high frequency of chromosome 16 deletions and upregulated cell cycle pathways. Strikingly, unlike other cancer types, we did not observe any association between T-cell populations and treatment response, but tumors from responders had a higher proportion of CXCL9+/CXCR3+ macrophages. Moreover, biomarkers discovered in previous immunotherapy trials were not predictive in the current cohort, suggesting a distinctive molecular landscape associated with differential responses to the combination therapy. CONCLUSIONS: This study unraveled extensive molecular changes underlying distinctive responses to the novel treatment and pinpointed new directions for harnessing combination therapy in patients with advanced HCC.

Application of Artificial Intelligence to In Vitro Tumor Modeling and Characterization of the Tumor Microenvironment.

In vitro tumor models have played vital roles in enhancing the understanding of the cellular and molecular composition of tumors, as well as their biochemical and biophysical characteristics. Advances in technology have enabled the evolution of tumor models from two-dimensional cell cultures to three-dimensional printed tumor models with increased levels of complexity and diverse output parameters. With the increase in complexity, the new generation of models is able to replicate the architecture and heterogeneity of the tumor microenvironment more realistically than their predecessors. In recent years, artificial intelligence (AI) has been used extensively in healthcare and research, and AI-based tools have also been applied to the precise development of tumor models. The incorporation of AI facilitates the use of high-throughput systems for real-time monitoring of tumorigenesis and biophysical tumor properties, raising the possibility of using AI alongside tumor modeling for personalized medicine. Here, the integration of AI tools within tumor modeling is reviewed, including microfluidic devices and cancer-on-chip models.

Early Triple-Negative Breast Cancers in a Singapore Cohort Exhibit High PIK3CA Mutation Rates Associated With Low PD-L1 Expression.

Mutations in the PI3K pathway, particularly PIK3CA, were reported to be intimately associated with triple-negative breast cancer (TNBC) progression and the development of treatment resistance. We profiled PIK3CA and other genes on 166 early-stage TNBC tumors from Singapore for comparison to publicly available TNBC cohorts. These tumors were profiled transcriptionally using a NanoString panel of immune genes and multiplex immunohistochemistry, then manually scored for PD-L1-positivity using 2 clinically relevant clones, SP142 and 22C3. We discovered a higher rate of PIK3CA mutations in our TNBC cohort than in non-Asian cohorts, along with TP53, BRCA1, PTPN11, and MAP3K1 alterations. PIK3CA mutations did not affect overall or recurrence-free survival, and when compared with PIK3CAWT tumors, there were no differences in immune infiltration. Using 2 clinically approved antibodies, PIK3CAmut tumors were associated with PD-L1 negativity. Analysis of comutation frequencies further revealed that PIK3CA mutations tended to be accompanied by MAP kinase pathway mutation. The mechanism and impact of PIK3CA alterations on the TNBC tumor immune microenvironment and PD-L1 positivity warrant further study.

Pitfalls in clinical genetics.

With the increasing availability of genetic tests, more doctors are offering and ordering such tests for their patients. Ordering a genetic test appears to be a simple process of filling in paperwork, drawing 3 mL of blood in an ethylenediaminetetraacetic acid tube and receiving a test report. This is identical to sending off a full blood count. However, it is far more complex than that. There are many potential pitfalls, as shown by the increasing number of complaints and lawsuits filed against doctors and allied health staff. Furthermore, clinical genetics involves more than just ordering tests; in fact, focusing on genetic tests alone is a potential pitfall. In this review, we discuss the common pitfalls in clinical genetics and how doctors can avoid these pitfalls to ensure patient safety and to safeguard their practice.

Corrigendum: Case report: Persistence of residual antigen and RNA of the SARS-CoV-2 virus in tissues of two patients with long COVID.

[This corrects the article DOI: 10.3389/fimmu.2022.939989.].

Using gene panels in the diagnosis of neuromuscular disorders: A mini-review.

The diagnosis of inherited neuromuscular disorders is challenging due to their genetic and phenotypic variability. Traditionally, neurophysiology and histopathology were primarily used in the initial diagnostic approach to these conditions. Sanger sequencing for molecular diagnosis was less frequently utilized as its application was a time-consuming and cost-intensive process. The advent and accessibility of next-generation sequencing (NGS) has revolutionized the evaluation process of genetically heterogenous neuromuscular disorders. Current NGS diagnostic testing approaches include gene panels, whole exome sequencing (WES), and whole genome sequencing (WGS). Gene panels are often the most widely used, being more accessible due to availability and affordability. In this mini-review, we describe the benefits and risks of clinical genetic testing. We also discuss the utility, benefits, challenges, and limitations of using gene panels in the evaluation of neuromuscular disorders.

Case report: Understanding the impact of persistent tissue-localization of SARS-CoV-2 on immune response activity via spatial transcriptomic analysis of two cancer patients with COVID-19 co-morbidity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected half a billion people, including vulnerable populations such as cancer patients. While increasing evidence supports the persistence of SARS-CoV-2 months after a negative nasopharyngeal swab test, the effects on long-term immune memory and cancer treatment are unclear. In this report, we examined post-COVID-19 tissue-localized immune responses in a hepatocellular carcinoma (HCC) patient and a colorectal cancer (CRC) patient. Using spatial whole-transcriptomic analysis, we demonstrated spatial profiles consistent with a lymphocyte-associated SARS-CoV-2 response (based on two public COVID-19 gene sets) in the tumors and adjacent normal tissues, despite intra-tumor heterogeneity. The use of RNAscope and multiplex immunohistochemistry revealed that the spatial localization of B cells was significantly associated with lymphocyte-associated SARS-CoV-2 responses within the spatial transcriptomic (ST) niches showing the highest levels of virus. Furthermore, single-cell RNA sequencing data obtained from previous (CRC) or new (HCC) ex vivo stimulation experiments showed that patient-specific SARS-CoV-2 memory B cells were the main contributors to this positive association. Finally, we evaluated the spatial associations between SARS-CoV-2-induced immunological effects and immunotherapy-related anti-tumor immune responses. Immuno-predictive scores (IMPRES) revealed consistent positive spatial correlations between T cells/cytotoxic lymphocytes and the predicted immune checkpoint blockade (ICB) response, particularly in the HCC tissues. However, the positive spatial correlation between B cells and IMPRES score was restricted to the high-virus ST niche. In addition, tumor immune dysfunction and exclusion (TIDE) analysis revealed marked T cell dysfunction and inflammation, alongside low T cell exclusion and M2 tumor-associated macrophage infiltration. Our results provide in situ evidence of SARS-CoV-2-generated persistent immunological memory, which could not only provide tissue protection against reinfection but may also modulate the tumor microenvironment, favoring ICB responsiveness. As the number of cancer patients with COVID-19 comorbidity continues to rise, improved understanding of the long-term immune response induced by SARS-CoV-2 and its impact on cancer treatment is much needed.

Generation of iPSC lines from hereditary spastic paraplegia 56 (SPG56) patients and family members carrying CYP2U1 mutations.

Hereditary spastic paraplegia 56 (SPG56) is an extremely rare autosomal recessive disorder caused by mutations in the CYP2U1 gene, involved in fatty acid metabolism. SPG56 causes progressive spasticity in upper and lower limbs, though due to the rarity of this subtype of spastic paraplegia, the molecular causes remain unclear and no treatment or cure exists. Here we describe the generation and validation of induced pluripotent stem cell (iPSC) lines from two unrelated patients with SPG56 and two heterozygous family members. These lines can be used to investigate the mechanisms driving progressive spasticity and evaluate the potential for gene replacement therapies.

Reduced resource utilization with early use of next-generation sequencing in rare genetic diseases in an Asian cohort.

Children with genetic diseases endure a prolonged and costly "diagnostic odyssey." The use of whole exome sequencing (WES) and whole genome sequencing (WGS) has improved the diagnosis rate, ending the odyssey. However, the additional costs associated WES/WGS has impeded their adoption in Asian settings. We aim to estimate the expected change to the mean number of diagnostic tests used, and the associated costs from a decision to use WES early in the diagnostic pathways of pediatric phenotypes, as compared to Existing Practice. Retrospective data from a patient cohort recruited under the Singapore Undiagnosed Disease Program from a tertiary hospital in Singapore, for the period October 2004 to September 2020, was analyzed. Four phenotype-specific subgroups were used: multiple congenital anomalies (MCA) without developmental delay; global developmental delay (GDD); neuromuscular disorder (NMD) and primary immunodeficiency disorder (PID). Patients had undergone a traditional diagnostic pathway and received a diagnosis either through clinical exome or WES or WGS. A costs only analysis was performed, by tabulating the outcomes "test quantity" and "test costs" incurred by patients. The outcomes were compared with alternate diagnostic pathways which incorporates the early introduction of WES trio testing. To include uncertainty in cost outcomes, simulation studies were done on uncertain parameters. Cost outcomes are reported in Singapore dollars (S$). The 92 included patients had MCA (n = 48), GDD (n = 29), NMD (n = 10), or PID (n = 5). Patients were aged between 18 days and 26 years, 52.2% were males. The majority were of Chinese ethnicity (81.5%). If patients had access to WES directly, test quantity reduced by 97.38% for MCA, 96.98% for GDD, 96.56% for NMD, and 99.84% for PID. The expected cost savings per patient were $5940 for MCA (US$4433), $5342 for GDD (US$3986), $4622 for NMD (US$3449), and $58,497 for PID (US$43,654). Uncertainty assessment for MCA and GDD patients showed a respective likelihood of 86.9% and 97.4% for cost savings. Adoption of alternate diagnostic pathways with early WES in selected pediatric subgroups are likelt to reduce costs, when compared to Existing Practice. Benefits arising from earlier diagnosis, and the potential cost savings could mitigate the large initial cost of implementing WES in Asian settings.

Bioactive PCL-Peptide and PLA-Peptide Brush Copolymers for Bone Tissue Engineering.

We report the modular synthesis of bioactive brush-type polycaprolactone-peptide and polylactide-peptide copolymers for applications in bone tissue engineering. The brush copolymers containing pendant side chains of polycaprolactone (PCL) or polylactide (PLA) and PEGylated peptides, including linear Arg-Gly-Asp and collagen-like peptide (Gly-Pro-Hyp)3, were synthesized by ring-opening metathesis polymerization with high conversions and low dispersities (<1.5). These PCL-peptide and PLA-peptide copolymers exhibited good thermal stability for material processing using melt-extrusion-based methods. The copolymers were blended with commercial PCL or PLA, extruded into filaments, and 3D printed using fused filament fabrication methods. These bioactive PCL and PLA materials promoted osteogenic differentiation in vitro and showed good biocompatibility in in vivo murine model study. The promising results presented herein will serve as a useful guide for the design and functionalization of PCL or PLA materials for use in bone tissue engineering.

Case report: Persistence of residual antigen and RNA of the SARS-CoV-2 virus in tissues of two patients with long COVID.

The World Health Organization has defined long COVID-19 (LC) as a condition that occurs in individuals with a history of SARS-CoV-2 infection who exhibit persistent symptoms after its acute phase that last for at least two months and cannot be explained by an alternative diagnosis. Since we had previously reported residual viral antigens in tissues of convalescent patients, we aimed to assess the presence of such antigens in long COVID tissues. Here, we established the presence of the residual virus in the appendix, skin, and breast tissues of 2 patients who exhibited LC symptoms 163 and 426 days after symptom onset. With multiplex immunohistochemistry, we detected viral nucleocapsid protein in all three tissues. The nucleocapsid protein was further observed to colocalize with macrophage marker CD68, suggesting that immune cells were direct targets of SARS-CoV-2. Additionally, using RNAscope, the presence of viral RNA was also detected. Our positive finding in the breast tissue is corroborated by the recent reports of immunocompromised patients experiencing LC symptoms and persistent viral replication. Overall, our findings and emerging LC studies raise the possibility that the gastrointestinal tract may function as a reservoir for SARS-CoV-2.

Clinical characteristics and genetic testing outcome of suspected hereditary peripheral nerve sheath tumours in a tertiary cancer institution in Singapore.

BACKGROUND: Peripheral Nerve Sheath Tumors (PNST) are a diverse group of mostly benign tumours uncommon in the general population. About 5-10% of PNSTs are hereditary, predominantly arising from germline variants in NF1, NF2, SMARCB1, or LZTR1 gene. METHODS: We reviewed the clinical characteristics and genetic testing results of patients referred to the NCIS Adult Cancer Genetics Clinic for suspected hereditary PNST. RESULTS: 3,001 patients suspected to have various hereditary cancer syndromes were evaluated between year 2000 to March 2021. 13 (0.4%) were clinically diagnosed to have hereditary PNSTs. The majority were male (54%), with a median age at presentation to the genetics clinic of 29 years (range 19-48). 11/13 (85%) patients had multiple PNSTs, 12/13 (92%) had young onset PNSTs, 5/13 (38.5%) had personal and family history of PNST. 11/13 patients (85%) had clinical features of neurofibromatosis type 1 (NF1) including one patient who also fulfilled clinical criteria of neurofibromatosis type 2 (NF2); 2/13 (14%) had multiple schwannomas. Four patients underwent multi-gene panel testing, including one patient with clinical NF1, one patient who met both clinical NF1 and NF2 criteria, and two patients with multiple schwannomas. The patient with clinical features of NF1 was heterozygous for a pathogenic c. 2033dup variant in the NF1 gene. The patient with both NF1/NF2 features was heterozygous for a novel c.732 T > A nonsense variant in the NF2 gene. The two patients with multiple schwannomas were heterozygous for a pathogenic/likely pathogenic variant in the LZTR1 gene and are the first LZTR1-positive schwannomatosis patients reported in Asia. CONCLUSION: Hereditary PNSTs are rare referrals to an adult cancer genetics clinic. NF1 is the most common PNST seen. LZTR1 variants may be the underlying cause in Asian patients with multiple schwannomatosis.

Clinical examinations for medical students during the COVID-19 outbreak: a simulated patient programme perspective.

Background: The COVID-19 pandemic had a profound impact on how our university had to administer the high stakes, final year medical undergraduate clinical examinations without real patients, while maintaining its validity and rigour. Method: 11 out of the 21 stations of the medical, surgical and orthopaedic clinical examinations needed to be converted to simulated patient (SP)-based or task-based stations. Cases were developed based on an assessment blueprint, with consideration for SP demographics and availability of equipment. Infection control measures were strictly enforced to avoid transmission of COVID-19. Planning had to include consideration for physical distancing, cohorting and segregation of students and examiners. Student and SP anxiety had to be addressed. Results: The examination was executed successfully for 300 medical students. Everyone worked professionally and dealt with the changes and precautions that were required with flexibility. Discussion: An infectious disease outbreak can derail plans for major clinical examinations. Factors that facilitated a rapid and effective response included decisive leadership, open communication, willingness to collaborate, mobilising resources, adaptability and flexibility. Our school managed uncertainty by erring on the side of caution. This experience may serve as a reference for others in similar situations, particularly when COVID-19 restrictions start to be lifted.

Cholestatic Jaundice in Sulphite Oxidase Deficiency - An Unusual Association.

Sulphite oxidase deficiency is an extremely rare inborn error of metabolism of sulphur containing amino acids. There are no reports of liver involvement in this condition. The authors describe a 9-y-old boy with known sulphite oxidase deficiency who presented with worsening cholestatic hepatitis which may be possibly related to underlying metabolic disorder. Although there is no current evidence that treating liver disease and ensuring normal hepatic function in sulphite oxidase deficiency would likely benefit patients, this could potentially contribute to optimising growth and development as well as improving the overall prognosis.