Comprehensive Molecular Analysis Research Articles

A novel missense ALMS1 variant causes aberrant splicing identified in a cohort of patients with Alström syndrome.

Purpose: Alström syndrome (AS) is a rare autosomal recessive disorder caused by variants of ALMS1. The objectives of this study were to describe the clinical and genetic characteristics of 19 Chinese patients with biallelic variants in ALMS1. Methods: We recruited 19 probands with biallelic disease-causing ALMS1 variants. All patients underwent ophthalmic and systematic evaluations and comprehensive molecular genetic analysis. Reverse transcriptase-polymerase chain reaction (RT-PCR) assays were performed to observe the effect of a novel missense variant on ALMS1 pre-mRNA splicing. Results: We identified 33 causative variants in ALMS1, including 15 frameshift small indels, 14 non-sense variants, two gross deletions, one splicing variant, and one missense variant. RT-PCR showed that the missense variant c.9542G>A (p.R3181Q) altered pre-mRNA splicing to generate a truncated protein p. (Ser3082Asnfs*6). Retinal dystrophy (RD) was noted in all the patients, followed by metabolism disturbance (obesity or acanthosis nigricans) in 66.7% and hearing impairment in 61.1% of the patients. Patient systemic symptom numbers and their age at evaluation showed a significant positive correlation, and BCVA and age at the last examination showed a moderate correlation. All patients exhibited early-onset RD and severe visual impairment. The exception was one patient carrying homozygous p. R3181Q, who showed a mild visual defect and atypical retinal phenotype. Conclusion: Our findings expand the pathogenic variant spectrum of ALMS1 and provide the first verification of a novel missense variant caused AS by aberrant pre-mRNA splicing. Patients with AS might demonstrate varied clinical spectra; therefore, genetic analysis is vital for the early and accurate diagnosis of patients with atypical AS.

Virus Evolution on Fitness Landscapes.

The landscape paradigm is revisited in the light of evolution in simple systems. A brief overview of different classes of fitness landscapes is followed by a more detailed discussion of the RNA model, which is currently the only evolutionary model that allows for a comprehensive molecular analysis of a fitness landscape. Neutral networks of genotypes are indispensable for the success of evolution. Important insights into the evolutionary mechanism are gained by considering the topology of sequence and shape spaces. The dynamic concept of molecular quasispecies is viewed in the light of the landscape paradigm. The distribution of fitness values in state space is mirrored by the population structures of mutant distributions. Two classes of thresholds for replication error or mutations are important: (i) the-conventional-genotypic error threshold, which separates ordered replication from random drift on neutral networks, and (ii) a phenotypic error threshold above which the molecular phenotype is lost. Empirical landscapes are reviewed and finally, the implications of the landscape concept for virus evolution are discussed.

Comprehensive molecular analysis to predict the efficacy of chemotherapy containing bevacizumab in patients with metastatic colorectal cancer.

Although bevacizumab is an important treatment for metastatic colorectal cancer (CRC), not all patients with CRC benefit from it; in unselected patient populations, only modest survival benefits have been reported. We evaluated clinical outcomes in 110 patients using comprehensive molecular characterization to identify biomarkers for a response to bevacizumab-containing treatment. The molecular analysis comprised whole-exome sequencing, ribonucleic acid sequencing, and a methylation array on patient tissues. Genomic and molecular characterization was successfully conducted in 103 patients. Six of 103 CRC samples were hypermutated, and none of the non-hypermutant tumors were microsatellite unstable. Among those 103 patients, 89 had adenocarcinoma (ADC), 15 were diagnosed with mucinous ADC, and six had signet-ring cell carcinoma (SRCC). Consensus molecular subtype (CMS) 2 was unique to ADC. Of the four SRCCs, two were CMS1, one was CMS4, and the other was CMS3. APC mutation status was a significantly enriched factor in responders to bevacizumab treatment. Fibroblast growth factor receptor (FGFR) 1/2 signaling was upregulated in non-responders, whereas cell cycle, transfer ribonucleic acid processing, nucleotide excision repair, and oxidative phosphorylation pathways were enriched in responders. In addition, IGF1 was differentially expressed in non-responders (log2 fold change = -1.43, p = 4.11 × 10-5, false discovery rate = 0.098), and FLT1 was highly methylated in non-responders (p = 7.55 × 10-3). When the molecular pathways were reanalyzed separately according to the backbone chemotherapy (FOLFOX vs. FOLFIRI), the significance of the molecular pathways varied according to the backbone chemotherapy. This study sought a subset of CRC patients with a distinct clinical response to chemotherapy containing bevacizumab. Our results need to be validated in a large group of homogenous patient cohort and examined according to the different chemotherapy backbones to create personalized therapeutic opportunities in CRC.

Drug sensitivity profiling of 3D tumor tissue cultures in the pediatric precision oncology program INFORM

The international precision oncology program INFORM enrolls relapsed/refractory pediatric cancer patients for comprehensive molecular analysis. We report a two-year pilot study implementing ex vivo drug sensitivity profiling (DSP) using a library of 75–78 clinically relevant drugs. We included 132 viable tumor samples from 35 pediatric oncology centers in seven countries. DSP was conducted on multicellular fresh tumor tissue spheroid cultures in 384-well plates with an overall mean processing time of three weeks. In 89 cases (67%), sufficient viable tissue was received; 69 (78%) passed internal quality controls. The DSP results matched the identified molecular targets, including BRAF, ALK, MET, and TP53 status. Drug vulnerabilities were identified in 80% of cases lacking actionable (very) high-evidence molecular events, adding value to the molecular data. Striking parallels between clinical courses and the DSP results were observed in selected patients. Overall, DSP in clinical real-time is feasible in international multicenter precision oncology programs.

Molecular Classification of Appendiceal Adenocarcinoma.

Appendiceal adenocarcinomas (ACs) are rare, histologically diverse malignancies treated as colorectal cancers despite having distinct biology and clinical behavior. To guide clinical decision making, we defined molecular subtypes of AC associated with patient survival, metastatic burden, and chemotherapy response. A comprehensive molecular analysis was performed in patients with AC to define molecular subtypes. Associations between molecular subtype and overall survival, intraoperative peritoneal cancer index, and first-line chemotherapy response were assessed adjusting for histopathologic and clinical variables using multivariable Cox proportional hazards, linear regression, and logistic regression models. We defined distinct molecular lineages of mucinous appendiceal adenocarcinoma (MAAP) from co-occurring mutations in GNAS, RAS, and TP53. Of 164 MAAP tumors, 24 were RAS-mutant (mut) predominant (RAS-mut/GNAS-wild-type [wt]/TP53-wt) with significantly decreased mutations and chromosomal alterations compared with tumors with GNAS mutations (GNAS-mut predominant) or TP53 mutations (TP53-mut predominant). No patient with RAS-mut predominant subtype metastatic MAAP died of cancer, and overall survival in this subgroup was significantly improved compared with patients with GNAS-mut (P = .05) and TP53-mut (P = .004) predominant subtypes. TP53-mut predominant subtypes were highly aneuploid; increased tumor aneuploidy was independently (P = .001) associated with poor prognosis. The findings retained significance in patients with any metastatic AC. RAS-mut predominant metastases exhibited reduced peritoneal tumor bulk (P = .04) and stromal invasion (P < .001) compared with GNAS-mut or TP53-mut predominant tumors, respectively. Patients with RAS-mut predominant MAAP responded more to first-line chemotherapy (50%) compared with patients with GNAS-mut predominant tumors (6%, P = .03). AC molecular patterns identify distinct molecular subtypes: a clinically indolent RAS-mut/GNAS-wt/TP53-wt subtype; a chemotherapy-resistant GNAS-mut predominant subtype; and an aggressive, highly aneuploid TP53-mut predominant subtype. Each subtype exhibits conserved clinical behavior irrespective of histopathology.

COT-8 DEVELOPMENT OF TARGETED GENE PANEL FOR RAPID MOLECULAR DIAGNOSIS OF BRAIN TUMORS

Abstract Background Brain tumors are diagnosed based on pathological and genetic features defined by WHO classification. Although targeted gene panels are clinically available, most of them do not cover all the necessary genes for the diagnosis of brain tumors. Moreover, broad copy number analysis, which the current WHO classification requires, usually lacks in the gene panel. Another problem is that those panels demand a high burden of time and cost, which disturbs rapid diagnosis and broad application. To overcome those problems, we developed a rapid and cost-effective workflow of molecular diagnosis for brain tumors. Methods Our panel contains 109 genes of which 68 are necessary for fundamental molecular diagnosis and 41 are other common driver genes. To detect copy number changes and structural variants, which generate a fused gene, additional probes are placed on common SNPs and introns containing common breakpoints. MGMT methylation status is examined at the same time using bisulfite-converted DNA amplification. Sequencing data is analyzed using a supercomputer. Results The analysis time is within 4 days: 2 days for library preparation, 1 day for sequencing, and 12 hours for analysis. Detected driver alterations were validated by whole genome sequencing data. MGMT methylation status was correlated between the results of our workflow and pyrosequencing. Conclusions We have developed a rapid comprehensive molecular analysis workflow that detects genetic alterations and MGMT methylation. Our method allows a cost-effective molecular diagnosis with high accuracy, which would improve molecular diagnosis for brain tumors.

Clinical and Molecular Profiling of AML Patients with Chromosome 7 or 7q Deletions Incontext of TP53 Alterations and Venetoclax Treatment

Background Deletions in chromosome 7 (del(7)) or its long arm (del(7q)) constitute the most common adverse cytogenetic events, seen in approximately 20-30% of newly diagnosed AML and portend worse outcomes (Grimwade et al., 1998). These deletions often co-occur in the context of complex karyotype and TP53 alterations (Rücker et al., 2012). The European Leukemia Network (ELN) 2017 classification in adult AML patients (Döhner et al., 2017) considers AML patients with del7q as an intermediate risk. However, del(7) was classified as adverse risk. Yet, for the purpose of clinical studies and therapeutic decisions, AML with del(7) and del(7q) are often consolidated as one cytogenetic group and able patients are generally recommended to undergo stem cell transplantation in first remission to mitigate the risk of relapse (DiNardo et al., 2020; Kalwinsky et al., 1990). However, in-depth clinical and molecular characterization comparing AML patients with del(7) versus del(7q) have been limited to small patient cohorts (Poiré et al., 2019). We herein aimed at conducting in-depth clinical and molecular characterization of AML patients with del(7) or del(7q) in the context of venetoclax therapy and TP53 mutations. Methods We retrospectively analyzed 243 treatment-naive patients with AML and del(7) (168/243; 69%) or del(7q) (75/243; 31%) who did not receive any myeloid-directed therapy prior to AML diagnosis. Fifty-one percent of patients had targeted next-generation genomic sequencing, while another 46% had at least one other myeloid-related gene sequenced. The mutation profile yielded 374 mutations in 40 genes, and 169/243 (69.5%) patients had ≥1 mutation (Figure 1). Patients were stratified based on co-occuring TP53 alterations and venetoclax therapy. Results Patients with del(7q) had similar demographic and molecular profiles compared to patients with del(7). The median OS duration for AML patients with del(7) compared to del(7q) was 7.4 vs 8.4 months respectively (p=0.20) and the median remission duration was 5.3 vs 6.2 months respectively (p=0.16). These data suggest that AML patients with chromosome 7 and 7q have similar clinical features and outcomes. Since mutations in TP53 were the most common in our AML cohort, we investigated the significance of TP53 alterations on the outcomes of AML patients with del(7) or del(7q). Except for higher rate of complex cytogenetics in AML patients with TP53 alterations (p<0.0001), the clinical and demographic characteristics were similar among patients with del(7) or del(7q) with or without TP53 alterations. However, patients with del(7) or del(7q) who harbored TP53 alterations had significantly worse OS (5.5 vs 10.5 months p < 0.0001) and remission duration (4.7 vs 6.8 months p=0.0035) but not RFS (3.5 vs 3.4 months p=0.16) compared to those with TP53 wildtype. We next investigated the clinical outcomes of patients with del(7) and del(7q) treated with venetoclax-based therapies. A total of 39/243 (16%) of patients received VEN-based therapy as part of their frontline therapy (31/168 (18%) in del(7) versus 8/75 (11%) in del(7q), p= 0.13). Remarkably, AML patients with del(7) who received VEN-based therapy (Rx + Ven) had significantly worst outcomes than patients treated without VEN-based (Rx) regimens with or without TP53 mutation. Specifically, the median OS, RFS and remission duration for del(7) Rx + Ven were the shortest at 5.8 months (p=0.47), 0.1 months (p<0.0001) and 2.2 months (p=0.0015), respectively, compared to the other treatment groups (Figure 2). Among patients with TP53 alterations in del(7) and del(7q) subgroups, the addition of venetoclax did not improve clinical outcomes. The median OS for del(7) TP53-alt Rx vs del(7) TP53-alt Rx+Ven was 5.2 months vs 4.5 months, respectively (p = 0.6). The median OS for del(7q) TP53-alt Rx vs del(7q) TP53-alt Rx+Ven was 6.4 months vs 7.9 months respectively (p = 0.69). Conclusion We conducted a comprehensive clinical and molecular analysis of AML patients with del(7) and del(7q) revealing differences in relapse-free survival in these patients, but not in OS or remission duration. This suggests that del(7q) should also be considered an adverse event. The co-occurrence of TP53 alterations conferred worse outcomes in del(7) and del(7q) patients, while addition of venetoclax did not improve outcomes. There is an unmet need to identify therapies that would improve outcomes in AML patients with del(7) or del(7q). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Differentially Expressed Proteins in Transfusion-Dependent Thalassemia (TDT) and Their Role in Tdt Disease Severity

Introduction: The management of transfusion-dependent thalassemia (TDT) has seen essential improvements; however, still several challenges to controlling the incidence worldwide. Limitations of available current treatment modalities in resource-limited countries are the driving force to investigate the novel diagnostic and therapeutic options for better clinical management of these patients. Proteomics may be a potential technique to identify novel biomarkers of disease severity and understand the pathophysiological mechanisms of TDT, as proven in many other diseases. Understanding the inter-relationships of proteins affected by the disease process, such as oxidative stress in TDT patients, can help to elucidate the mechanisms involved in the disease pathophysiology. However, plasma proteome profile studies in TDT are limited. This study aimed to investigate the differentially expressed proteins (novel proteins) in serum from TDT to understand the pathophysiology of TDT and determine their role in TDT severity by performing quantitative serum proteomics profiling. Methods: The sample size was calculated based on the sensitivity and specificity of the protein biomarkers identified in the pilot study. A total of 64 patients were included in this study. A serum sample was collected from 32 TDT patients and 32 non-thalassemia controls. Before the serum samples could be processed for label-free quantification using the liquid chromatography-mass spectrometry/mass spectrometry (LCMS/MS) approach, high abundance proteins such as serum albumin and immunoglobulins were depleted using Pierce™ Albumin/IgG Removal Kit (Thermo Fisher Scientific, USA.) according to the manufacturer's protocols. The protein content in samples after depletion of high abundance proteins was determined using the Bradford assay using the protocol recommended by the manufacturer protocol (BioRad, USA). Each protein sample was prepared for LC-MS/MS analysis using EasyPep™ Mini MS Sample Prep Kit (Thermo Fisher Scientific, USA). Analysis of sera was performed on the Agilent 6560C ion mobility Q-TOF LC/MS system (Agilent, USA). To validate the LCMS/MS results, ELISA was done. Differentially regulated proteins with p-value <0.05 were identified from the dataset and uploaded to online databases PANTHER to classify and identify the proteins' functions. The functional gene ontology (GO) annotation of the identified protein in terms of molecular function, biological process, and cellular component was evaluated using this curated biological database. The statistically significant expressed proteins were submitted to STRING, and a combined score of >0.4 (medium confidence) was considered experimentally validated and statistically significant interactions between proteins. The cluster with the largest number of proteins with strong connections was identified for the pathway enrichment analysis. The identified protein cluster from the STRING database was uploaded to the KEGG online database to acquire the enriched pathway. Results: 51 dysregulated proteins were found between cases and controls (Table). However, only 13 showed statistically significant differential expression (p <0.05) with a fold change >1, and mostly were downregulated. Gene ontology functional annotation analysis showed these proteins were involved in molecular functions, mainly catalytic activities (37%), followed by molecular function regulation (15%). The biological processes primarily involved the cellular (35.5%) and metabolic processes (32.3%). The pathway analysis showed that these proteins were involved mainly in hemostasis. In KEGG pathway analysis, the proteins were involved primarily in fat digestion and absorption and vitamin digestion & absorption. Conclusion: We elucidated the possible biological processes modulated by oxidative stress using the differentially expressed novel proteins and the associated pathways using comprehensive molecular and biocomputational analysis. Interestingly, most of the differentially expressed proteins in this study have not been yet reported in previous studies to be modulated by oxidative stress in TDT patients. Therefore, the identified dysregulated proteins can serve as a potential biomarker to monitor a patient's condition, determine the effect, need for blood transfusion, and help develop novel prognostic and diagnostic methods. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pseudoprogression and Sarcoidosis-like Phenomena after CART-Cells and Bispecific Antibodies in Multiple Myeloma

Introduction: CAR T-cell therapy is establishing itself as a new standard of care in relapsed and refractory multiple myeloma (RRMM). Yet, there are a number of open issues including the lack of data on optimal follow-up assessments post CAR T therapy. Appreciating the high incidence of extramedullary lesions and patchy disease patterns seen in the RRMM setting, whole body 18F-Fluorodeoxyglucose (FDG) PET/CT imaging is increasingly used. Whether FDG is the best tracer in this setting has yet to be determined, as the technique is being based not only on the quantification of increased glucose uptake by tumor cells but also by others such as inflammatory cells. A phenomenon linked to this issue is pseudoprogression, which refers to an increase in the target lesion size due to influx of immune cells that may be misinterpreted as disease relapse. Pseudoprogression is a well-known event after checkpoint blockade therapy, seen in up to 10% of patients with solid cancers. Whether myeloma patients treated with CAR T or bispecific antibodies are at risk for pseudoprogression is, to the best of our knowledge, unknown. Methods: We present a case study of a RRMM patient who experienced pseudoprogression twice during his course of disease treatment that included BCMA CAR T cells Idecabtagene vicleucel and GPRC5DxCD3 bispecific antibody talquetamab. We performed a comprehensive molecular analysis including single cell RNAseq from bronchoalveolar lavage as well as PET imaging using the three PET tracers 18F-FDG, 68Ga-DOTATOC, and 68Ga-Pentixafor (binds to CXCR4) to unravel the biology underlying pseudoprogression in this case. Results: A 61-year-old Caucasian male was diagnosed with biopsy-proven pulmonary MM and treated with BCMA targeting CAR T cells in his 4th line of therapy. Ten days after CAR T infusion, the patient started violent coughing and a FDG-PET revealed full resolution of focal bone lesions but disseminated FDG uptake at the lung and mediastinal lymph nodes reminiscent to sarcoidosis. We performed a PET using 68Ga-DOTATOC that usually detects sarcoidosis with high sensitivity and specificity, which was negative. Negative results were also seen for 68Ga-Pentixafor, which is an alternative tracer to FDG in the detection of MM. Bronchoalveolar lavage (BAL) along with endobronchial ultrasound guided biopsy was done, showing noncaseating granuloma formation in the biopsy without evidence of residual MM cells. Single cell RNA-seq on the BAL sample revealed CD4-positive T cells exhibiting simultaneous expression of genes associated with Th1 T-cells (TBX21, CXCR3, IFNG, TNF) and genes associated with Th17 T-cells (RORC, IL23R, CCR6, CCL20, KLRB1), indicative for a Th1-polarized Th17 phenotype. Furthermore, these cells expressed IL4I1, ABCB1, and CSF210-12 , while IL17A, IL17B, IL17E (IL25), and or IL17F was not expressed, highlighting presence of Th17.1 cells. This cell type has been described to be pro-inflammatory and pathogenic and implicated in several auto-immune diseases, including sarcoidosis. Together, our data suggest a non-infectious, Th17.1 T-cell driven sarcoidosis-like auto-immune phenomenon located in the lungs following anti-BCMA CAR T-cell therapy. Respiratory symptoms completely resolved following high-dose inhalative steroids. The patient relapsed from complete remission (CR), and FDG PET showed the known sarcoidosis-like pulmonary changes, but also revealed a number of new soft-tissue lesions, suspicious for extramedullary relapse. In contrast to the previous scans, these lesions were CXCR4 positive, suggesting a role of using multiple PET tracers in this setting. Later, the patient was enrolled on a trial investigating talquetamab. One week after dosing, the patient experienced severe pain crisis and a diffusion-weighted MRI revealed increase in size of multiple extramedullary lesions, but no new lesions. M- protein levels were decreasing at that time suggesting either mixed response or pseudoprogression. The patient was treated with painkillers and all lesions disappeared on a follow-up scan 6 weeks later, confirming pseudoprogression, and he is in an ongoing CR for 6 months at the time of writing. Conclusion: We present two educating episodes of pseudoprogression in one patient following immune oncology drugs in MM. While one event was transient and self-limiting, the pulmonary manifestations were similar to sarcoidosis and required treatment with steroids.

Proliferating Pilar Tumors Are Characterized by Recurrent 15q, 6q, and 6p22.2 Alterations.

Proliferating pilar tumors (PPTs) are rare neoplasms of external root sheath derivation, which most commonly occur on the scalp of elderly women. Although typically showing classic histologic features such as trichilemmal type keratinization, a lobular architecture and peripheral palisading, squamous cell carcinoma (SCC) remains a common diagnostic pitfall. Therefore, we sought to explore the molecular pathogenesis of PPTs and compare it with that of cutaneous squamous cell carcinoma (cSCC). Herein, we describe the use of a next-generation DNA sequencing platform to provide the most comprehensive molecular genetic analysis to date of a cohort of 5 PPTs and compare them to 5 head and neck cutaneous SCCs. Recurrent broad arm-level gains of 15q and concurrent single-copy losses of 6q and 6p22.2 were observed in 4 of 5 (80%) PPT cases. Other recurrent mutations or alterations of significance were not found in PPTs. Notably, these chromosomal changes were not identified in any of the 5 cutaneous SCCs, which instead showed recurrent alterations in the known SCC driver genes TP53 , CDKN2A , and NOTCH1 . Here, we show for the first time that PPTs are molecularly distinct from cutaneous SCC and provide evidence that recurrent alterations in chromosome 15 and chromosome 6 are central to the pathogenesis of PPTs.

Comprehensive Molecular Profiling of Sinonasal Teratocarcinosarcoma Highlights Recurrent SMARCA4 Inactivation and CTNNB1 Mutations.

Sinonasal teratocarcinosarcoma (TCS) is a rare tumor defined by intermixed neuroepithelial, mesenchymal, and epithelial elements. While its etiology was historically ambiguous, we recently reported frequent SMARCA4 loss by immunohistochemistry, suggesting that TCS might be related to SMARCA4-deficient sinonasal carcinomas. However, other molecular alterations including CTNNB1 mutation have been reported in TCS, and its full genetic underpinnings are unclear. Here, we performed the first comprehensive molecular analysis of sinonasal TCS to better understand its pathogenesis and classification. We collected 30 TCS including 22 cases from our initial study. Immunohistochemical loss of SMARCA4 was seen in 22 cases (73%), with total loss in 18 cases (60%). β-catenin showed nuclear localization in 14 cases (64%) of the subset tested. We selected 17 TCS for next-generation sequencing with enrichment for partial or intact SMARCA4 immunoexpression. We identified inactivating SMARCA4 mutations in 11 cases (65%) and activating CTNNB1 mutations in 6 cases (35%), including 5 cases with both. Of 5 cases that lacked SMARCA4 or CTNNB1 mutation, 2 harbored other SWI/SNF complex and Wnt pathway alterations, including 1 with SMARCB1 inactivation and 1 with concomitant APC and ARID1A mutations, and 3 had other findings, including DICER1 hotspot mutation. These findings confirm that SMARCA4 inactivation is the dominant genetic event in sinonasal TCS with frequent simultaneous CTNNB1 mutations. They further underscore a possible relationship between TCS and sinonasal carcinomas with neuroendocrine/neuroectodermal differentiation. However, while SMARCA4 and β-catenin immunohistochemistry may help confirm a challenging diagnosis, TCS should not be regarded as a molecularly defined entity.

Comprehensive Molecular Analysis Identified an SRSF Family-Based Score for Prognosis and Therapy Efficiency Prediction in Hepatocellular Carcinoma.

Simple SummaryHepatocellular carcinoma (HCC) remains one of the most common malignancies worldwide. Despite significant progress in the comprehensive management of HCC, continued efforts are still necessary to identify prognostic assessment approaches for patient-specific survival so that more appropriate treatment and management regimens can be proposed for different HCC subpopulations. Here, we comprehensively exploited the expression characteristics of 12 SRSF family members across multiple datasets. We further identified an SRSF score consisting of 18 SRSF-related genes which were associated with overall survival and drug sensitivity in HCC. Moreover, the predictive power of the SRSF score was validated in independent HCC cohorts and different HCC subgroups. We further explored the effect of SRSF11 expression on HCC cell proliferation and drug sensitivity. Overall, our study provides a novel predictive model to assess the prognosis and drug sensitivity of HCC.The serine/arginine-rich splicing factors (SRSF)-mediated alternative splicing plays an essential role in the occurrence and progression of hepatocellular carcinoma (HCC). However, the SRSF-based signature that can predict the prognosis and therapy efficiency is yet to be investigated in HCC. Here, we comprehensively assessed the landscape and prognostic significance of the SRSF family genes in HCC. Then, we screened the SRSF family-related genes for signature construction and explored their biological characteristics. We further established an SRSF score consisting of 18 SRSF-associated genes and evaluated its correlation with prognosis and drug sensitivity in HCC. The predictive power of the SRSF score was validated in independent HCC cohorts and different HCC subgroups. Moreover, we further investigated that knockdown of SRSF11, a pivotal gene in the SRSF score, inhibited CDK1-dependent proliferation and enhanced the drug sensitivity of HCC cells. Overall, our study identified a novel SRSF family-based predictive model, and we demonstrated that SRSF11 is a promising therapeutic target for HCC, which enhances our understanding of the SRSF family genes and provides valuable insights into the clinical treatment and molecular mechanisms of HCC.

Teenage-Onset Colorectal Cancers in a Digenic Cancer Predisposition Syndrome Provide Clues for the Interaction between Mismatch Repair and Polymerase δ Proofreading Deficiency in Tumorigenesis.

Simple SummaryColorectal cancer (CRC) in adolescents and young adults (AYA) is rare. Genetic causes include autosomal recessive and dominant monogenic disorders due to pathogenic variants (PVs) in genes involved in DNA repair. However, the genetic etiology of the majority of AYA-CRC remains unidentified. In two teenage siblings with CRC, we show to our knowledge for the first time that AYA-CRC cases can be caused by digenic inheritance of each a heterozygous pathogenic variant (PV) in the mismatch-repair (MMR) gene PMS2 and the proofreading polymerase (PP) Pol δ gene POLD1. With the aim to elucidate how the constitutional polymerase proofreading defect and the high propensity to MMR deficiency (MMRd) interact, we performed a comprehensive tumor analysis of the two siblings’ tumors. Results indicate that tumorigenesis is initiated by MMRd and the inherited POLD1 PV contributes to fast tumor progression reflected by an ultra-high tumor mutational burden (TMB) and specific mutational signatures.Colorectal cancer (CRC) in adolescents and young adults (AYA) is very rare. Known predisposition syndromes include Lynch syndrome (LS) due to highly penetrant MLH1 and MSH2 alleles, familial adenomatous polyposis (FAP), constitutional mismatch-repair deficiency (CMMRD), and polymerase proofreading-associated polyposis (PPAP). Yet, 60% of AYA-CRC cases remain unexplained. In two teenage siblings with multiple adenomas and CRC, we identified a maternally inherited heterozygous PMS2 exon 12 deletion, NM_000535.7:c.2007-786_2174+493del1447, and a paternally inherited POLD1 variant, NP_002682.2:p.Asp316Asn. Comprehensive molecular tumor analysis revealed ultra-mutation (>100 Mut/Mb) and a large contribution of COSMIC signature SBS20 in both siblings’ CRCs, confirming their predisposition to AYA-CRC results from a high propensity for somatic MMR deficiency (MMRd) compounded by a constitutional Pol δ proofreading defect. COSMIC signature SBS20 as well as SBS26 in the index patient’s CRC were associated with an early mutation burst, suggesting MMRd was an early event in tumorigenesis. The somatic second hits in PMS2 were through loss of heterozygosity (LOH) in both tumors, suggesting PPd-independent acquisition of MMRd. Taken together, these patients represent the first cases of cancer predisposition due to heterozygous variants in PMS2 and POLD1. Analysis of their CRCs supports that POLD1-mutated tumors acquire hypermutation only with concurrent MMRd.

Clinical and molecular profiling of AML patients with chromosome 7 or 7q deletions in the context of TP53 alterations and venetoclax treatment

Deletions in chromosome 7 (del(7)) or its long arm (del(7q)) constitute the most common adverse cytogenetic events in acute myeloid leukemia (AML). We retrospectively analyzed 243 treatment-naive patients with AML and del(7) (168/243; 69%) or del(7q) (75/243; 31%) who did not receive any myeloid-directed therapy prior to AML diagnosis. This is the largest comprehensive clinical and molecular analysis of AML patients with del(7) and del(7q). Our results show that relapse-free survival was significantly longer for AML patients with del(7q) compared to del(7), but the overall survival and remission duration were similar. TP53 mutations and del5/5q were the most frequent co-occurring mutations and cytogenetic abnormalities, and conferred worse outcomes in del(7) and del(7q) patients. Venetoclax-based treatments were associated with worse outcomes in TP53 mutated AML patients with del(7) or del(7q), as well as del(7) with TP53 wildtype status, requiring further investigation.

847P Precision oncology for resistant acral, mucosal and cutaneous melanomas: A prospective broad high throughput genomics feasibility study

There are limited therapies for metastatic cutaneous, mucosal and acral melanomas (m) progressing after checkpoint (CPI) and BRAF/MEK (TKI) inhibitors. The Treat20 Plus study aimed to identify oncogenic drivers through high throughput genomics that could inform treatment recommendations ((Trec). From 1.2016 - 1.2019, 54 resistant m were prospectively subjected to a comprehensive molecular analysis (WGS, WES, RNA seq), that allowed a molecular tumor board to make individualized treatment recommendations. Primary sites: cutaneous (34), mucosal (16), acral (4), PS 1(0-3), abnormal LDH (37), # of therapies 5(2-22) (surgery 2(0-18), irradiation 1 (0-5), CPI 2(1-5), TKI 0 (0-2), chemo 0(0-2). No Trec in 10 p (low purity 7, early PD 3). Trec in 44 p (81%) with 4 (0-5) / p, for a total of 108 Trec including inhibitors of MEK 27%, CDK4/6 23%, TKI 7%, MET 6%, MTOR 6%, EGFR, FGFR, RAS 5 %, ALK 4%, NOTCH and PARP 2%, IGFR, NTRK, IDO and ER 1%. Treatment realization made in 21/44 p (48 %). Non-realization was early PD (14), still on CPI (6), on chemo (1), no druggable mutation (2). Among the 21 treated p 1 CR, 4 PR (RR: 24 %; 9 % of whole cohort (wc)), 3 SD (Clinical benefit: 38 %; 15% of wc) and 13 PD. Duration of the clinical benefit ranged from 147-678+ d. Resistance to prior TKI were observed in 8/9 BRAF V600E p by BRAF fusion (1), splicing (1), NRAS mutation (4), PTEN (2), RB1 (1), CDKN2A loss (2). CPI resistance mutations were shown in B2M (1), SOCS1 (1), MDM2 (1) and MDM2 focal amplification (2), and homozygous deletion in PTEN (4), but no relevant JAK1, JAK2, IRF1, IFNGR1/2, PIAS4, EGFR, MDM4, EZH2 alterations. Responding p had BRAF 599delinsTT (1), BRAF G469S (1), cKIT (2) mutations and NTRK fusion (1). Stabilization: IGFR (1) MET overexpression (1), CDKN2A loss (1). Among the seven p without BRAF V600E, NRAS and NF1 mutation (triple-negative profile) six had a clinical benefit. PFS for the treated p: 2.26 months (95%CI: 0.00-4.98) and the OS for the 54 p: 8.7 months (95 % CI: 1.56-15.97). For resistant acral, mucosal and cutaneous m, precision oncology was feasible with Trec in 81 % of p. It uncovered targetable alterations that led to a clinical benefit in 38 % of p mainly presenting with absence of BRAF V600E, NRAS and NF1 mutation.

Immune Microenvironment and Lineage Tracing Help to Decipher Rosette-Forming Glioneuronal Tumors: A Multi-Omics Analysis.

Rosette-forming glioneuronal tumors (RGNT) are rare low-grade primary central nervous system (CNS) tumors. The methylation class (MC) RGNT (MC-RGNT) delineates RGNT from other neurocytic CNS tumors with similar histological features. We performed a comprehensive molecular analysis including whole-exome sequencing, RNAseq, and methylome on 9 tumors with similar histology, focusing on the immune microenvironment and cell of origin of RGNT. Three RGNT in this cohort were plotted within the MC-RGNT and characterized by FGFR1 mutation plus PIK3CA or NF1 mutations. RNAseq analysis, validated by immunohistochemistry, identified 2 transcriptomic groups with distinct immune microenvironments. The "cold" group was distinguishable by a low immune infiltration and included the 3 MC-RGNT and 1 MC-pilocytic astrocytoma; the "hot" group included other tumors with a rich immune infiltration. Gene set enrichment analysis showed that the "cold" group had upregulated NOTCH pathway and mainly oligodendrocyte precursor cell and neuronal phenotypes, while the "hot" group exhibited predominantly astrocytic and neural stem cell phenotypes. In silico deconvolution identified the cerebellar granule cell lineage as a putative cell of origin of RGNT. Our study identified distinct tumor biology and immune microenvironments as key features relevant to the pathogenesis and management of RGNT.

Phylogenomic Evidence for the Origin of Obligate Anaerobic Anammox Bacteria Around the Great Oxidation Event.

The anaerobic ammonium oxidation (anammox) bacteria can transform ammonium and nitrite to dinitrogen gas, and this obligate anaerobic process accounts for up to half of the global nitrogen loss in surface environments. Yet its origin and evolution, which may give important insights into the biogeochemistry of early Earth, remain enigmatic. Here, we performed a comprehensive phylogenomic and molecular clock analysis of anammox bacteria within the phylum Planctomycetes. After accommodating the uncertainties and factors influencing time estimates, which include implementing both a traditional cyanobacteria-based and a recently developed mitochondria-based molecular dating approach, we estimated a consistent origin of anammox bacteria at early Proterozoic and most likely around the so-called Great Oxidation Event (GOE; 2.32–2.5 Ga) which fundamentally changed global biogeochemical cycles. We further showed that during the origin of anammox bacteria, genes involved in oxidative stress adaptation, bioenergetics, and anammox granules formation were recruited, which might have contributed to their survival on an increasingly oxic Earth. Our findings suggest the rising levels of atmospheric oxygen, which made nitrite increasingly available, was a potential driving force for the emergence of anammox bacteria. This is one of the first studies that link the GOE to the evolution of obligate anaerobic bacteria.

Comprehensive molecular analysis of immortalization hallmarks in thyroid cancer reveals new prognostic markers.

BackgroundComprehensive molecular studies on tumours are needed to delineate immortalization process steps and identify sensitive prognostic biomarkers in thyroid cancer.Methods and ResultsIn this study, we extensively characterize telomere‐related alterations in a series of 106 thyroid tumours with heterogeneous clinical outcomes. Using a custom‐designed RNA‐seq panel, we identified five telomerase holoenzyme‐complex genes upregulated in clinically aggressive tumours compared to tumours from long‐term disease‐free patients, being TERT and TERC denoted as independent prognostic markers by multivariate regression model analysis. Characterization of alterations related to TERT re‐expression revealed that promoter mutations, methylation and/or copy gains exclusively co‐occurred in clinically aggressive tumours. Quantitative‐FISH (fluorescence in situ hybridization) analysis of telomere lengths showed a significant shortening in these carcinomas, which matched with a high proliferative rate measured by Ki‐67 immunohistochemistry. RNA‐seq data analysis indicated that short‐telomere tumours exhibit an increased transcriptional activity in the 5‐Mb‐subtelomeric regions, site of several telomerase‐complex genes. Gene upregulation enrichment was significant for specific chromosome‐ends such as the 5p, where TERT is located. Co‐FISH analysis of 5p‐end and TERT loci showed a more relaxed chromatin configuration in short telomere‐length tumours compared to normal telomere‐length tumours.ConclusionsOverall, our findings support that telomere shortening leads to a 5p subtelomeric region reorganization, facilitating the transcription and accumulation of alterations at TERT‐locus.

Sugarcane Genotypes with Contrasting Biological Nitrogen Fixation Efficiencies Differentially Modulate Nitrogen Metabolism, Auxin Signaling, and Microorganism Perception Pathways.

Sugarcane is an economically important crop that is used for the production of fuel ethanol. Diazotrophic bacteria have been isolated from sugarcane tissues, without causing visible plant anatomical changes or disease symptoms. These bacteria can be beneficial to the plant by promoting root growth and an increase in plant yield. Different rates of Biological Nitrogen Fixation (BNF) were observed in different genotypes. The aim of this work was to conduct a comprehensive molecular and physiological analysis of two model genotypes for contrasting BNF efficiency in order to unravel plant genes that are differentially regulated during a natural association with diazotrophic bacteria. A next-generation sequencing of RNA samples from the genotypes SP70-1143 (high-BNF) and Chunee (low-BNF) was performed. A differential transcriptome analysis showed that several pathways were differentially regulated among the two BNF-contrasting genotypes, including nitrogen metabolism, hormone regulation and bacteria recognition. Physiological analyses, such as nitrogenase and GS activity quantification, bacterial colonization, auxin response and root architecture evaluation, supported the transcriptome expression analyses. The differences observed between the genotypes may explain, at least in part, the differences in BNF contributions. Some of the identified genes might be involved in key regulatory processes for a beneficial association and could be further used as tools for obtaining more efficient BNF genotypes.

Comprehensive quantum chemical calculations and molecular docking analysis of uracil mustard by first principle

Uracil mustard belongs to the nitrogen mustard family and is primarily used in anticancer drugs. The research that follows, investigates many quantum chemical features such as the computation of global minimum energies with no negative wavenumber values using the Density Functional Theory (DFT) with Becke three functional and 6-311G (d, p)/6–311++G (d, p) basis sets. All the vibrational modes have been calibrated and justified in comparison to their experimental counterparts. Mustard's polarizability and hyperpolarizability components, Natural Bond Analysis (NBO), electronic properties, Fukui function analysis, various global parameters, Quantum Theory of Atoms In Molecule (QTAIM) analysis, ADMET analysis, and docking analysis have all been investigated using the same theory and basis sets, indicating its biochemical significance. The biological activity of the molecule is reported by using PASS software. The Full fitness score and binding affinity parameters are utilized to determine the binding strength with 6cq3 protein. The acidity of the title molecule is calculated in water solvent by polarizable continuum model (PCM) solvent effects (estimated in water). The HOMO, LUMO, and MESP plots are used to explore the nature of binding and surfaces. The Fukui functions are computed using Mulliken atomic charges for neutral atoms, cations, and anions. The Ultraviolet–visible (UV–vis) of the molecule is computed employing the TD-DFT method.