Predictive significance of FGFR4 p.G388R polymorphism in metastatic colorectal cancer patients receiving trifluridine/tipiracil (TAS-102) treatment.

BACKGROUND: TAS-102 (Lonsurf®) is an oral fluoropyrimidine consisting of a combination of trifluridine (a thymidine analog) and tipiracil (a thymidine phosphorylation inhibitor). The drug is effective in metastatic colorectal cancer (mCRC) patients refractory to fluorouracil, irinotecan and oxaliplatin. This study is a real-world analysis, investigating the interplay of genotype/phenotype in relation to TAS-102 sensitivity. METHODS: Forty-seven consecutive mCRC patients were treated with TAS-102 at the National Cancer Institute of Naples from March 2019 to March 2021, at a dosage of 35 mg/m2, twice a day, in cycles of 28 days (from day 1 to 5 and from day 8 to 12). Clinical-pathological parameters were described. Activity was evaluated with RECIST criteria (v1.1) and toxicity with NCI-CTC (v5.0). Survival was depicted through the Kaplan-Meyer curves. Genetic features of patients were evaluated with Next Generation Sequencing (NGS) through the Illumina NovaSeq 6000 platform and TruSigt™Oncology 500 kit. RESULTS: Median age of patients was 65 years (range: 46-77). Forty-one patients had 2 or more metastatic sites and 38 patients underwent to more than 2 previous lines of therapies. ECOG (Eastern Cooperative Oncology Group) Performance Status (PS) was 2 in 19 patients. The median number of TAS-102 cycles was 4 (range: 2-12). The most frequent toxic event was neutropenia (G3/G4 in 16 patients). There were no severe (> 3) non-haematological toxicities or treatment-related deaths. Twenty-six patients experienced progressive disease (PD), 21 stable disease (SD). Three patients with long-lasting disease control (DC: complete, partial responses or stable disease) shared an FGFR4 (p.Gly388Arg) mutation. Patients experiencing DC had more frequently a low tumour growth rate (P = 0.0306) and an FGFR4 p.G388R variant (P < 0.0001). The FGFR4 Arg388 genotype was associated with better survival (median: 6.4 months) compared to the Gly388 genotype (median: 4 months); the HR was 0.25 (95% CI 0.12- 0.51; P = 0.0001 at Log-Rank test). CONCLUSIONS: This phenotype/genotype investigation suggests that the FGFR4 p.G388R variant may serve as a new marker for identifying patients who are responsive to TAS-102. A mechanistic hypothesis is proposed to interpret these findings.

Analysis of microsatellite instability (MSI) in pediatric gonadal and extra-gonadal germ cell tumors.

Gonadal and extragonadal pediatric germ cell tumors (GCTs) are rare neoplasms with different clinical behavior. Although surgery and cisplatin-based chemotherapy are resolutive in most cases, some patients do not respond to chemotherapy and have a worse outcome. Microsatellite instability (MSI) was correlated to resistance to chemotherapy and sensitivity to immunotherapy in different neoplasms. A series of 21 pediatric GCTs were tested by immuno-histochemistry and PCR to evaluate MSI status. Next generation sequencing was applied to further evaluate cases with discordant results between immunohistochemistry and PCR. Twenty-one cases of pediatric GCT were included in the series. The mean age ranged between 1 and 10 years. Nine cases were gonadal GCTs and the remaining 12 were extra-gonadal GCTs. By immunohistochemistry, one case showed a deficit of Mismatch repair (MMR) proteins. This case was a 1-year-old children affected by gonadal yolk sac tumor. However, all cases resulted microsatellite stable (MSS) by PCR and NGS. MSI was not detected in our series of pediatric GCTs, as well as the data present in literature about adult patients with GCTs. Molecular techniques could have a role to confirm the MSI status in case of dMMR by immunohistochemistry.

From Chaos to Opportunity: Decoding Cancer Heterogeneity for Enhanced Treatment Strategies.

Cancer manifests as a multifaceted disease, characterized by aberrant cellular proliferation, survival, migration, and invasion. Tumors exhibit variances across diverse dimensions, encompassing genetic, epigenetic, and transcriptional realms. This heterogeneity poses significant challenges in prognosis and treatment, affording tumors advantages through an increased propensity to accumulate mutations linked to immune system evasion and drug resistance. In this review, we offer insights into tumor heterogeneity as a crucial characteristic of cancer, exploring the difficulties associated with measuring and quantifying such heterogeneity from clinical and biological perspectives. By emphasizing the critical nature of understanding tumor heterogeneity, this work contributes to raising awareness about the importance of developing effective cancer therapies that target this distinct and elusive trait of cancer.

KRAS p.G12C Mutation in Metastatic Colorectal Cancer: Prognostic Implications and Advancements in Targeted Therapies.

KRAS is frequently mutated in tumors. It is mutated in approximately 30% of all cancer cases and in nearly 50% of cases of metastatic colorectal cancer (CRC), which is the third leading cause of cancer-related deaths worldwide. Recent advancements in understanding CRC biology and genetics have highlighted the significance of KRAS mutations in the progression of CRC. The KRAS gene encodes a small GTPase (Guanosine TriPhosphatases) that plays a key role in signaling pathways associated with important proteins involved in amplifying growth factor and receptor signals. Mutations in KRAS are frequently observed in codons 12 and 13, and these mutations have oncogenic properties. Abnormal activation of KRAS proteins strongly stimulates signals associated with various cancer-related processes in CRC, including cell proliferation, migration and neoangiogenesis. In this review, we explore the distinct prognostic implications of KRAS mutations. Specifically, the KRAS p.G12C mutation is associated with a worse prognosis in metastatic CRC. The correlation between structure, conformation and mutations is visually presented to emphasize how alterations in individual amino acids at the same position in a single protein can unexpectedly exhibit complex involvement in cancer. Last, KRAS p.G12C is discussed as an emerging and promising therapeutic target in metastatic CRC, providing a concise overview of available clinical data regarding the use of new inhibitors.

Origin, Genetic Variation and Molecular Epidemiology of SARS-CoV-2 Strains Circulating in Sardinia (Italy) during the First and Second COVID-19 Epidemic Waves.

Understanding how geography and human mobility shape the patterns and spread of infectious diseases such as COVID-19 is key to control future epidemics. An interesting example is provided by the second wave of the COVID-19 epidemic in Europe, which was facilitated by the intense movement of tourists around the Mediterranean coast in summer 2020. The Italian island of Sardinia is a major tourist destination and is widely believed to be the origin of the second Italian wave. In this study, we characterize the genetic variation among SARS-CoV-2 strains circulating in northern Sardinia during the first and second Italian waves using both Illumina and Oxford Nanopore Technologies Next Generation Sequencing methods. Most viruses were placed into a single clade, implying that despite substantial virus inflow, most outbreaks did not spread widely. The second epidemic wave on the island was actually driven by local transmission of a single B.1.177 subclade. Phylogeographic analyses further suggest that those viral strains circulating on the island were not a relevant source for the second epidemic wave in Italy. This result, however, does not rule out the possibility of intense mixing and transmission of the virus among tourists as a major contributor to the second Italian wave.

Pulmonary leiomyosarcoma arising in pulmonary hamartoma: an exceptional occurrence in a rare tumor.

A solitary peripheral lung nodule was found in the left lung of a 52-year-old man. It was located in the lower lobe and measured 18.5 cm of major axis on chest computed tomography. A tru-cut core biopsy was obtained and a proliferation of bland, monomorphic, spindle cells in interlacing fascicles was observed. Accordingly, a surgical resection of the neoplasm was subsequently carried out. Macroscopically, the tumor appeared as a well-circumscribed nodule with a firm and whitish cut surface. Histologically, the neoplasm was predominantly composed of bland and monomorphic spindle cells, with a predominantly fascicular growth pattern, in which many tubular and cleft-like spaces of entrapped normal respiratory epithelium were involved. Myxoid change, stromal hyalinization and scattered bizarre mononucleated and multinucleated cells were also observed. Based on clinico-morphological, immunophenotypical and molecular features, we made a diagnosis of malignant transformation of pulmonary adenoleiomyomatous hamartoma into pulmonary leiomyosarcoma. As far as we know, this is the first described case of this exceptionally rare occurrence in an already rare neoplasm.

Genetic landscape of colorectal cancer patients manifesting tumor shrinkage during SARS-Cov-2 infection.

We previously described three patients affected by metastatic colorectal cancer (mCRC) who experienced spontaneous tumour shrinkage during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Thereafter, the patients were closely monitored and no systemic treatments were applied. Here, we report follow-up clinical information about these patients as well as genetic characterization of their primary tumours through the TruSigt™Oncology 500 Next Generation Sequencing test targeting 523 cancer-relevant genes. An Illumina NovaSeq 6000 platform was used to perform sequencing. Time-to-progression was 23 and 2 months, respectively, in Patients 2 and 3 while it was not reached in Patient 1. Patients 1 and 2 had the greatest anti-SARS-CoV-2 IgG titres. Assessment of genetic landscapes evidenced common mutation in BARD1 gene (p.Val507Met) in Patients 1 and 2. Although our report is descriptive in its nature, we suggest that complex and unexplored interactions between genetic background and components of the immune response to SARS-CoV-2 infection could be responsible of unexpected rare mCRC shrinkage.

Preliminary Experience of Liquid Biopsy in Lung Cancer Compared to Conventional Assessment: Light and Shadows.

Purpose: To assess the qualitative relationship between liquid biopsy and conventional tissue biopsy. As a secondary target, we evaluated the relationship between the liquid biopsy results and the T stage, N stage, M stage, and compared to grading. Methods: The Local Ethics Committee of the "Università degli Studi della Campania Luigi Vanvitelli", with the internal resolution number 24997/2020 of 12.11.2020, approved this spontaneous prospective study. According to the approved protocol, patients with lung cancer who underwent Fine-Needle Aspiration Cytology (FNAC), CT-guided biopsy, and liquid biopsy were enrolled. A Yates chi-square test was employed to analyze differences in percentage values of categorical variables. A p-value < 0.05 was considered statistically significant. Data analysis was performed using the Matlab Statistic Toolbox (The MathWorks, Inc., Natick, MA, USA). Results: When a genetic mutation is present on the pathological examination, this was also detected on the liquid biopsy. ROS1 and PDL1 mutations were found in 2/29 patients, while EGFR Exon 21 was identified in a single patient. At liquid biopsy, 26 mutations were identified in the analyzed samples. The mutations with the highest prevalence rate in the study populations were: ALK (Ile1461Val), found in 28/29 patients (96.6%), EML4 (Lys398Arg), identified in 16/29 (55.2%) patients, ALK (Asp1529Glu), found in 14/29 (48.3%) patients, EGFR (Arg521Lys), found in 12/29 (41.4%) patients, ROS (Lys2228Gln), identified in 11/29 (37.9%) patients, ROS (Arg167Gln) and ROS (Ser2229Cys), identified in 10/29 (34.5%) patients, ALK (Lys1491Arg) and PIK3CA (Ile391Met), identified in 8/29 (27.6%) patients, ROS (Thr145Pro), identified in 6/29 (20.7%) patients, and ROS (Ser1109Leu), identified in 4/29 (13.8%) patients. No statistically significant differences can be observed in the mutation rate between the adenocarcinoma population and the squamous carcinoma population (p > 0.05, Yates chi-square test). Conclusions: We showed that, when a genetic mutation was detected in pathological examination, this was always detected by liquid biopsy, demonstrating a very high concordance rate of genomic testing between tissues and their corresponding mutations obtained by liquid biopsy, without cases of false-negative results. In addition, in our study, liquid biopsy highlighted 26 mutations, with the prevalence of ALK mutation in 96.6% of patients, supporting the idea that this approach could be an effective tool in cases with insufficient tumor tissue specimens or in cases where tissue specimens are not obtainable.

Multisite assessment of the impact of cell-free DNA-based screening for rare autosomal aneuploidies on pregnancy management and outcomes.

Cell-free (cf) DNA screening is a noninvasive prenatal screening approach that is typically used to screen for common fetal trisomies, with optional screening for sex chromosomal aneuploidies and fetal sex. Genome-wide cfDNA screening can screen for a wide variety of additional anomalies, including rare autosomal aneuploidies (RAAs) and copy number variants. Here, we describe a multi-cohort, global retrospective study that looked at the clinical outcomes of cases with a high-risk cfDNA screening result for a RAA. Our study cohort included a total of 109 cases from five different sites, with diagnostic outcome information available for 68% (74/109) of patients. Based on confirmatory diagnostic testing, we found a concordance rate of 20.3% for presence of a RAA (15/74) in our study population. Pregnancy outcome was also available for 77% (84/109) of cases in our cohort. Many of the patients experienced adverse pregnancy outcomes, including intrauterine fetal demise, fetal growth restriction, and preterm birth. These adverse outcomes were observed both in patients with fetal or placental confirmation of the presence of a RAA, as well as patients that did not undergo fetal and/or placental diagnostic testing. In addition, we have proposed some suggestions for pregnancy management and counseling considerations for situations where a RAA is noted on a cfDNA screen. In conclusion, our study has shown that genome-wide cfDNA screening for the presence of rare autosomal aneuploidies can be beneficial for both patients and their healthcare practitioners. This can provide a possible explanation for an adverse pregnancy outcome or result in a change in pregnancy management, such as increased monitoring for adverse outcomes.

Microsatellite Status Detection in Gastrointestinal Cancers: PCR/NGS Is Mandatory in Negative/Patchy MMR Immunohistochemistry.

BACKGROUND: Microsatellite instability (MSI) is a predictive biomarker for immune checkpoint inhibitors. The main goal was to investigate the discordance between IHC and PCR/NGS for MSI testing in gastrointestinal cancers. METHODS: Two series were analyzed through IHC for mismatch-repair-system proteins (MMRP) and PCR, with one series of 444 colorectal cancers (CRC) and the other of 176 gastric cancers (GC). All cases with discordant results between IHC and PCR were analyzed by NGS. IHC staining was evaluated as follows: proficient MMR (pMMR), with all MMR positive; deficient MMR (dMMR), with the loss of one heterodimer; and cases with the loss/patchy expression of one MMR (lo-paMMR). Cases with instability in at least two markers by PCR were MSI-high (MSI-H) and with instability in one marker, MSI-low (MSI-L). Cases without instability were evaluated as microsatellite-stable (MSS). RESULTS: In the CRC cohort, 15 out of 444 cases were dMMR and 46 lo-paMMR. Among the 15 dMMR, 13 were MSI-H and 2 MSS. Among the 46 lo-paMMR, 13 were MSI-H and 33 were MSS. In the GC cohort, 13 out of 176 cases were dMMR and 6 cases lo-paMMR. Among the 13 dMMR, 12 were MSI-H and only 1 was MSS. All six lo-paMMR cases were MSS. All NGS results were in agreement with PCR. CONCLUSIONS: In clinical practice, MMR-IHC could be used as a screening test and additional molecular analysis is mandatory exclusively in cases carrying loss/patchy MMR-IHC.

Detection of 46, XY Disorder of Sex Development (DSD) Based on Plasma Cell-Free DNA and Targeted Next-Generation Sequencing.

Mutations in the HSD17B3 gene cause HSD17B3 deficiency and result in 46, XY Disorders of Sex Development (46, XY DSD). The diagnosis of 46, XY DSD is very challenging and not rarely is confirmed only at older ages, when an affected XY female presents with primary amenorrhea or develops progressive virilization. The patient described in this paper represents a case of discrepancies between non-invasive prenatal testing (NIPT) and ultrasound based fetal sex determination detected during prenatal screening. Exome sequencing was performed on the cell free fetal DNA (cffDNA), amniotic fluid, and the parents. Libraries were generated according to the manufacturer's protocols using TruSight One Kits (Illumina Inc., San Diego, CA, USA). Sequencing was carried out on NEXT Seq 500 (Illumina) to mean sequencing depth of at least 100×. A panel of sexual disease genes was used in order to search for a causative variant. The finding of a mutation (c.645 A>T, p.Glu215Asp) in HSD17B3 gene in amniotic fluid as well as in cffDNA and both parents supported the hypothesis of the HSD17B3 deficiency. In conclusion, we used clinical exome sequencing and non-invasive prenatal detection, providing a solution for NIPT of a single-gene disorder. Early genetic diagnoses are useful for patients and clinicians, contribute to clinical knowledge of DSD, and are invaluable for genetic counseling of couples contemplating future pregnancies.

First Genomic Evidence of Dual African Swine Fever Virus Infection: Case Report from Recent and Historical Outbreaks in Sardinia.

African swine fever virus (ASFV) is one of the pathogens of highest concern worldwide. Despite different virus lineages co-circulating in several areas, dual infections in the same animal have been rarely observed, suggesting that ASF superinfections are infrequent events. Here we present the first genome-wide detection and analysis of two intragenotype dual ASFV infections. The dual infections have been detected in a hunted wild boar and in a pig carcass, both infected by ASFV genotype I in Sardinia in 1984 and 2018, respectively. We characterize the genetic differences between the two sequences, their intra-host frequency, and their phylogenetic relationship among fully sequenced ASFV strains from Sardinia. Both dual infections involve pairs of closely related but different viruses that were circulating in Sardinia in the same period. The results imply that dual ASFV infections or similar ASFV strains are more common than expected, especially in ASF endemic areas, albeit difficult to detect.

A Deeper Insight into Evolutionary Patterns and Phylogenetic History of ASFV Epidemics in Sardinia (Italy) through Extensive Genomic Sequencing.

African swine fever virus (ASFV) is the etiological agent of the devastating disease African swine fever (ASF), for which there is currently no licensed vaccine or treatment available. ASF is defined as one of the most serious animal diseases identified to date, due to its global spread in regions of Africa, Europe and Asia, causing massive economic losses. On the Italian island of Sardinia, the disease has been endemic since 1978, although the last control measures put in place achieved a significant reduction in ASF, and the virus has been absent from circulation since April 2019. Like many large DNA viruses, ASFV mutates at a relatively slow rate. However, the limited availability of whole-genome sequences from spatial-localized outbreaks makes it difficult to explore the small-scale genetic structure of these ASFV outbreaks. It is also unclear if the genetic variability within outbreaks can be captured in a handful of sequences, or if larger sequencing efforts can improve phylogenetic reconstruction and evolutionary or epidemiological inference. The aim of this study was to investigate the phylogenetic patterns of ASFV outbreaks between 1978 and 2018 in Sardinia, in order to characterize the epidemiological dynamics of the viral strains circulating in this Mediterranean island. To reach this goal, 58 new whole genomes of ASFV isolates were obtained, which represents the largest ASFV whole-genome sequencing effort to date. We provided a complete description of the genomic diversity of ASFV in terms of nucleotide mutations and small and large indels among the isolates collected during the outbreaks. The new sequences capture more than twice the genomic and phylogenetic diversity of all the previously published Sardinian sequences. The extra genomic diversity increases the resolution of the phylogenetic reconstruction, enabling us to dissect, for the first time, the genetic substructure of the outbreak. We found multiple ASFV subclusters within the phylogeny of the Sardinian epidemic, some of which coexisted in space and time.

Functional Transcriptome Analysis in ARSACS KO Cell Model Reveals a Role of Sacsin in Autophagy.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a rare early-onset neurological disease caused by mutations in SACS, which encodes sacsin. The complex architecture of sacsin suggests that it could be a key player in cellular protein quality control system. Molecular chaperones that operate in protein folding/unfolding and assembly/disassembly patterns have been described as essential modulators of selectivity during the autophagy process. We performed RNA-sequencing analysis to generate a whole-genome molecular signature profile of sacsin knockout cells. Using data analysis of biological processes significantly disrupted due to loss of sacsin, we confirmed the presence of decreased mitochondrial function associated with increased oxidative stress, and also provided a demonstration of a defective autophagic pathway in sacsin-depleted cells. Western blotting assays revealed decreased expression of LC3 and increased levels of p62 even after treatment with the lysosomal inhibitor bafilomycin A1, indicating impairment of the autophagic flux. Moreover, we found reduced co-immunolocalization of the autophagosome marker LC3 with lysosomal and mitochondrial markers suggesting fusion inhibition of autophagic compartments and subsequent failed cargo degradation, in particular failed degradation of damaged mitochondria. Pharmacological up-regulation of autophagy restored correct autophagic flux in sacsin knockout cells. These results corroborate the hypothesis that sacsin may play a role in autophagy. Chemical manipulation of this pathway might represent a new target to alleviate clinical and pathological symptoms, delaying the processes of neurodegeneration in ARSACS.

Positive selection in Europeans and East-Asians at the ABCA12 gene.

Natural selection acts on genetic variants by increasing the frequency of alleles responsible for a cellular function that is favorable in a certain environment. In a previous genome-wide scan for positive selection in contemporary humans, we identified a signal of positive selection in European and Asians at the genetic variant rs10180970. The variant is located in the second intron of the ABCA12 gene, which is implicated in the lipid barrier formation and down-regulated by UVB radiation. We studied the signal of selection in the genomic region surrounding rs10180970 in a larger dataset that includes DNA sequences from ancient samples. We also investigated the functional consequences of gene expression of the alleles of rs10180970 and another genetic variant in its proximity in healthy volunteers exposed to similar UV radiation. We confirmed the selection signal and refine its location that extends over 35 kb and includes the first intron, the first two exons and the transcription starting site of ABCA12. We found no obvious effect of rs10180970 alleles on ABCA12 gene expression. We reconstructed the trajectory of the T allele over the last 80,000 years to discover that it was specific to H. sapiens and present in non-Africans 45,000 years ago.