Genotyping-by-sequencing in an orphan plant species Physocarpus opulifolius helps identify the evolutionary origins of the genus Prunus

Background: The Rosaceae family encompasses numerous genera exhibiting morphological diversification in fruit types and plant habit as well as a wide variety of chromosome numbers. Comparative genomics between various Rosaceous genera has led to the hypothesis that the ancestral genome of the family contained nine chromosomes, however, the synteny studies performed in the Rosaceae to date encompass species with base chromosome numbers x = 7 (Fragaria), x = 8 (Prunus), and x = 17 (Malus), and no study has included species from one of the many Rosaceous genera containing a base chromosome number of x = 9. Results: A genetic linkage map of the species Physocarpus opulifolius (x = 9) was populated with sequence characterised SNP markers using genotyping by sequencing. This allowed for the first time, the extent of the genome diversification of a Rosaceous genus with a base chromosome number of x = 9 to be performed. Orthologous loci distributed throughout the nine chromosomes of Physocarpus and the eight chromosomes of Prunus were identified which permitted a meaningful comparison of the genomes of these two genera to be made. Conclusions: The study revealed a high level of macro-synteny between the two genomes, and relatively few chromosomal rearrangements, as has been observed in studies of other Rosaceous genomes, lending further support for a relatively simple model of genomic evolution in Rosaceae

In Silico Identification of MYB and bHLH Families Reveals Candidate Transcription Factors for Secondary Metabolic Pathways in Cannabis sativa L

Plant secondary metabolic pathways are finely regulated by the activity of transcription factors, among which members of the bHLH and MYB subfamilies play a main role. Cannabis sativa L. is a unique officinal plant species with over 600 synthesized phytochemicals having diverse scale-up industrial and pharmaceutical usage. Despite comprehensive knowledge of cannabinoids\u2019 metabolic pathways, very little is known about their regulation, while the literature on flavonoids\u2019 metabolic pathways is still scarce. In this study, we provide the first genome-wide analysis of bHLH and MYB families in C. sativa reference cultivar CBDRx and identification of candidate coding sequences for these transcription factors. Cannabis sativa bHLHs and MYBs were then classified into functional subfamilies through comparative phylogenetic analysis with A. thaliana transcription factors. Analyses of gene structure and motif distribution confirmed that CsbHLHs and CsMYBs belonging to the same evolutionary clade share common features at both gene and amino acidic level. Candidate regulatory genes for key metabolic pathways leading to flavonoid and cannabinoid synthesis in Cannabis were also retrieved. Furthermore, a candidate gene approach was used to identify structural enzyme-coding genes for flavonoid and cannabinoid synthesis. Taken as a whole, this work represents a valuable resource of candidate genes for further investigation of the C. sativa cannabinoid and flavonoid metabolic pathways for genomic studies and breeding programs

Molecular genetics and genomics of the Rosoideae: state of the art and future perspectives

The Rosoideae is a subfamily of the Rosaceae that contains a number of species of economic importance, including the soft fruit species strawberry (Fragaria x ananassa), red (Rubus idaeus) and black (Rubus occidentalis) raspberries, blackberries (Rubus spp.) and one of the most economically important cut flower genera, the roses (Rosa spp.). Molecular genetics and genomics resources for the Rosoideae have developed rapidly over the past two decades, beginning with the development and application of a number of molecular marker types including restriction fragment length polymorphisms, amplified fragment length polymorphisms and microsatellites, and culminating in the recent publication of the genome sequence of the woodland strawberry, Fragaria vesca, and the development of high throughput single nucleotide polymorphism (SNP)-genotyping resources for Fragaria, Rosa and Rubus. These tools have been used to identify genes and other functional elements that control traits of economic importance, to study the evolution of plant genome structure within the subfamily, and are beginning to facilitate genomic-assisted breeding through the development and deployment of markers linked to traits such as aspects of fruit quality, disease resistance and the timing of flowering. In this review, we report on the developments that have been made over the last 20 years in the field of molecular genetics and structural genomics within the Rosoideae, comment on how the knowledge gained will improve the efficiency of cultivar development and discuss how these advances will enhance our understanding of the biological processes determining agronomically important traits in all Rosoideae species

The genome sequence and transcriptome of Potentilla micrantha and their comparison to Fragaria vesca (the woodland strawberry)

Background The genus Potentilla is closely related to that of Fragaria, the economically important strawberry genus. Potentilla micrantha is a species that does not develop berries, but shares numerous morphological and ecological characteristics with F. vesca. These similarities make P. micrantha an attractive choice for comparative genomics studies with F. vesca Findings In this study, the Potentilla micrantha genome was sequenced and annotated, and RNA-Seq data from the different developmental stages of flowering and fruiting were used to develop a set of gene predictions. A 327 Mbp sequence and annotation of the genome of P. micrantha, spanning 2,674 sequence contigs, with an N50 size of 335,712, estimated to cover 80% of the total genome size of the species was developed. The genus Potentilla has a characteristically larger genome size than Fragaria, but the recovered sequence scaffolds were remarkably collinear at the micro-syntenic level with the genome of F. vesca, its closest sequenced relative. A total of 33,602 genes were predicted, and 95.1% of BUSCO genes were complete within the presented sequence. Thus, we argue that the majority of the gene-rich regions of the genome have been sequenced Conclusions Comparisons of RNA-Seq data from the stages of floral and fruit development revealed genes differentially expressed between P. micrantha and F. vesca. The data presented are a valuable resource for future studies of berry development in Fragaria and the Rosaceae and they also shed light on the evolution of genome size and organization in this family

Prognostic value of normal sodium levels in patients with metastatic renal cell carcinoma receiving tyrosine kinase inhibitors

Background: Although serum sodium concentration, particularly hyponatremia, has been shown to be a prognostic marker of survival in metastatic renal cell carcinoma (mRCC), the impact of normal sodium levels has not been investigated. Herein, we investigate the influence of normonatremia in mRCC patients treated with tyrosine kinase inhibitors (TKIs). Materials and methods: For this retrospective study, the clinical and biochemical data of patients treated with first-line TKIs for mRCC were available from seven Italian cancer centers. We collected natremia levels at baseline and first evaluation after treatment excluding patients with sodium levels outside the normal range (<135 or >145 mEq/L). The remaining patients were subdivided into two groups according to the median sodium value: natremia patients with <140 mEq/L (n = 132) and baseline natremia patients with ≥140 mEq/L (n = 185). Subsequently, we analyzed the impact of sodium levels on response rate (RR), disease control rate (DCR), progression-free survival (PFS), and overall survival (OS). PFS and OS were estimated through the Kaplan–Meier method, and differences between groups were examined by the log-rank test. Univariate and multivariate Cox regression analyses were applied to evaluate the prognostic factors for PFS and OS. Results: Of the 368 patients, 317 were included in the analysis, 73.1% were men, and the median age was 67 years (range 36–89). When comparing patients with baseline natremia ≥140 mEq/L (n = 185) to patients with natremia <140 mEq/L (n = 132), the PFS was 15 vs. 10 months (p < 0.01) and the OS was 63 vs. 36 months, respectively (p = 0.02). On the first evaluation, patients with serum sodium ≥140 mEq/L had longer PFS (15 vs. 10 months, p < 0.01) and OS (70 vs. 32 months, p < 0.01) than patients with levels <140 mEq/L. Moreover, clinical outcomes showed a significant improvement in patients with natremia ≥140 mEq/L compared with patients with levels <140 mEq/L both at baseline and first evaluation: PFS was 19 vs. 11 months (p < 0.01) and OS was 70 vs. 36 months (p < 0.01), respectively. Conclusions: To the best of our knowledge, this is the first study to investigate the impact of normonatremia in mRCC. We found that serum sodium levels <140 mEq/L at baseline and first assessment are independently associated with worse PFS and OS in mRCC patients treated with TKIs in the first-line setting

Application of the Meet-URO score to metastatic renal cell carcinoma patients treated with second- and third-line cabozantinib

Background: The addition of neutrophil-to-lymphocyte ratio (NLR) and bone metastases to the International Metastatic RCC Database Consortium (IMDC) score (by the Meet-URO score) has been shown to better stratify pretreated metastatic renal cell carcinoma (mRCC) patients receiving nivolumab. This study aimed to validate the Meet-URO score in patients receiving cabozantinib to assess its predictivity and prognostic role. Methods: A multicenter retrospective analysis evaluated mRCC patients receiving ⩾second-line cabozantinib. NLR, IMDC score and bone metastases were assessed before the start of cabozantinib. The primary endpoint was overall survival (OS). Harrell's c-index was calculated to compare the accuracy of the prediction of the two scores. Results: Overall, 174 mRCC patients received cabozantinib as second and third line (51.7% and 48.3%, respectively) with a median follow-up of 6.8 months. A shorter median overall survival (mOS) was observed for the IMDC poor-risk group, NLR ⩾3.2 and the presence of bone metastases, while the IMDC intermediate-risk group had a similar mOS to the favourable-risk one. Applying the Meet-URO score, three risk groups were identified: group 1 (55.2% of patients) with a score of 0-3, group 2 (38.5%) with a score of 4-8 and group 3 (6.3%) with a score of 9. Compared to group 1 (mOS: 39.4 months), a statistically significant worse mOS was observed in group 2 (11.2 months) and group 3 (3.2 months) patients, respectively. The Meet-URO c-index score was 0.640, showing a higher discriminative ability than the IMDC score (c-index: 0.568). Conclusion: This analysis showed that the Meet-URO score provides a more accurate prognostic stratification than the IMDC score in mRCC patients treated with ⩾second-line cabozantinib besides nivolumab. Moreover, it is an easy-to-use tool with no additional costs for clinical practice (web-calculator is available at: https://proviso.shinyapps.io/Meet-URO15_score/). Future investigations will include the application of the Meet-URO score to the first-line immunotherapy-based combination therapies

Application of the Meet-URO score to metastatic renal cell carcinoma patients treated with second- and third-line cabozantinib

open18Background: The addition of neutrophil-to-lymphocyte ratio (NLR) and bone metastases to the International Metastatic RCC Database Consortium (IMDC) score (by the Meet-URO score) has been shown to better stratify pretreated metastatic renal cell carcinoma (mRCC) patients receiving nivolumab. This study aimed to validate the Meet-URO score in patients receiving cabozantinib to assess its predictivity and prognostic role. Methods: A multicenter retrospective analysis evaluated mRCC patients receiving ⩾second-line cabozantinib. NLR, IMDC score and bone metastases were assessed before the start of cabozantinib. The primary endpoint was overall survival (OS). Harrell's c-index was calculated to compare the accuracy of the prediction of the two scores. Results: Overall, 174 mRCC patients received cabozantinib as second and third line (51.7% and 48.3%, respectively) with a median follow-up of 6.8 months. A shorter median overall survival (mOS) was observed for the IMDC poor-risk group, NLR ⩾3.2 and the presence of bone metastases, while the IMDC intermediate-risk group had a similar mOS to the favourable-risk one. Applying the Meet-URO score, three risk groups were identified: group 1 (55.2% of patients) with a score of 0-3, group 2 (38.5%) with a score of 4-8 and group 3 (6.3%) with a score of 9. Compared to group 1 (mOS: 39.4 months), a statistically significant worse mOS was observed in group 2 (11.2 months) and group 3 (3.2 months) patients, respectively. The Meet-URO c-index score was 0.640, showing a higher discriminative ability than the IMDC score (c-index: 0.568). Conclusion: This analysis showed that the Meet-URO score provides a more accurate prognostic stratification than the IMDC score in mRCC patients treated with ⩾second-line cabozantinib besides nivolumab. Moreover, it is an easy-to-use tool with no additional costs for clinical practice (web-calculator is available at: https://proviso.shinyapps.io/Meet-URO15_score/). Future investigations will include the application of the Meet-URO score to the first-line immunotherapy-based combination therapies.openRebuzzi, Sara Elena; Cerbone, Luigi; Signori, Alessio; Santoni, Matteo; Murianni, Veronica; De Giorgi, Ugo; Procopio, Giuseppe; Porta, Camillo; Milella, Michele; Basso, Umberto; Massari, Francesco; Maruzzo, Marco; Iacovelli, Roberto; Battelli, Nicola; Carmisciano, Luca; Banna, Giuseppe Luigi; Buti, Sebastiano; Fornarini, GiuseppeRebuzzi, Sara Elena; Cerbone, Luigi; Signori, Alessio; Santoni, Matteo; Murianni, Veronica; De Giorgi, Ugo; Procopio, Giuseppe; Porta, Camillo; Milella, Michele; Basso, Umberto; Massari, Francesco; Maruzzo, Marco; Iacovelli, Roberto; Battelli, Nicola; Carmisciano, Luca; Banna, Giuseppe Luigi; Buti, Sebastiano; Fornarini, Giusepp

First line avelumab in PD-L1+ve metastatic or locally advanced urothelial cancer (aUC) patients unfit for cisplatin (cis): The ARIES trial

Background: Avelumab (ave) was approved as maintenance therapy after platinum-based first line (1L) therapy for patients (pts) with aUC based on ph. 3 Javelin Bladder 100 study (NCT02603432), showing significant overall survival (OS) improvement. Here we tested the activity of ave as 1L of therapy in cis-unfit pts with aUC and PD-L1+ve expression. Methods: ARIES is a single-arm, multi-site, open-label phase II trial. Enrolled pts had aUC, were cis-unfit (at least one of: ECOG-PS = 2, CrCl < 60 mL/min, grade ≥2 peripheral neuropathy/hearing loss, progression within 6-mos before the end of neo/adj chemo), had not previously received chemo for aUC and PD-L1≥5% (SP263) centrally assessed. Pts received ave 10 mg/Kg IV Q2W until progression, unacceptable toxicity and withdrawal, whichever occurred first. The primary endpoint was the 1-year OS. Key secondary endpoints were median-OS, -PFS, ORR and safety. Results: A total of 198 eligible cis-unfit pts have been tested for PD-L1 and 71 (35.6%) have been found positive. Among enrolled patients (N = 71), median age was 75 y, 35 (49.3%) had visceral disease, and 22 (31.0%) had ECOG-PS = 2; 50 (70.4%) had CrCl < 60 mL/min and 9 (12.7%) progressed within 6-mos from the end of neo/adj chemo. At the cut-off data (Oct 7, 2021), median follow up was 9.0 mo and 13 patients are still on treatment. The median OS was 10.0 mos (95% CI, 5.7-14.3), and 40.8% of patients were alive at 1-year. The ORR for all patients was 22.5%; complete response, 1.4% (n = 1); partial response, 21.1% (n = 15). Clinical benefit was 43.6% (n = 31). Median PFS was 2.0 mos (95% CI, 1.4-2.6). Among the 56 pts who received at least 3 cycles (29 days) of therapy the median OS was 16.0 vs 1.0 mos. Five (7.0%) grade 3 ave-related adverse events, and no treatment-related death were reported. Conclusions: Ave is active and safe in pts with cis-unfit, PD-L1+ve aUC and poor baseline characteristics

Early primary tumor response in metastatic RCC patients treated with immune checkpoint inhibitors-based combinations

Background: 25-30% of renal cell carcinoma presents with metastases (mRCC) at diagnosis. The activity of immune checkpoint inhibitor (ICI)-combinations on the primary tumor (PT) is debated. Patients andMethods: mRCC patients (pts) with PT who received first-line nivolumab plus ipilimumab (N/I) or pembrolizumab plus axitinib (P/A) were included. We investigated the early primary tumor response (EPTR) at the first radiological assessment. Results: 73 pts were included. The median early reduction of the PT longest diameter was 12.4% with P/A versus 6.2% with N/I (p = 0.42). We evaluated if the type of EPTR could affect the metastases response. Among pts with PT stable disease (SD), 8.3% had metastatic disease progression (PD) with P/A and 34.8% with N/I. Early PT partial response (PR) was associated with no metastatic PD with both N/I and P/A. The 2 pts with PT PD had also metastatic PD to P/A. Of the 3 PT with PD to N/I, 1 had metastatic SD and 2 PD. In the overall population, of the 94.1% without PT progression (PR+SD), 47.5% had metastatic PR, 35.6% SD, 16.9% PD. Conclusions: ICIs-combinations achieved an early PT PR in about 10-20%, without any complete responses. Only a small percentage of PT had an early PD, mainly associated with metastatic PD. However, among those PT without an early progression, metastatic PR can be achieved in approximately 50% of cases