Clinical Predictors of Response to Anti-PD-1 First-Line Treatment in a Single-Centre Patient Cohort: A Real-World Study.

AIMS: Cutaneous melanoma is one of the most immunogenic tumours. Immunotherapy with checkpoint inhibitors, such as anti-PD-1 antibodies, has significantly improved the prognosis in metastatic melanoma. However, only half of the patients respond to this therapy and have a favourable outcome. Identifying factors associated with treatment failure and early identification of responders are both important to select the best treatment approach for each patient. The aim of our study was to investigate clinical biomarkers of response to treatment with anti-PD-1 antibodies. MATERIALS AND METHODS: We selected all patients with stage IV melanoma (n = 147), subjected to first-line treatment with anti-PD-1 in the last 10 years. We investigated the associations between patients' different clinical features and progression-free survival, using the Cox proportional hazards models. RESULTS: In the multivariate analysis, an increased risk of disease progression was observed among patients with stage M1d metastases (hazard ratio 3.30; 95% confidence interval 1.58-6.91), compared with patients with stage M1a-M1b. Moreover, the risk of progression was greater in patients with the Eastern Cooperative Oncology Group Performance Status (ECOG PS) 1 (hazard ratio 2.04; 95% confidence interval 1.02-4.06) and in patients with ECOG PS ≥ 2 (hazard ratio 2.19; 95% confidence interval 1.05-4.55) compared with ECOG PS 0. High levels of lactate dehydrogenase (hazard ratio 2.06; 95% confidence interval 1.18-3.59) and the presence of respiratory diseases (hazard ratio 4.14; 95% confidence interval 1.42-12.0) at the beginning of anti-PD-1 treatment were also associated with an increased risk of disease progression. In a subgroup analysis, neutrophil count and neutrophil/lymphocyte ratio before anti-PD-1 treatment were higher in patients who underwent disease progression. CONCLUSION: In our study population, independent predictors of disease progression among patients treated with first-line anti-PD-1 were as follows: ECOG PS, staging, lactate dehydrogenase and the presence of respiratory diseases.

GTP-cyclohydrolase I gene mutations in patients with autosomal dominant and recessive GTP-CH1 deficiency: identification and functional characterization of four novel mutations.

GTP-cyclohydrolase I (GTP-CH1, EC 3.5.4.16) is encoded by the GCH1 gene. Mutations in the GCH1 gene cause both dopa-responsive dystonia (McKusick 128230) and recessive GTP-CH1 deficiency (McKusick 600225). The exact molecular mechanism resulting in decreased GTP-CH1 activity in the patients is still obscure. We report the clinical features and molecular and functional study of the GCH1 gene in eight Italian patients affected by dominant and recessive GTP-CH1 deficiency. All the studied patients had mutations in the GCH1 gene. Three missense mutations (V205G, K224R, P199A), a frameshift mutation (Delta G693), and a splice-site mutation (ivs5 + 1g > c) were found. Except for K224R these are all novel mutations. To analyse the defect caused by the novel mutations, an in vivo functional assay in a Saccharomyces cerevisiae strain lacking the endogenous gene encoding GTP-CH1 ( FOL2 ) was performed. Complementation analysis showed that the Delta G693 and V205G mutations abolish the enzymatic function, while the P199A mutation causes a conditional defect. In conclusion, the clinical phenotypes displayed by our patients confirm the wide clinical spectrum of the disease and further support the lack of correlation between a given mutation and a clinical phenotype. Complementation analysis in yeast is a useful tool for confirming the pathogenetic effect of GCH1 mutations.

The Saccharomyces cerevisiae SDA1 gene is required for actin cytoskeleton organization and cell cycle progression.

The organization of the actin cytoskeleton is essential for several cellular processes. Here we report the characterization of a Saccharomyces cerevisiae novel gene, SDA1, encoding a highly conserved protein, which is essential for cell viability and is localized in the nucleus. Depletion or inactivation of Sda1 cause cell cycle arrest in G(1) by blocking both budding and DNA replication, without loss of viability. Furthermore, sda1-1 temperature-sensitive mutant cells arrest at the non-permissive temperature mostly without detectable structures of polymerized actin, although a normal actin protein level is maintained, indicating that Sda1 is required for proper organization of the actin cytoskeleton. To our knowledge, this is the first mutation shown to cause such a phenotype. Recovery of Sda1 activity restores proper assembly of actin structures, as well as budding and DNA replication. Furthermore we show that direct actin perturbation, either in sda1-1 or in cdc28-13 cells released from G(1) block, prevents recovery of budding and DNA replication. We also show that the block in G(1) caused by loss of Sda1 function is independent of Swe1. Altogether our results suggest that disruption of F-actin structure can block cell cycle progression in G(1) and that Sda1 is involved in the control of the actin cytoskeleton.

Complementation of the fol2 deletion in Saccharomyces cerevisiae by human and Escherichia coli genes encoding GTP cyclohydrolase I.

Saccharomyces cerevisiae is so far the only organism where a knock-out mutant in the gene encoding GTP cyclohydrolase I (FOL2) has been obtained. GTP cyclohydrolase I controls the de novo biosynthetic pathway of tetrahydrobiopterin and folic acid. Since deletion of yeast FOL2 leads to a recessive auxotrophy for folinic acid, we used a yeast fol2Delta mutant for an in vivo functional assay of heterologous GTP cyclohydrolases I. We show that the GTP cyclohydrolase I, encoded either by the E. coli folE gene or by the human cDNA, complements the yeast fol2Delta mutation by restoring folate prototrophy. Furthermore the folE-3x allele of the E. coli gene, carrying three base substitutions, failed to complement the yeast fol2Delta defect. This allele behaved as a negative semidominant to the wild type folE and, when overexpressed, completely abolished complementation of fol2Delta by folE. Thus, the yeast fol2 null mutant is a suitable system to characterize mutations in genes encoding GTP cyclohydrolase I.

A 9359 bp fragment from the right arm of Saccharomyces cerevisiae chromosome VII includes the FOL2 and YTA7 genes and three unknown open reading frames.

In the framework of the EU programme for systematic sequencing of the Saccharomyces cervisiae genome we determined the sequence of a 9359 bp fragment of the right arm of chromosome VII. Five open reading frames (ORFs) of at least 300 nucleotides were found in this region. YGR267c encodes a protein with significant similarity to the enzyme GTP-cyclohydrolase I, that controls the first step in the biosynthetic pathway leading to various pterins and shows a high degree of sequence conservation from bacteria to mammals. We have recently demonstrated (Nardese et al., 1996) that YGR267c corresponds to the FOL2 gene, previously localized in the same chromosomal region by genetic mapping. The protein deduced from YGR270w belongs to the superfamily of putative ATPases associated with diverse cellular activities. It corresponds to the YTA7 gene, a member of a set of yeast genes coding for putative ATPases with high similarity to constituents of the 26S protease. The three ORFs YGR266w, YGR268c and YGR269w encode putative products of unknown function, with neither significant similarity to proteins in databases nor recognizable domains. YGR268c and YGR269w are partially overlapping ORFs: YGR268c seems to correspond to a real gene. whereas YGR269w is probably a fortuitous ORF.

The nucleotide sequence of Saccharomyces cerevisiae chromosome VII.

The complete nucleotide sequence of Saccharomyces cerevisiae chromosome VII has 572 predicted open reading frames (ORFs), of which 341 are new. No correlation was found between G+C content and gene density along the chromosome, and their variations are random. Of the ORFs, 17% show high similarity to human proteins. Almost half of the ORFs could be classified in functional categories, and there is a slight increase in the number of transcription (7.0%) and translation (5.2%) factors when compared with the complete S. cerevisiae genome. Accurate verification procedures demonstrate that there are less than two errors per 10,000 base pairs in the published sequence.

A simple signal element mediates transcription termination and mRNA 3' end formation in the DEG1 gene of Saccharomyces cerevisiae.

DEG1 is a weakly transcribed gene of Saccharomyces cerevisiae, closely associated with CEN6. We mapped its major poly(A) site only 24 nucleotides (nt) downstream of the stop codon, and only 26 nt upstream of the CDEI centromere element. The deletion of this 50 nt stretch completely abolishes formation of the mRNA 3' end. A shorter deletion of a 16 nt sequence in the 3'-untranslated region has the same effect on transcription termination and 3'-maturation function. A TATATA sequence within this 16 nt region is essential for both functions, while a TGTATA sequence has a weak compensating activity in 3' end maturation if the TATATA stretch is deleted. We assume that the 3' end formation signals of the DEG1 gene have this simple structure: a single essential element (TATATA, whether alone or with the few surrounding nucleotides), probably, but not necessarily, cooperating with the sequence at the poly(A) site. This simple structure differs from the emerging model for 3' end-processing signals in that (i) it is shorter: 24 nt long at the most, while the model suggests 39 nt; (ii) there is no element located downstream of the TATATA signal to position the poly(A) site; and (iii) unlike the other naturally occurring signals studied, no cooperation among multiple TATATA-like elements is observed. We found that the same TATATA sequence also directs transcription termination, irrespective of promoter strength, and presumably without the cooperation of a downstream polymerase II pausing site. Taken together, these findings support the hypothesis that the DEG1 3' end-forming signals are more condensed than in other yeast genes, probably because of their proximity to CEN6.

Analysis of a 17.9 kb region from Saccharomyces cerevisiae chromosome VII reveals the presence of eight open reading frames, including BRF1 (TFIIIB70) and GCN5 genes.

We report the nucleotide sequence of a 17,893 bp DNA segment from the right arm of Saccharomyces cerevisiae chromosome VII. This fragment begins at 482 kb from the centromere. The sequence includes the BRF1 gene, encoding TFIIIB70, the 5' portion of the GCN5 gene, an open reading frame (ORF) previously identified as ORF MGA1, whose translation product shows similarity to heat-shock transcription factors and five new ORFs. Among these, YGR250 encodes a polypeptide that harbours a domain present in several polyA binding proteins. YGR245 is similar to a putative Schizosaccharomyces pombe gene, YGR248 shows significant similarity with three ORFs of S. cerevisiae situated on different chromosomes, while the remaining two ORFs, YGR247 and YGR251, do not show significant similarity to sequences present in databases.

Disruption of the GTP-cyclohydrolase I gene in Saccharomyces cerevisiae.

GTP-cyclohydrolase I is the first enzyme in the biosynthetic pathway leading to folic acid and tetrahydrobiopterin. We determined the complete sequence of the GTP-cyclohydrolase I gene from the yeast Saccharomyces cerevisiae. The gene, which is located in the subtelomeric region of the right arm of chromosome VII, gives a major transcript of about 1000 nt and encodes a protein of 243 amino acids, which is highly homologous to the GTP-cyclohydrolase I from bacteria to man. We obtained by gene replacement a knock-out mutant that shows a recessive conditional lethality due to folinic acid auxotrophy, and lacks any detectable specific enzymatic activity. The gene was identified as FOL2, previously genetically mapped in the same region (J. Game, personal communication).

Nucleotide sequence of 9.2 kb left of CRY1 on yeast chromosome III from strain AB972: evidence for a Ty insertion and functional analysis of open reading frame YCR28.

We report the 9210 bp sequence from a segment of yeast chromosome III cloned from strain AB972 in lambda PM3270. Analysis of this sequence and its comparison with the one derived from the corresponding segment of strain XJ24-24A revealed that the AB972 region contains a duplication of about 2 kb and a Ty element, which are not found in XJ24-24A and cause a quite significant rearrangement of the whole region. We performed functional analysis of YCR28, the largest open reading frame we found in both AB972 and XJ24-24A. YCR28 encodes a putative protein of 512 amino acids with some similarities to yeast allontoate permease. Its disruption does not cause any detectable phenotype on rich medium or on allantoate medium, while we observed a strain-dependent effect on sensitivity to amino acid balance and to 3-aminotriazole, when cells were grown in synthetic medium.

The complete DNA sequence of yeast chromosome III.

The entire DNA sequence of chromosome III of the yeast Saccharomyces cerevisiae has been determined. This is the first complete sequence analysis of an entire chromosome from any organism. The 315-kilobase sequence reveals 182 open reading frames for proteins longer than 100 amino acids, of which 37 correspond to known genes and 29 more show some similarity to sequences in databases. Of 55 new open reading frames analysed by gene disruption, three are essential genes; of 42 non-essential genes that were tested, 14 show some discernible effect on phenotype and the remaining 28 have no overt function.

A gene tightly linked to CEN6 is important for growth of Saccharomyces cerevisiae.

Transcriptional analysis of the region flanking the left boundary of the centromere of chromosome VI revealed the presence of a gene immediately adjacent to CEN6. The transcription of the gene is directed toward the centromere, and nucleotide sequence analysis showed that the coding region terminates only 50 bp away from CEN6. Our results extend to chromosome VI the observation that centromere-flanking regions of S. cerevisiae are transcriptionally active. Disruption of the coding region of the gene showed that its product, whilst not essential for cell viability, is important for normal cell growth. The gene has been termed DEG1 (DEpressed Growth rate). Comparison of the deduced amino acid sequence of DEG1 with a protein sequence databank revealed homology with the enzyme tRNA pseudouridine synthase I of E. coli.

Caffeine interactions with methyl methanesulphonate, hycanthone, benlate, and cadmium chloride in chromosomal meiotic segregation of Saccharomyces cerevisiae.

Interactions of caffeine with chemicals known for their effects on chromosomal segregation during meiosis of Saccharomyces cerevisiae were studied. It appears that caffeine does interfere with the action of other compounds during the different phases of meiosis. Treatments with methyl methanesulphonate (MMS) and cadmium chloride (CdCl2) resulted in a synergistic effect consisting of an increase in the frequency of recombination. The greatest effects were found on the induction of diploid spores: MMS, hycanthone, and distamycin demonstrated strong, benlate little synergistic action. CdCl2 demonstrated antagonism to caffeine by counter-inhibiting its effect on the induction of diploids. Concerning disomic induction: caffeine reduced (or left unchanged) the effect on non-disjunction when MMS and hycanthone were used. Simple additive effects were caused in conjunction with distamycin, benlate, and (in small doses) CdCl2. 2 mg of caffeine/ml in treatments with CdCl2 resulted in a very high frequency of disomic clones.

Disomic and diploid meiotic products induced in Saccharomyces cerevisiae by the salts of 27 elements.

The effects of salts of 27 elements on recombination and on the production of disomic and/or diploid spores during meiosis of Saccharomyces cerevisiae has been investigated. Be(NO3)2, MgSO4, FeSO4, CuSO4, AgNO3, Na2HAsO4 were inactive on the events studied during meiotic cell division. AuCl4, CdCl2, C4H6O4Pb, SnCl2, K2Cr2O7, RbCl induced both disomic and diploid spores. LiCl acted similarly and also affected recombination. Activity in the induction of disomic spores was shown by MnSO4, HgCl2 and SrCl2. CsCl, CaCl2, Na2MoO4, NiCl2, K2PtCl4 increased the frequency of diploid spores, while NaWO4, VOSO4, KCl, BaCl2 already increased recombination frequency. NaBiO3 showed an effect on meiotic recombination only. A decrease in the occurrence of both diploid and disomic spores was suggested by the data obtained with CoCl2.

Drug synergism or antagonism in the induction of diploid meiotic products in Saccharomyces cerevisiae.

The induction of yeast diploid meiotic products by treatment with bleomycin and mitomycin C is reduced when sporulating cells are treated in combination with propranolol, and increased when they are treated in combination with caffeine. We show that bleomycin and mitomycin C act by blocking the second meiotic division. The frequency of this event appears to be directly related to the intracellular cAMP concentration which is known to be influenced in opposite ways by caffeine and propranolol.

Mutants resistant to manganese in Saccharomyces cerevisiae.

Several mutants resistant to Mn(2+) have been isolated and characterized in Saccharomyces cerevisiae. All the mutations are semidominant and allelic to a single nuclear gene (MNRI). Mg(2+) in the growth medium reverses the inhibitory effect of Mn(2+) in a competitive way. This appears to be due to the inhibition of the uptake of Mn(2+) by the cells, not to an increase of the amount of Mg(2+) inside the cells.The analysis of the distribution of Mn(2+) taken up by growing cells shows that the amount of the ion present in insoluble form is far higher in resistant than in sensitive cells. We therefore believe that yeast cells have a sequestering system for Mn(2+) and that the major difference between mutants and wild-type strains lies in the much higher efficiency of this system.

Nuclear inheritance of resistance to antimycin A in Saccharomyces cerevisiae.

A group of 30 independent mutants of Saccharomyces cerevisiae, resistant to the respiratory inhibitor antimycin A, was investigated from a genetical and biochemical point of view. All the mutants can be grouped into two nuclear loci: AMY1 maps on the VII chromosome, between leu 1 and trp 5; AMY2 is close to its centromere on either chromosome XVIII or XIX. Both genes do not affect mitochondrial structures or functions.

Effect of mutation in the aromatic amino acid pathway on sporulation of Saccharomyces cerevisiae.

Mutations in ARO1 and ARO2 genes coding for enzymes involved in the common part of the aromatic amino acid pathway completely block the sporulation of Saccharomyces cerevisiae when in a homozygous state, whereas mutations in all the other genes of the same pathway do not. This effect is not due to the lack of any intermediate metabolite but rather to the accumulation of a metabolite preceding chorismic acid. Shikimic acid or one of its precursors was identified as the possible inhibitor. The presence of the three aromatic amino acids in the sporulation medium restores the ability to undergo meiosis. This seems not to be due to a feedback inhibition of the first enzymes of the pathway but rather to a competition between aromatic amino acids and the inhibitor on a site specific for the meiotic process. The inhibition of sporulation seems to occur at a very early step in meiosis, as indicated by the lack of premeiotic DNA synthesis in aro1 and aro2 mutants.