We recommend further research into buffering agents in highly trained female athletes that control or account for the MC, studies that replicate the demands of athletes’ heat and altitude camps, and investigations of highly trained athletes’ use of combined buffering agents. In a practical context, we recommend developing evidence-based buffering protocols for individual athletes which feature co-supplementation with other evidence-based products, reduce the likelihood of side-effects, and optimise key moderating factors: supplement dose and timing, and exercise duration and intensity.This narrative review evaluated the evidence for buffering agents , with specific consideration of three discrete scenarios: female athletes, extreme environments and combined buffering agents. Studies were screened according to exclusion and inclusion criteria and were analysed on three levels: (1) moderating variables , (2) design factors  and (3) athlete-specific factors . Only 19% of the included studies for the three buffering agents reported a performance benefit, and only 10% recruited highly trained athletes. This low transferability of research findings to athletes’ real-world practices may be due to factors including the small number of sodium citrate studies in females ( Furta−] ions , 92. Alta−] ions . Despitein males . Neverthin males , 93, andn = 29), only 21% reported a performance benefit , none used a supplement dose or timing that was consistent with the current evidence-based guidelines  in females n = 9, only 2es Table  50, 94], 94n = 2Future investigations on the efficacy of buffering agents in females should focus on quantifying menstrual status, using methods to define MC phases based on hormonal profiles and/or controlling menstrual status, reporting types of hormonal contraceptive method used, tracking MC phase and standardising the phase for exercise testing and allocating participants using hormonal contraceptives to a control group . Such in−] and [H+]  . In addiand [H+] –104. MeaV˙O2max) . FurtherV˙O2max) , 106. Afea level . Acute ealtitude . Some coaltitude  and altialtitude . HoweverOf the 18 studies investigating buffering agents in extreme environments, five studies were in hot conditions (mean ± SD temperature 31.4 ± 2.2 °C and relative humidity 48.0 ± 8.4%) and 13 studies were in hypoxia (mean ± SD elevation 3181 ± 1024 m). Of these studies, only 22% reported a performance benefit  and a compensatory increase in ventilation [More recently, ketone ester supplements have been proposed to enhance endurance performance via effects associated with an exogenously derived increase in plasma ketone bodies , 122. Hotilation . Howevertilation . The auttilation . AlthougAn important consideration for athletes wishing to utilise any supplement within training or competition is the presence of side-effects or risks associated with the product. There is accumulating evidence of the safety of beta-alanine supplementation when it is ingested in the protocols (dose and duration) that have been scientifically investigated. The longest study to date showed that 24 weeks of beta-alanine supplementation at 6.4 g/day did not lead to any changes in clinical markers of renal, hepatic or muscle health in healthy individuals . The samDespite the clear safety of chronic beta-alanine ingestion, a common side-effect that is experienced acutely is paraesthesia, which is described as an uncomfortable prickly sensation on the surface of the skin. This sensation of paraesthesia occurs due to the binding of beta-alanine to peripheral neuronal receptors . AlthougThe unpleasant side-effects that follow sodium bicarbonate ingestion are well known and can include abdominal pain, stomach ache, flatulence, nausea, diarrhoea, vomiting and headache , 140. SiMethods to minimise/avoid the undesired side-effects associated with sodium bicarbonate ingestion have been investigated. One previous study  investigSodium citrate is often considered an attractive alternative to sodium bicarbonate due to the perceived lower risk and intensity of side-effects , 157. ItThe side-effects associated with sodium bicarbonate and sodium citrate appear similar in symptoms, incidence and intensity. Nonetheless, comparison between studies is somewhat limited by the lack of a standardised method of obtaining and reporting these symptoms. Furthermore, although many reported side-effects are unlikely or less likely to substantially influence exercise performance (e.g. burping or flatulence), others may be detrimental to performance (e.g. diarrhoea or vomiting). Further, some symptoms are likely to be more problematic in some sports (e.g. running events) than others (e.g. cycling), and in some cases, athletes might well be willing to experience some of these symptoms, particularly if they are mild and if it means performance gains. The intensity of these symptoms and moment at which they are reported in relation to exercise are also key factors. Further research should strive to determine whether the combination of strategies  that minimises symptoms leads to further reductions in their number and intensity. Athletes should experiment with these supplements throughout training and adapt the dose and timing of these supplements accordingly based upon recommendations  can be used to inform any modifications to supplement dose (e.g. decreases within the evidence-based range) and timing . Additional strategies  may provide additional benefit [We recommend that when athletes are deemed suitable to undertake trials of the use of buffering agents, this work is undertaken in conjunction with performance support practitioners (e.g. sport scientists and sports dietitians). Activities should focus on key modifying factors that can impact the efficacy of buffering agents (supplement dose and timing), with these first being performed under resting conditions to avoid the additional variables associated with training sessions. Evidence-based supplement dose and timing should be used Fig. , support benefit , 24.This phase provides an opportunity to implement trials of buffering agents within training sessions that provide event-specific characteristics likely to benefit from manipulation of acid–base balance  , 3, 5, 7Additional refinement of athletes’ buffering practices can be achieved by the introduction of buffering agents  during minor competitions in the lead-up to the major events [In this narrative review, we have evaluated the current evidence for the use of buffering agents in specific populations , extreme environments  and in combined supplementation protocols . We have also considered the moderating factors used in research studies , factors specific to the translation of findings to real-world performance  and other factors with a high level of specificity to athletes’ performance (e.g. recruiting highly trained or elite-level athletes as research participants). Within these contexts, the current evidence of performance improvements from the use of sodium bicarbonate, sodium citrate and beta-alanine in highly trained or elite athletes is limited; indeed, only 11/58 of the included studies (19%) reported a performance benefit. This outcome, however, may have been adversely impacted by some of the limitations within the existing literature . We have highlighted priorities for future research, including (i) buffering studies in highly trained females, which include quantification of MC factors, (ii) buffering studies conducted at altitude with performance tests simulating real-world competitive events, and (iii) the use of various combinations of buffering agents by highly trained and elite research participants. When integrating the use of buffering agents into athletes’ preparation for major events, potential strategies include identifying opportunities for co-supplementation with other evidence-based supplements, reducing the likelihood of side-effects, and optimising key moderating factors—supplement dose and timing, and exercise duration and intensity– to develop evidence-based buffering protocols for individual athletes.


Schizophrenia affects people worldwide. In Europe, the advantages of second-generation and long-acting injectable antipsychotics (SG-LAIs) are known and used, supported by scientific evidence. However, somewhere there is limited evidence on this topic.Highlight the improvements in antipsychotic treatment and raise awareness of the scar between Europe and Africa, showing the results of the evidence with a case of cooperation with Cameroon.About a case of a 42-year-old Cameroonian woman with 25 years of schizophrenia, treated with first-generation antipsychotic polypharmacy (APP) oral and depot, with several psychotic relapses, disorganized behaviors, motor and cognitive impairment and isolation (telemedicine consultation received through a NGO platform).A search on PubMed was performed, selecting two systematic reviews including “antipsychotic” AND “Africa”, one systematic review for SGAs and four reviews for LAIs.Seven articles were reviewed, finding that APP use is highly prevalent in Africa with a lack of research on this, SGAs show an improved safety and tolerability profile and LAIs are among the most effective treatments in psychiatry improving adherence and overall patient outcomes.In our case, we recommend progressively adjusting treatment to SG-LAI monotherapy, visiting the patient six months later in Cameroon, observing sustained stability of positive symptoms with an improvement of negative symptoms and good adherence and tolerability to treatment without extrapyramidal effects.Our case is an example of the evidence that supports the improvement that SG-LAIs represent in psychiatric treatment and how international cooperation can help bridge the gap between Africa and Europe. Nevertheless, more research is needed to build bridges.None Declared


GJB2, encoding connexin-26, are a major cause of NSHI (the DFNB1 type), but few other genes have significant epidemiological contributions. Mutations in the STRC gene result in the DFNB16 type of autosomal recessive NSHI, a common cause of moderate hearing loss. STRC is located in a tandem duplicated region that includes the STRCP1 pseudogene, and so it is prone to rearrangements causing structural variations. Firstly, we screened a cohort of 122 Spanish familial cases of non-DFNB1 NSHI with at least two affected siblings and unaffected parents, and with different degrees of hearing loss (mild to profound). Secondly, we screened a cohort of 64 Spanish sporadic non-DFNB1 cases, and a cohort of 35 Argentinean non-DFNB1 cases, all of them with moderate hearing loss. Amplification of marker D15S784, massively parallel DNA sequencing, multiplex ligation-dependent probe amplification and long-range gene-specific PCR followed by Sanger sequencing were used to search and confirm single-nucleotide variants (SNVs) and deletions involving STRC. Causative variants were found in 13 Spanish familial cases (10.7%), 5 Spanish simplex cases (7.8%) and 2 Argentinean cases (5.7%). In all, 34 deleted alleles and 6 SNVs, 5 of which are novel. All affected subjects had moderate hearing impairment. Our results further support this strong genotype–phenotype correlation and highlight the significant contribution of STRC mutations to moderate NSHI in the Spanish population.Non-syndromic hearing impairment (NSHI) is a very heterogeneous genetic condition, involving over 130 genes. Mutations in  Hearing impairment exerts a strong impact on the lives of the affected subjects. In developed countries, the majority of cases have a genetic etiology . Non-synGJB2, encoding connexin-26, are a major cause of NSHI . Few other genes have significant epidemiological contributions. Remarkably, mutations in the STRC gene, which result in the DFNB16 type of autosomal recessive NSHI . Thi. ThiSTRCSTRC variants in 20 unrelated cases, all of whom exhibited moderate hearing impairment. Our results further support this solid genotype–phenotype correlation and the significant contribution of STRC pathogenic variants to moderate NSHI in all studied populations.In this work, we have used these diverse experimental approaches to analyze several cohorts of hearing-impaired subjects from Spain and Argentina. We identified causative GJB2 gene (coding region and splice sites) by Sanger sequencing, as well as testing for the common del(GJB6-D13S1830) and del(GJB6-D13S1854) deletions in the DFNB1 locus, revealed causative genetic variants in 99 families. The remaining 122 families were investigated in this study. In this cohort, the degree of hearing loss was profound in 25 cases, severe in 18 cases, moderate in 75 cases and mild in 4 cases. We enrolled a cohort of 221 Spanish unrelated familial cases of autosomal recessive NSHI. All cases in this cohort had at least two affected siblings with unaffected parents. Before this work, a preliminary screening of the Subsequently, we also included in this study a cohort of 64 Spanish simplex non-DFNB1 cases, and a cohort of 35 Argentinean non-DFNB1 cases . All affected subjects in these two cohorts had moderate hearing loss. A diagram of the study setup is shown in Hearing was evaluated by pure-tone audiometry, testing for air conduction (frequencies 250–8000 Hz) and bone conduction (frequencies 250–4000 Hz). Hearing impairment was classified as mild (21–40 dB HL), moderate (41–70 dB HL), severe (71–95 dB HL) and profound (>95 dB HL), according to the pure tone average (PTA) threshold levels at 0.5, 1, 2 and 4 kHz.DNA was extracted from peripheral blood samples by using the Chemagic Magnetic Separation Module I automated system .Microsatellite markers D15S780, D15S778, D15S1039 and D15S126 were amplified using fluorescently labeled primers and PCR conditions as previously reported . AmplifiGJB6: upper primer 5′- CGTCTTTGGGGGTGTTGCTT-3′, lower primer 5′-CATGAAGAGGGCGTACAAGTTAGAA-3′). Two bands are expected in normal individuals: a 168 bp band corresponding to the D15S784 marker  and a 333 bp band corresponding to exon 4 of GJB6.Amplification of marker D15S784  was performed through a multiplex PCR that includes an amplification control  assay is reported in STRC, and for its neighboring genes  was used to detect copy number variations in the DFNB16 locus. It includes probes for exons 19, 20, 23, 24, 25 and 28 of ng genes .STRC-specific amplification, we developed two long-range PCR assays that generate two overlapping amplicons that cover the complete STRC sequence . To achieve specificity, primers were located on sequence stretches containing unique SNPs within the gene  , as implSTRC on 15q15.3 (order of markers: cen-D15S780-D15S778-STRC-D15S1039-D15S126-tel). In 13 of the 37 families, haplotype analysis could not exclude genetic linkage to DFNB16 . In patients with deletion of both alleles of STRC, only the small band corresponding to the pseudogene is obtained (STRC.Since the D15S784 marker is not located within obtained . This teCKMT1B (1 probe), STRC (7 probes) and CATSPER2 (5 probes) detected zero copies, whereas the probe on PPIP5K1 and the probe on CKMT1A detected two copies  was performed on probands from the 13 families in which genetic linkage to DFNB16 had not been excluded, to confirm the deletions in the probands from families HRC1 and HRC3-HRC6, to investigate hypothetical heterozygous deletions in the remaining cases, and to study the segregation of the deletions in parents and unaffected siblings. All biallelic deletions were confirmed in the affected subjects, their parents being heterozygous. Moreover, a heterozygous deletion was detected in the proband of family HRC2, which he had inherited from his father . In the o copies .STRC and STRCP1 for exons 1–15, it is not possible to screen the gene for SNVs through sequencing exon by exon directly from genomic DNA. To circumvent this problem, we developed two gene-specific PCR assays, which generate two overlapping amplicons that cover the complete STRC sequence . Sanger sequencing was performed exon by exon on these two amplicons from the proband of family HRC2. This approach revealed the novel c.4483_4484del, p.(Phe1495Cysfs*9) variant in exon 23 of STRC . In familial case IHT1, a heterozygous deletion encompassing only STRC was found. Further analysis through Sanger sequencing on the two STRC-specific amplicons revealed a novel nonsense variant, c.1030C > T, p.(Arg344*) in exon 4. The two affected subjects in the family were compound heterozygous for this pathogenic SNV and the deletion.In parallel, a cohort of 35 unrelated Argentinean non-DFNB1 cases  with moderate NSHI was screened for deletions in the DFNB16 locus, through the PCR-based D15S784 test, followed by MLPA. In simplex case IHT2, both alleles of STRC-specific Sanger sequencing , and a cohort of 64 Spanish simplex cases with moderate NSHI. Sequence variants identified through the gene panel were confirmed and their segregation was studied using MLPA and quencing . We eluc in STRC . In all Three of these four SNVs were novel. These included two nonsense variants (p.(Glu1275*) and p.Gly1366*), in exons 19 and 20, respectively) and two missense variants: p.Pro1520Arg) in exon 24, previously reported , and the66*, in e20Arg in STRC. All of them present with moderate hearing impairment , which generate STRC-specific templates on which Sanger sequencing can be performed exon by exon. Even in these times of massively parallel sequencing, it is still needed to confirm whether the sequence variants that are found are really located in the gene and not in the pseudogene. In fact, many SNVs in the literature have not been confirmed to be STRC-specific [Since the discovery of its involvement in autosomal recessive NSHI in 2001 , screeniquencing ,17. Diffquencing ,19,20,21specific . STRC variants, DFNB16 has been considered a major cause of NSHI, being second in frequency only to DFNB1 in most tested populations [STRC, two figures that are very close to that of the meta-analysis. These results confirm DFNB16 as a major contributor to NSHI also in the Spanish population. In Argentinean cases, 2 out of 35 cases (5.7%) carried biallelic pathogenic variants in STRC. This apparently lower frequency is likely a consequence of the cohort that was tested, smaller in size and predominantly composed of simplex cases (32 out of 35). In fact, one of the positive cases was familial. Anyway, comparison of the prevalence of the diverse types of NSHI across different populations is hampered by the variety of methodological approaches that are used for the screenings, and by the heterogeneity in size and composition of the cohorts  [In several studies on large cohorts of hearing-impaired subjects who were investigated specifically for ulations ,22,23,24ulations , and refSTRC gene, makes it prone to structural rearrangements, that are thought to be generated by non-allelic homologous recombination. Consequently, the most frequent type of pathogenic variants are deletions, which may account for about 86% of the mutant alleles [CKMT1B, STRC and CATSPER2 . In another study, 77% of the deletions belonged to this type [STRC and CATSPER2 results in the deafness and male infertility syndrome. Eleven hearing impaired subjects of our cohort who carry biallelic type 1 deletions are males, a fact to be considered when providing genetic counseling and clinical follow-up.The tandemly duplicated structure of the region on 15q15.3 that contains, among others, the  alleles . In our his type . This isAlthough we have grouped in a same type all deletions lacking the same MLPA probes, it should be noted that it does not mean that they are identical and patients are homozygous. The recombination events that gave rise to each deletion could have taken place in any position within the duplicated region, as it is likely that they occur recurrently. In fact, our analysis on families HRC1-HRC6 with type 1 deletions show that all of them are associated with different haplotypes . In a stSTRC pathogenic variants of our study from whom we could obtain audiological data fit within this model (The DFNB16 hearing impairment is considered to be moderate, with a characteristic slightly down-sloping profile in the audiogram ,25,26,27is model . In someis model . Cases wFinally, two recent studies have revealed that some DFNB16 hearing-impaired subjects present with recurrent benign paroxysmal positional vertigo ,29. In oSTRC gene lies poses special problems to molecular testing. Stratifying the patients according to the different genetic etiologies of NSHI is the first step towards the application of specific treatments, such as gene therapy, which are being investigated and will be implemented in a near future [The tools and screening strategies we have developed and used in this study are of help to the routine diagnosis of DFNB16 NSHI, as the region where the r future .


Aloe species are widespread and diverse in African ecosystems, and this commonly correlates to their habitual use as reservoirs of herbal medicine. The side effects associated with chemotherapy and the development of antimicrobial resistance to empirically used antimicrobial drugs are substantial, paving the way for novel phytotherapeutic approaches. This comprehensive study aimed to evaluate and present Aloe secundiflora (A. secundiflora) as a compelling alternative with potential benefits in colorectal cancer (CRC) treatment. Important databases were systematically searched for relevant literature, and out of a large collection of 6421 titles and abstracts, only 68 full-text articles met the inclusion criteria. A. secundiflora possesses an abundant presence of bioactive phytoconstituents in the leaves and roots, including anthraquinones, naphthoquinones, phenols, alkaloids, saponins, tannins, and flavonoids, among others. These metabolites have proven diverse efficacy in inhibiting cancer growth. The presence of innumerable biomolecules in A. secundiflora signifies the beneficial effects of incorporating the plant as a potential anti-CRC agent. Nonetheless, we recommend further research to determine the optimal concentrations necessary to elicit beneficial effects in the management of CRC. Furthermore, they should be investigated as potential raw ingredients for making conventional medications. Aloe species are abundant and diverse in African landscapes, and this frequently translates to their regular use as a source of phytotherapeutic medicine to support human health and wellbeing. They have also occasionally been mentioned as having utility in ethnoveterinary medicine by African ethnic groups ..58].Flavonoids have been found to have a wide range of anticancer actions, including modulating the activity of enzymes that scavenge reactive oxygen species (ROS), participating in cell-cycle arrest, inducing apoptosis and autophagy, and reducing the proliferation and invasiveness of cancer cells . In termDue to the presence of phenolic hydroxyl groups, flavonoids have the capacity to stabilize free radicals and can directly scavenge ROS and chelate metal ions . The actA. secundiflora has similar pharmacotherapeutic characteristics to other species of the same genus, Aloe. They can thus be utilized for potential intervention and management of CRC in predetermined concentrations based on the extensive documented evidence on the pharmacological activity of the plant genus Aloe. We advise additional research to establish the proper concentrations and levels required to evoke positive effects in CRC management in humans, owing to the lack of proven amounts and specified concentration measurements.It is crucial to understand that A. secundiflora contains similar pharmacotherapeutic characteristics to other identified species of the genus Aloe. It can thus be utilized for potential intervention and management of CRC in predetermined concentrations based on the extensive documented evidence on the pharmacological activity of the plant genus. The presence of innumerable biomolecules in A. secundiflora signifies the usefulness of incorporating this plant as a potential anti-CRC agent with certain phytotherapeutic effects. Despite the positive effects recorded in ethnoveterinary applications of A. secundiflora—and particularly in chickens—this is the first unique review detailing its potential application in human health, with specific emphasis on colorectal cancer. This is of course due to the abundant presence of bioactive phytoconstituents common in the leaves and roots of the perennial, evergreen, and succulent plants.The wellbeing of mankind is significantly impacted by the molecules in natural products, which have an evolutionary structure. Numerous secondary metabolites are synthesized by the biosynthetic machinery of nature, and each of them exhibits unique biological characteristics that make it desirable to use them as pharmaceutical structural models or as health products. It is crucial to understand that A. secundiflora for clinical use, due to the lack of established quantities and concentration measurements, we recommend further research to determine the minimal or optimal concentrations/levels necessary to elicit beneficial effects in CRC management in humans, whether individually or in combination. Additionally, this important plant may be investigated as a potential source of raw ingredients for making conventional medications.Nonetheless, before recommending


PIK3CA mutations, low basal pERK, and a group of differentially expressed genes (DEGs). The combination of DIACC3010 and trastuzumab was evaluated in Her2-positive cell lines and models. Potential biomarkers for the synergistic efficacy of the combination of DIACC3010 + trastuzumab also included DEGs as well as a lack of up-regulation of pERK. Of 27 GC patient-derived xenograft (PDX) models tested in BALB/c nu/nu mice, 59% were sensitive to DIACC3010 + trastuzumab. Of the 21 HER2-negative PDX models, DIACC3010 significantly inhibited the growth of 38%. Altogether, these results provide a path forward to validate the potential biomarkers of DIACC3010 sensitivity in GC and support clinical evaluation of DIACC3010 monotherapy and combination with trastuzumab in patients with HER2- negative and positive advanced GCs, respectively.The PI3K-Akt-mTOR (PAM) pathway is implicated in tumor progression in many tumor types, including metastatic gastric cancer (GC). The initial promise of PAM inhibitors has been unrealized in the clinic, presumably due, in part, to the up-regulation of Akt signaling that occurs when the pathway is inhibited. Here we present that DIACC3010 (formerly M2698), an inhibitor of two nodes in the PAM pathway, p70S6K and Akt 1/3, blocks the pathway in in vitro and in vivo preclinical models of GC while providing a mechanism that inhibits signaling from subsequent Akt up-regulation. Utilizing GC cell lines and xenograft models, we identified potential markers of DIACC3010-sensitivity in Her2-negative tumors, i.e.,  For pre-metastatic, early-stage GCs, the current standards of care (surgery and chemotherapy) provide relatively good outcomes . Refined chemotherapeutic treatments and the approval of targeted therapies, such as trastuzumab, ramucirumab3, pembrolizumab, and nivolumab5, have helped to improve GC patients’ outcomes. However, patients with late-stage, metastatic disease, which represents most cases due to its relatively late detection, still have poor prognoses2 with global 5-year survival rates below 20%6. Innate or acquired resistance is also a significant issue, reducing effectiveness of approved therapies in this disease. Undoubtedly, better treatment options are needed for GC patients.Gastric cancer (GC) is the fifth most common malignancy and the third leading cause of cancer death worldwide7. The mTOR inhibitor everolimus showed promising results in a Phase II clinical trial8 but failed to provide significantly better outcomes than best supportive care in the GRANITE-1 Phase III trial9. Akt is another promising target for GC given the relatively high rate (78%)- of Akt activation in this disease10. However, neither pan-Akt inhibitors ipatasertib11 or MK-220612 had significant activity in Phase II clinical trials of patients with GC.PI3K/Akt/mTOR (PAM) pathway components have been explored as targets for GC, due to its involvement in both the origin and progression of GC13. A drug that overcomes these toxicities, while still blocking the PAM pathway, would be a promising candidate for GC treatment. DIACC3010 (formerly M2698) is a selective ATP-competitive dual inhibitor of p70S6K and Akt1/3, which can overcome the compensatory Akt feedback loop induced by PAM pathway inhibition, while avoiding the toxic effects of pan-Akt inhibition15. In a Phase I study (NCT01971515) in patients with advanced solid tumors, DIACC3010 was well tolerated with evidence of clinical benefit, most notably in patients whose tumors lacked confounding tumor alterations  but harbored PAM pathway alterations16. It is noteworthy that no limiting hyperglycemia was observed in this clinical trial with DIACC3010 in contrast to that seen with other pan Akt inhibitors17.The unrealized potential of PAM pathway inhibitors may be due, in part, to toxicity concerns and the potential hyperactivation of Akt isoforms occurring upon PAM pathway blockageIn addition, given that trastuzumab is an approved drug for treating HER2+ GC and PAM pathway activation has been described as a marker of resistance to anti-HER2 therapy in GC, any investigation of the potential of DIACC3010 as a GC treatment should also include combination with trastuzumab.PIK3CA mutations18 and pERK expression19) and potential biomarkers of sensitivity and resistance to PAM pathway inhibition. Altogether, these results support further clinical evaluation of DIACC3010 in patients with advanced GCs, both alone in HER2-negative GC and in combination with anti-HER2 treatment in HER2-amplified GC.Therefore, we characterized the potential of DIACC3010 to treat human GC by employing both in vitro and in vivo preclinical models and investigated how the preclinical efficacy of DIACC3010, alone and combined with trastuzumab, correlated with known ; GCIY, GSU, HGC-27, KE-39, MKN-7, and SH-10-TC (Riken Bioresources Center); NCI-N87 (American Type Culture Collection); OE-19 (European Collection of Authenticated Cell Cultures); and 44As3 .2 at 37 °C. The growth-inhibitory effects of DIACC3010, trastuzumab , and the combination of these single agents were examined using a tetrazolium salt-based proliferation assay . Each cell line was seeded into 96-well plates (1.5 × 103 cells/100 μL/well), incubated for 24 h (h) at 37 °C and then treated with DIACC3010 (0–10 μM) alone or with trastuzumab (0–10 μg/mL) for 72 h. Medium was removed and replaced with WST-8 solution (10 μL) and fresh medium (90 μL), and the plates were incubated for 2 h at 37 °C.Cells were maintained in the medium recommended by suppliers, supplemented with 10% fetal bovine serum (Cell Culture Technologies), 100 units/mL penicillin, 100 μg/mL streptomycin, and 25 μg/mL amphotericin B (Sigma-Aldrich) in a humidified atmosphere containing 5% CO50) was determined from dose–response plots. Mean values and variances for each cell line were calculated for cell viability assays. Negative values shown in the dose–response plots indicate a decrease in cell count from baseline . Bliss independence models were used to examine effects of DIACC3010 + trastuzumab. The Bliss expectation was calculated as (A + B) − A × B, where A and B were the fractional growth inhibitions induced by agents A and B at a given dose. Bliss sums were calculated for each cell line from the Bliss excess scores 20.Growth-inhibitory effects were assessed spectrophotometrically with a SpectraMax 190 (Molecular Devices), and 50% growth inhibition (GI6 cells/well) were treated in vitro with DIACC3010 (0 or 1 μM) for 2, 6, and 24 h to assess DIACC3010 monotherapy effects on chosen biomarkers. For the combination studies, OE-19 and NCI-N87 cells were treated with DIACC3010 (0 or 1 μM) and/or trastuzumab (0 or 10 μg/mL) for 24 h.Of the 13 GC cell lines, 7 were further investigated because of their potential use in xenograft models . These 7 cell lines , pHER2 , pan-Akt (#4685), PTEN (#9188), PRAS40 (#2691), pPRAS40 , p70S6K (#2708), pP70S6K , S6 (#2217), pS6 , pAkt , ERK1/2 (#4695) and pERK1/2 . Secondary antibodies were HRP conjugated donkey anti-mouse IgG (#715-036-150) and donkey anti-rabbit IgG (#711-036-152), purchased from Jackson Immuno Research.1221/1222) (1:200), Akt (1:200), PTEN (1:500), PRAS40 (1:500), phosphorylated-PRAS40 (1:500), P70S6 kinase (1:500), phosphorylated-P70S6 kinase (1:500), S6 (1:500), phosphorylated-S6 (1:500), phosphorylated-Akt (1:400), ERK1/2 (1:200) and phosphorylated-ERK1/2 (1:400) as indicated. Subsequently, membranes were washed with a tris-buffered saline containing 0.1% Tween 20, and proteins were visualized using ECL prime substrate . Western blots were imaged using the ChemiDoc XRS Plus System (Bio-Rad) and band images were quantified by Image J (https://imagej.nih.gov/ij). The exposure time for detecting the signal in individual western blots differed among the cell lines evaluated and was determined at the optimum exposure time, given that the intensity of the baseline signal of each marker varied among the cell lines.Cells were lysed in RIPA buffer (Wako) and equal amounts of extracted protein (2 μg) were separated by SDS-PAGE and transferred to PVDF membranes using a Trans-Blot Turbo transfer machine (Bio-Rad). Immunostaining of the membranes was performed using the iBind western System (Life Technology) according to the manufacturer's protocol. Primary antibodies and secondary antibodies were diluted to appropriate concentrations in iBind buffer. Antibodies were used at the following dilutions: against HER2 (1:500), phosphorylated HER2 (Tyr3 cells/well). After 24 h, the medium was changed for wells containing DIACC3010  and/or trastuzumab (1 µg/mL) and the cells were incubated in a humidified chamber (at 37 °C) for 10 days. Cells were washed with PBS, fixed with 4% paraformaldehyde, and stained with crystal violet in water for at least 2 h at room temperature before imaging. The area covered by the colonies was quantified with ColonyArea, as described by Guzmán et al.21.Cell survival was determined by inoculating 6-well plates with different GC cell lines (1.5–3 × 10DNA was extracted from cell lines using the QIAamp DNA Mini Kit (QIAGEN) and stored at − 20 °C. Mutational hotspots in 48 cancer related genes were characterized using the TruSeq Amplicon Cancer Panel  cells suspended in 0.1 or 0.2 mL PBS, respectively, were injected subcutaneously into the right flanks of 5-week-old BALB/c nu/nu mice. BALB/c nu/nu mice were obtained from Charles River Laboratories, Japan. Mice were maintained under specific pathogen-free conditions and provided with standard feed and sterilized water. Mice were monitored daily, and tumor volume (TV) was measured once or twice weekly using calipers; TVs were calculated as length/2 × width2. Endpoints calculated at the end of each study were tumor/control (T/C) and tumor growth inhibition (TGI); t/TVΔc)], where t is treated and c is control.HGC-27 and OE-19 . Mice with HGC-27 tumors were treated orally (po) with 10, 20, or 30 mg/kg DIACC3010 daily (QD) × 14 (10 mL/kg) and mice with OE-19 tumors were treated with DIACC3010 30 mg/kg po QD × 21, trastuzumab 15 mg/kg/week by intravenous injection (QW i.v.), or DIACC3010 + trastuzumab. Control mice were treated with the vehicle for DIACC3010 QD po .When TVs reached approximately 200 mm3). For DIACC3010 monotherapy, mice with HGC-27 tumors were treated with vehicle or DIACC3010 in vehicle at 20 mg/kg po QD × 4. Mice bearing OE-19 tumors were treated with DIACC3010 (30 mg/kg po QD × 4) and trastuzumab in PBS (15 mg/kg i.v. × 1). Tumor samples were collected upon euthanasia at 4 and 24 h after the final DIACC3010 treatment (n = 3/time point).In a separate group of mice implanted with the same tumor cohort, HGC-27 and OE-19 tumors were allowed to grow for 14 days before mice were assigned to treatment groups (average TV of approximately 300–400 mmTwenty-seven patient-derived xenograft (PDX) models  of GC were used. They were derived from male and female Chinese patients (28–75 years of age) with either poorly or moderately differentiated gastric adenocarcinoma, except for one patient who was classified as having a Signet ring cell carcinoma , trastuzumab (15 mg/kg QW i.v.)22, or DIACC3010 + trastuzumab. TVs and body weights . The other half was cut into halves again and snap frozen in liquid nitrogen and stored at − 80 °C. HER2 status of the PDX models was assessed in FFPE mounted sections by immunohistochemistry , and scored from 0 to 3+. Models that scored 2+ or 3+ were investigated for HER2 amplification FISH, using the PathVysion HER2 DNA Probe Kit (Abbott Laboratories).Tumors from naïve, untreated mice of each PDX model were dissected when they had reached 400–600 mmg for 30 min at 4 °C. The supernatants were transferred again to new 1.5 mL centrifuge tubes, 1 µl benzonase  was added to each tube, and all tubes were sonicated for 20 s in a sonicating 37 °C water bath. An additional centrifugation was performed at 14,000g for 15 min at 4 °C, and lysates were transferred again to 1.5 mL centrifuge tubes and protein concentrations were measured by Pierce BCA assay (Thermo Fisher) according to manufacturer’s instructions, and each sample was diluted to a standard concentration of 0.5 mg/ mL. Lysates were then aliquoted (10 µl per well) into multiple 96-well plates and stored at − 20 °C until analysis.Phosphorylated (p) and total (t) ERK protein was analyzed in snap frozen PDX tumor fragments from untreated animals. Tumor pieces were placed in individual Percellys 2 mL homogenizing tubes containing ceramic beads  with 500 µl 10 × RIPA buffer  diluted to 1 × with water supplemented with phosphatase and protease inhibitors , Pierce Protease Inhibitor Mini Tablets , and phenylmethanesulfonyl fluoride  at a working concentration of 100 µg/mL from a stock solution of 2.5 mg/mL dissolved in isopropyl alcohol. Tubes were placed in a Percellys 24 Bead Mill Homogenizer located within a 4°C cold room. Tumor fragments were homogenized 3 times for 15 s each at 5000 rpm followed by a 30 s pause and another homogenization 3 times for 15 s each at 5000 rpm. The lysates were transferred to 1.5 mL centrifuge tubes and centrifuged at 14,000Immunomodulatory analysis was performed following the ProteinSimple  Jess 2 Immunoassays RePlex Module protocol provided with the EZ standard pack 1 of 12-230 kDa kit. Tumor lysates were probed with pERK1/2 antibody  and tERK1/2 antibody , according to manufacturer’s protocol, at a dilution of 1:1000 per well. Data analysis was performed using the Compass for SW software version 5.0. Using the high dynamic range detection profile exposure, area results for pERK were divided by area results for tERK to yield a corrected area.For mutational status, genomic DNA was isolated from snap frozen tumor fragments from untreated xenograft mice using the AllPrep DNA/RNA FFPE Kit (Qiagen); 20 to 27 mg of tissue was used as input for the DNA isolation. The genomic DNA concentrations were quantified using the fluorescent dye-based QubitTM double stranded DNA HS Assay Kit (Thermo Fisher Scientific). Library preparation from 30 ng DNA per sample was performed with the Illumina AmpliSeq® Cancer HotSpot Panel v2 . Purified RNA was quantified using a Qubit® 3.0 Fluorometer (Thermo Fisher Scientific). Gene expression was analyzed in 50 ng input RNA using the PanCancer IO360 Panel (nanoString IO 360 V1.0); raw counts were corrected by subtracting the mean + 2 standard deviations (SDs) from negative controls and then normalizing against the geometric means of both positive controls and reference genes. Gene expression amplification was evaluated using the multiplex digital nCounter® platform and analyzed with nSolver v4.0 software . The lower limit of detection was set at 20 counts, therefore transcripts where more than 95% of samples had counts under 20 were excluded from subsequent analyses.Gene expression normalization was performed relative to housekeeping genes using the GeoMean algorithm. Of the 20 housekeeping genes on the panel, those with the lowest standard deviations after normalization were used (SD < 0.45), employing the geNorm algorithm for each comparison. Mean squared error analysis was used to identify potential samples of low data quality; no samples were removed. All normalized data was then transformed on a log2 scale to undergo further analysis.All animal studies using cell line xenograft models were approved by the Committee for Animal Experimentation of the National Cancer Center, Japan . These guidelines meet the ethical standards required by law and comply with the guidelines for the use of experimental animals in Japan.The PDX models were previously established at ChemPartner. At the time of model establishment tumor tissues were obtained in hospitals from patients with informed consent, in accordance with protocols approved by the hospital and ChemPartner Institutional Ethics Committees. PDX studies were conducted at ChemPartner in compliance with the guidelines for the care and use of animals established by the Institutional Animal Care and Use Committee at EMD Serono Research and Development Institute, Inc., Billerica, MA, USA, an affiliate of Merck KGaA.All in vivo experiments were carried out in accordance with ARRIVE guidelines .: Statistical significance was analyzed for R and p values between GI50 values of DIACC3010 and phosphor-protein biomarker expression in the 13 cell lines by linear regression. In colony forming assay (CFA), p values were calculated based on one-way analysis of variance (ANOVA) with Tukey HSD (honestly significant difference).In vitroIn vivo: In the efficacy and pharmacodynamic (PD) biomarker studies, statistical significance was analyzed using ANOVA followed by Tukey’s post-hoc pairwise comparisons for the OE-19 xenograft or Dunnett’s pairwise comparisons with controls for the HGC-27 tumor model. TV data from PDX models were log-transformed and analyzed by Repeated Measures Analysis of Covariance (RM-ANCOVA) with the starting tumor volume as the covariate, followed by Tukey’s post-hoc pairwise comparisons.For statistical analysis of pERK/tERK protein, models were grouped according to treatment response, i.e., statistically significant treatment effects compared to vehicle. Group A: models that responded equally to DIACC3010 and its combination with trastuzumab; Group B: models that responded equally to trastuzumab and its combination with DIACC3010; Group C: models that responded equally to DIACC3010, trastuzumab, and the combination; Group D: models that only responded to the combination of DIACC3010 and trastuzumab; and Group E: models that did not respond to any of the treatments. Data from Group A were compared to those from combined Group B-D by unpaired t-test with Welch’s two-tailed correction.The aforementioned statistical analyses were performed using IBM® SPSS® Statistics version 21, R version 2.1.0, or GraphPad PRISM version 9.1.2. Significance of α = 0.05 was used for all tests except PDX tumor volume data from efficacy studies, where α = 0.10.p value  q-value) threshold of 0.05 was used for statistical significance.All statistical analyses of nanoString data were performed on log2 transformed normalized counts. Differential expression analyses were carried out using nSolver4.0 and Advanced Analysis package 2.0 to determine differentially abundant transcripts with a preset threshold of statistical significance. To control for multiple testing, a corrected for multiple comparisons using Benjamini–Yekutieli FDR adjusted PIK3CA mutations and additional pathologic mutations  values of 84 and 160 nM, respectively  levels. Given that both MKN-1 and KE39 were resistant to DIACC3010, the presence of the putative sensitivity marker PIK3CAmut along with the absence of the potential resistance marker pERK may render GC cells susceptible to growth inhibition by DIACC3010.The basal levels of potential resistance markers were next examined through protein expression in the cells at steady-state via western blot analysis Fig. a,b. The 50 values showed that expression of the markers examined at steady state, pERK/ERK, pPRAS40 (Thr246)/PRAS40 , and pS6 (Ser240/244)/S6 (a substrate for p70S6K), did not correlate with the sensitivity of the cells to DIACC3010-mediated growth inhibition  resulted in significantly smaller tumors compared with vehicle control treatment, (p = 0.14), there appears to have been activation of the Akt feedback loop, which was confirmed when pAkt was normalized to β-actin (p = 0.04). Despite activation of pAkt, pPRAS40 was not increased, demonstrating the dual nature of DIACC3010 to inhibit the pathway at p70S6K and abrogate signaling through activated Akt. Neither pERK/β-actin nor pERK/total ERK ratios were affected by DIACC3010 , mid (MKN-7), and high  mean Bliss sums with varying levels of HER2 expression were tested in a colony forming assay (CFA) with DIACC3010 and trastuzumab. Overall, the CFA results supported the Bliss data; OE-19, but not NCI-N87, exhibited synergistic growth inhibition following treatment with DIACC3010 and trastuzumab Fig. .To investigate the signaling in OE-19 and NCI-N87 HER2+ lines that had disparate synergy results with the DIACC3010 + trastuzumab combination, the PD effects of DIACC3010 + trastuzumab combination were examined by western blot analysis Fig. a. In botp < 0.05; Fig. p > 0.05), thus confirming the synergy seen in vitro. We observed no notable effects of treatment on the body weight in any group  were sensitive to DIACC3010 monotherapy.We assessed the effects of DIACC3010 and trastuzumab monotherapies and combination therapy in relation to the HER2 and mutational status in 27 GC PDX models derived from Chinese patient tumors. In the PDX models, sensitivity to a given treatment was determined by statistically significant differences compared with control. Overall, 9 of the 27 PDX models (33%) were sensitive to DIACC3010 monotherapy, 9/27 were sensitive to trastuzumab monotherapy (33%), and 16/27 (59%) were sensitive to the combination Fig. . In the We looked at the relative sensitivities of the models to the different treatments and parsed the models into five groups. Four of the 27 PDX models  were equally sensitive to DIACC3010 monotherapy and the combination . This was a similar rate of sensitivity to DIACC3010 as was seen in the cell line models . Additionally, none of the four models that responded to DIACC3010 were ERBB2-amplified, as determined by FISH . However, with 25% (1/4) of the Group A PDX models and both of the DIACC3010-sensitive cell line models harboring PIK3CA mutations, we consider this a possible response marker in a subset of tumors.Of the models sensitive to DIACC3010 (Group A), two had tumor growth inhibition that could be described as stasis Fig. a, one ofHER2 amplification as determined by FISH (Supplementary Table 29. The frequency of HER2+ models (HER2 (3+) or positive by FISH) was 22% (6/27), which was consistent with clinical findings30. Of the six ERBB2-amplified PDX models, four were sensitive to trastuzumab (67%), whereas none of the models that were sensitive to DIACC3010 were ERBB2-amplified and we considered that amplified ERBB2 may be an exclusion marker for sensitivity to DIACC3010 in GC. Without a relatively high rate of response to DIACC3010 in the models that were not ERBB2 amplified, we conducted several analyses of the untreated PDX tumors in an attempt to identify potential predictive markers of sensitivity to DIACC3010. First, sensitivity of the 27 GC PDX models to treatment with DIACC3010, trastuzumab or the combination was compared with mutational variants within these models, identified using the Illumina oncogene hotspot sequencing panel , KRAS , and APC . These mutation frequencies are comparable to those observed in larger GC genomic databases . Similar to what was seen for the cell lines, sensitivity to the treatments, whether monotherapy or the combination, could not be linked to the status of these mutations in the models. Based on the data from the cell line models, we then examined pERK protein in the PDX tumors. Variability in the pERK/tERK ratio across samples was relatively high . Our main interest was whether we could identify potential predictive response markers for DIACC3010 (Group A) or for the combination (Group D), and whether we could identify potential resistance markers for the insensitive models (Group E). We only considered genes that were differentially expressed at Log2-fold or more and that were significant with an adjusted CXCL10, CXCL11, IL1A, IL16, S100A12, CLEC7A, VEGFC, WNT4). Two DEGs were detected in models sensitive to the DIACC3010 + trastuzumab combination in the Group D comparison , both being involved in extracellular matrix (ECM) and invasion. No significant DEGs were detected for the Group E non-responder comparison . Two DEGs were identified for both the Group B/trastuzumab-sensitive models  of DEGs, with models that were sensitive to DIACC3010 having a greater expression of genes involved in chemokine/cytokine and immune-related signaling and angiogenesis/cancer cell microenvironment  expression with higher disease control rate and prolonged progression-free survival, thus representing a candidate predictive biomarker for everolimus35. However, we did not observe that pS6 was a potential biomarker in our GC studies. The recommended doses of these drug candidates are also limited by safety issues, preventing the continuous use of high drug exposure.The promise of PAM pathway inhibitors in treating GC has as yet been unrealized. This is despite the involvement of the PAM pathway in GC disease and the reported preliminary activity of inhibitors both preclinically and in clinical trials36, treatment with pan-Akt inhibitor capivasertib + paclitaxel in patients with tumor expressing PIK3CA mutations resulted in 33.3% overall response rate (ORR). The choice of mutated PIK3CA as a potential marker for response to PAM pathway inhibitors is consistent with previous evidence from breast cancer37, GC40, and other tumor types41. In the current study, we detected PIK3CA mutations in three GC cell lines; the two that responded to DIACC3010 monotherapy harbored the E542K mutation. This E542K mutation was present in GC tumors from patients with the greatest response to pan-Akt inhibitor capivasertib in the VIKTORY trial36, whereas the more resistant cell line in our study carried the E545K mutation, which was associated with less of a response in the VIKTORY trial. We also detected the E545G PIK3CA mutation in one PDX model that responded to DIACC3010 monotherapy with tumor stasis. This low prevalence of mutant PIK3CA in the GC PDX panel in the present study  is close to the 7% seen in GC tumors in patients28. Based on these preclinical data, we consider E542K and E545G PIK3CA mutations and lack of HER2 amplification as promising biomarkers of sensitivity to DIACC3010 in GC, in addition to the relatively low basal pERK as mentioned above. Treating a larger set of preclinical HER2 negative PIK3CA mutated models with DIACC3010 may provide preclinical proof-of-concept for PIK3CA mutations as predictive biomarkers of sensitivity to this compound. In addition, the DEGs involved in chemokine/cytokine signaling for the Group A models may represent a potential multi-gene biomarker of sensitivity to DIACC3010.In more recent GC clinical trials, investigators have been incorporating biomarkers to stratify patients. In the VIKTORY umbrella GC trial42. In our study, the HER2-overexpressing GC cell lines NCI-N87 and OE‑19 were insensitive to DIACC3010 monotherapy, possibly due to DIACC3010-induced pERK elevation, whereas DIACC3010 + trastuzumab synergistically inhibited in vitro growth of OE-19 cells, colony formation, and in vivo growth of OE-19 tumors in mice. Consistent with the in vitro findings, DIACC3010 + trastuzumab reduced pS6 and pERK expression in vivo, showing effective dual PAM and MAPK pathway inhibition. In HER2+ NCI-N87 cells, synergy was not observed, and DIACC3010 + trastuzumab did not block up-regulation of pERK. In a previous report, the dual PI3K/mTOR inhibitor, BEZ235, had antitumor activity against HER2+ GC in PDX models by inhibiting HER2 signaling pathways, indicated by pAkt and pS6 inhibition43. In our study, synergy was not seen when the up-regulation of pERK by DIACC3010 in HER2-overexpressing cell line-derived tumors was not held in check by the combination with trastuzumab.Anti-HER2-based therapy is the current standard of care for HER2+ GC in the first-line setting in many regions, and primary and acquired resistance are significant challenges for patientsERBB2-amplified was 22% in the current study, which is similar to the 14–24%45 reported for other GC PDX models and within the range seen in patient tumors49. We did not test by FISH any GC PDX models rated as 0 or 1+ by HER2 IHC, which can have amplified ERBB2 in the clinic52. Overall, we feel that the PDX models represented patients at a level that allows translational conclusions.We also examined the combination in GC PDX models. The percentage of GC PDX that were 53, but some patterns did emerge. Based on our data, we propose five types of potential responses to DIACC3010 and trastuzumab in GC. One is GC tumors that do not respond to DIACC3010, trastuzumab, or to their combination. We saw this in roughly 40% of the PDX models tested in vivo, which could be generally characterized by a lack of HER2 amplification and, based on cell line data, relatively high pERK. Two additional groups would be GC tumors that respond to either DIACC3010 or trastuzumab monotherapies, as discussed above. A fourth group would be sensitive to both DIACC3010 and trastuzumab monotherapies but do not respond in an additive manner to the combination. In these cases, HER2 signaling in these tumors may be through the “normal,” i.e. non-activated, PAM pathway54 linearly, such that blocking either HER2 upstream with trastuzumab or the PAM pathway downstream with DIACC3010 generates similar results.It is not surprising that responses among the models were varied considering the heterogeneity of GC, both within the tumors and among the tumors/patients55. DIACC3010 had no effects on tumor growth in either model. Both were also resistant to trastuzumab monotherapy despite being HER2 (3+) and ERBB2-amplified56, and the DIACC3010 + trastuzumab combination caused significant regression in both xenografts which was accompanied by a reduction in pERK in OE-19 tumors. Synergy of the combination was quantified in vitro for the OE-19 cells with Bliss and CFA analyses, and based on the similar in vivo results, we surmise that the GAX007 response to the combination was synergistic as well. Thus, these models represent potential GC patients whose HER2 (3+), trastuzumab-resistant tumors may be sensitive to treatment with the combination such that DIACC3010 resolves the resistance to trastuzumab.The final group would be GC tumors that respond to the DIACC3010 + trastuzumab combination yet are resistant to either agent alone. This type of response was seen in three PDX models, but in two of them, the combination was not statistically different from the monotherapies as their effects on tumor growth were intermediate between those of the vehicle and the combination. The response seen in the GAX007 model, however, was similar to that of the OE-19 cell line xenograft. Both models were established from moderately differentiated adenocarcinomas58. Thirdly, Her2-targeted therapy in clinical practice is primarily adopted in combination with cytotoxic agents, whereas in the current study, the focus was on tumor sensitivity to targeted therapies without chemotherapy60. Lastly, tumor heterogeneity is thought to contribute to resistance to molecular-targeted agents in gastric cancer, which poses a challenge in translating preclinical models into the clinical setting61.Although this study included a variety of analyses, from in vitro cell line studies to in vivo PDX studies, there are several limitations in consideration of translation to clinical application. First, the cell lines used in our study were limited to thirteen, making it difficult to draw conclusions about the correlation between drug sensitivity and gene mutation or protein expression level. For example, there are only 3 GC cells lines with PIK3CA mutations in this study, so that number would need to be expanded in future studies to solidify conclusions about significance of the mutation. Secondly, although DIACC3010 is selective kinase inhibitor against p70S6K and Akt1/3, our study did not account for the differential contribution of Akt2 and Akt1/3 to drug sensitivity. It has been reported that inhibition of the PAM pathway with mTOR inhibitors induces feedback activation of different AKT isoforms in cell line specific manner in GI cancer models, suggesting the importance of considering Akt isoform differences when evaluating drug sensitivityPIK3CA mutations, and low basal pERK as potential predictive markers of sensitivity. An additional next step would be to combine DIACC3010 with trastuzumab in models of HER2 (3+)/trastuzumab-resistant GC and confirm potential response markers such as a reduction in pERK and DEGs. Our combination findings are expected to be applicable to recently approved fam-trastuzumab-deruxtecan-nxki as well. Overall, this study shows that an “all comers” approach resulted in 59% of the PDX models responding to DIACC3010 + trastuzumab, and 38% of the models for which trastuzumab treatment was not applicable responded to DIACC3010 alone, which supports the use of DIACC3010 to treat GC in the clinic.To conclude, we have confirmed the dual-inhibitory mechanism of action of DIACC3010 on the PAM pathway in GC and propose it should be further investigated in this disease. We have provided a foundation for a path forward to the clinic and suggest that next steps include a targeted approach that would include testing of DIACC3010 in additional models of HER2-negative GC in combination with standard of care chemotherapeutics and investigation of DEGs or signature, Supplementary Information.


Format for Formulary Submissions in response to the evolving health care landscape. During our 20-year history of developing dossiers, the 1 consistency we have observed is the struggle to achieve balance between efficacy and safety, evidence and aspiration, and access and cost. As Lee et al. (2016) describes,1 the primary stakeholders in the AMCP Format  often have different goals for using the Format.We applaud the AMCP Format Executive Committee for updating the AMCP Format makes great strides towards achieving this balance, providing the necessary elements for sound evidence-based formulary decisions while not being overly prescriptive. While the new Format may require some “rethinking” of the current Table of Contents, it will result in a succinct dossier that provides clarity for a new technology’s comparative clinical and economic value. We would like to call attention to 3 specific points with regards to the new Format.We feel that the new version of the Format should not be a data repository for all clinical data—the relevance of evidence for the HCDM audience should be weighed before inclusion. Version 4.0 has updated the page recommendations and added suggested maximum limits.2 Having heard from HCDMs that they are often overwhelmed with the data in the dossier and the review time required,3 we feel that this will help balance the needs of both groups. The page recommendations are such that the dossier length may range from 45 pages to 90 pages for an “average” new product with a single indication and typical FDA submission package. This range permits the manufacturer to provide any additional evidence needed to support a product’s value.First, the Format; however, specifics are left to the discretion of the manufacturer and should be informed by the product/technology, disease state, and other individualized elements. For example, pharmacoeconomic data (exclusive of the model report in Section 4.0) may be handled in several ways. A retrospective claims analysis of 2 therapeutic options in multiple sclerosis may demonstrate clinical impact (number of relapses) and economic impact  and thus be considered for either Section 3.0 or 5.0. While this is an operational issue, manufacturers should still carefully consider the type of evidence to include. Version 4.0 also states that manufacturers should provide justification or rationale for the selection of the evidence, from product comparators to clinical guidelines to clinical studies. This is the first time that the rationale for selection of evidence will be explicitly described in the dossier and should help ease concerns raised by HCDMs regarding bias or “cherry-picking” of data.Second, interpretation and consideration of which evidence should be provided will still be required of the manufacturer. Version 4.0 provides clarity on the evidence that HCDMs want to see and guidance as to inclusion in the 3 We hope that providing transparency into the thought process of the evidence presented will help to alleviate some of these concerns. The vast majority of manufacturers genuinely seek to provide the evidence that is needed and requested in order to support evidence-based formulary decision making.Third, in addition to fostering an open dialogue between manufacturers and HCDMs, the dossier should be received and reviewed with an open mind. We, too, have often heard the criticism that manufacturer-provided dossiers are “biased.”All of these changes move towards open and ongoing communication between manufacturers and HCDMs. As dossier producers, we often see the chasm that exists between the 2 disparate stakeholders. We welcome Version 4.0 as the bridge to cross this gap and look forward to its implementation.


Evidence-informed policymaking integrates the best available evidence on programme outcomes to guide decisions at all stages of the policy process and its importance becomes more pronounced in resource-constrained settings. In this paper, we have reviewed the use of systematic review evidence in framing National Health Programme (NHP) guidelines in India. We searched official websites of the different NHPs, linked to the main website of the Ministry of Health and Family Welfare (MoHFW), in December 2020 and January 2021. NHP guideline documents with systematic review evidence were identified and information on the use of this evidence was extracted. We classified the identified systematic review evidence according to its use in the guideline documents and analysed the data to provide information on the different factors and patterns linked to the use of systematic review evidence in these documents. Systematic reviews were mostly visible in guideline documents addressing maternal and newborn health, communicable diseases and immunization. These systematic reviews were cited in the guidelines to justify the need for action, to justify recommendations for action and opportunities for local adaptation, and to highlight implementation challenges and justify implementation strategies. Guideline documents addressing implementation cited systematic reviews about the problems and policy options more often than citing systematic reviews about implementation. Systematic reviews were linked directly to support statements in few guideline documents, and sometimes the reviews were not appropriately cited. Most of the systematic reviews providing information on the nature and scale of the policy problem included Indian data. It was seen that since 2014, India has been increasingly using systematic review evidence for public health policymaking, particularly for some of its high-priority NHPs. This complements the increasing investment in research synthesis centres and procedures to support evidence-informed decision making, demonstrating the continued evolution of India’s evidence policy system. Key messagesOver the years, India has demonstrated a visible evolution in the evidence policy system that can be attributed to two key complementary components of the evidence system (1) development of systematic review generation centres, and (2) commitment to evidence-informed policy development.The visibility of systematic reviews in Indian policy documents has been growing since 2014 with international support. Systematic reviews are most visible in policies addressing maternal and newborn health, communicable diseases and immunization. These systematic reviews are sometimes linked directly to support statements in policy documents and other times they appear in a list of additional readings towards the end of the document.Systematic reviews are cited in Indian public health policy documents to: (1) justify the need for action; (2) identify key components, experiences, views, effects and costs of policy options to justify recommendations for action and highlight opportunities for local adaptation; and (3) highlight challenges and justify implementation strategies.Indian data were mostly used in systematic reviews defining the policy problem rather than assessing different policy options emphasizing the need for methodologies that transfer global evidence about policy options to local settings.This growing visibility of systematic reviews in policy documents reflects the earlier global enthusiasm for producing systematic reviews to address perinatal care and epidemiology, and the subsequent developments in systematic reviewing of health systems research and social science  more broadly.Evidence-informed policymaking is an approach to policymaking that involves the use of systematic and transparent research evidence . AdditioThis approach to policymaking often uses the evidence synthesized via ‘systematic reviews’ to answer policy relevant questions . GeneralIn India, the history of evidence for clinical decisions began in the 1990s when formal training on evidence-based medicine (EBM) was provided to the medical and nursing faculty of India, by the International Clinical Epidemiology Network (INCLEN) . The impEvidence-informed decision-making was also recommended for policy because of its transparency, inclusiveness and independence, with attention paid to formal priority setting and resource allocation to avoid political or other competing interests . ChallenNevertheless, evidence-informed policymaking has seen significant progress over the past few decades and, with systematic review methods now available to synthesize various types of studies, including the context of studies, the methodology is better suited to addressing challenging complex policy issues. Over the years, India has understood the importance of shared research knowledge and building linkages between researchers and decision makers, especially in health policy and systems research. As a result, in 2013 the Ministry of Health and Family Welfare (MoHFW) in India advocated the formation of a National Knowledge Platform to (1) enable knowledge sharing among researchers and policymakers, (2) enable capacity building in research, (3) facilitate research knowledge dissemination, and (4) support research initiatives in priority areas . More inWith these structures and commitments in place, the question remains whether India has converted this progress and understanding of evidence synthesis into policy development? Evaluation and review of some of the clinical guidelines in the past suggest that India is in a transition phase of guideline development and the country requires more local capacity in evidence search and synthesis to increase the methodological quality and rigour of the clinical guidelines . However, comprehensive information about this use is not available in one place. Although there is information available on the evaluation of some of the clinical guideline documents, there is a gap regarding information about the use of systematic reviews in other, non-clinical NHPs. In this paper, we have widened our scope beyond clinical guidelines to consider the use of systematic review evidence in the NHPs of India that are mentioned on the MoHFW website. A study design of stand-alone document analysis was applied to answer the question of whether India’s NHP guideline documents are explicitly informed by evidence syntheses. The methods followed recommendations from a qualitative systematic review of document analysis in health policy analysis studies in low- and middle-income countries (LMICs)  adoptingA targeted search was conducted to identify the documents providing information about the use of systematic reviews in NHPs of India. Documents sought for analysis were the guideline documents produced by the NHPs in India  systematic reviews justifying the importance and need for action; (2) systematic reviews justifying recommendations for action, and opportunities for local adaptation; and (3) systematic reviews informing implementation. Detailed information on the identified evidence is given in National Guidelines for Deworming in Pregnancy’ (2014), ‘India Newborn Action Plan’ (2014), ‘Final Report joint assessment of the tuberculosis diagnostic network of India’ (2017), ‘National framework for joint TB diabetes collaborative activities-RNTCP’ (2017) and ‘National guidelines for dengue case management during Covid-19 Pandemic’ (2020), justified the importance and need for action via systematic review evidence that provided information on the nature and scale of the problem , National Tuberculosis Elimination Programme (NTEP), Non-Communicable Diseases, Injury & Trauma and National Vector Borne Disease Control Programme (NVBDCP), mentioned or cited the use of systematic review evidence in their guideline documents for justifying the importance and need for action. Five published guideline documents from the aforementioned four NHPs, namely ‘ problem ,b; 2020aSixteen published guideline documents from four NHPs  used systematic review evidence for justifying recommendations for action and opportunities for local adaptation. These 16 published guideline documents cited systematic reviews that provided information on the different programme and policy options applicable for the specific NHPs . More inOperational Guidelines—Introduction of Rotavirus Vaccine in the Universal Immunization Programme’ (2019) justifies the implementation strategies for the process of scaling-up of the rotavirus vaccine, therefore, supporting implementation (Guidelines for midwifery services in India’ (2018) ’ (2007), and ‘Reducing risk factors for Non-Communicable Diseases (NCDs) in Primary Care: Training manual for Medical Officers’ (2016), cited systematic reviews that provided information on the extent of the problem and assessed different policy options , and UIP used systematic review evidence for informing implementation. In line with Lavis’ framework for evidence use in policy , the ‘Opentation . The ‘Gu’ (2018) , evidenc options ; 2016c. The section above classifies systematic review evidence according to its role in the published NHP guideline documents. In this section, we have elaborated more on the use of systematic review evidence by the NHPs of India. We found only six NHPs providing information about the use of systematic reviews in the guidelines see . AdditioSystematic review evidence was mostly visible in guidelines or policies addressing maternal and newborn health (RMNCH + A), communicable diseases and immunization. Most systematic reviews cited in the guideline documents provided information on assessing policy options and were used for justifying recommendations for actions and opportunities for local adaptation. Guideline or policy documents addressing implementation cited systematic reviews of epidemiology about the problems and policy options more often than systematic reviews about implementation.Only 9 of the 22 guideline documents (41%) consistently linked systematic reviews to the relevant statements in the document . Other tIndigenization and local adaptation of the systematic review evidence is a vital component in policymaking. We found that for clinical programmes and policies, global systematic review evidence was used and cited in the Indian guidelines and many of these included Indian data. However, for some of the non-clinical guidelines, where the context may be particularly influential in how interventions work, the lack of Indian data is a concern. The INDEX-TB guidelines  considerAdditionally, it was observed that none of the NHP guideline documents drew on synthesis of qualitative research that could justify recommendations for action and identify opportunities for local adaptation or inform implementation.Important global events and international funding paved the way for the creation of some of the NHPs and their use of systematic review evidence. The NACP was exceptionally early in citing systematic reviews in guideline documents. NACP I & II were initiated after World Bank funding in 1992 and 1999, long before systematic reviews were commonly used for policy . AnotherIn contrast, most of the guideline documents citing systematic reviews were published after 2014, with major clusters in 2016 and 2017 see . For insThe growing visibility of systematic reviews in Indian guideline documents since 2014 coincided with the growth of evidence-informed medicine and evidence synthesis centres in the country  and reflThis paper focuses on the use of systematic review evidence in formulating public health policies for NHPs in India and the overall evolution of the evidence policy system in India. Our findings show that over the years, India has consistently invested in developing new centres for producing systematic reviews and capacity building in evidence synthesis. Additionally, since 2014, the visibility of evidence has increased in the Indian guideline documents, specifically for six NHPs i.e. NTEP, NACP, NVBDCP, NPCDCS, UIP and RMNCH + A. Overall, citing systematic reviews in guideline or policy documents appears to be a recent development, possibly influenced by international sources of funding. However, proper referencing of the systematic reviews in the guideline documents remains an area of concern because only 9 documents out of 22 guideline documents, identified from the above six NHPs, provided systematic review citations linked to statements in the text. Generally, the systematic review evidence cited in the guideline documents was international, and many of these systematic reviews included Indian data. Indian data were used by more systematic reviews providing information about the problem than those assessing policy options. The lack of Indian data in some systematic reviews providing information on non-clinical, context specific research questions is a concern.Our findings show India’s evolving system for evidence-informed policy. This evolution can be attributed to the country’s long-term investment in two key complementary components of the evidence system: (1) systematic review generation centres; and (2) commitment to evidence-informed policy development. Theoretical literature offers different models for how these key components work together to support KTA . The knoThe demand-pull model of knowledge translation focuses on research users demanding ‘evidence’ for specific policy questions from the researchers, so that the policy decision is evidence informed. Supporting this principle of the demand-pull model of KTA is the growing commitment in India to developing evidence-informed guidance, expressed by guideline groups framing their questions and drawing on existing systematic reviews or commissioning new systematic reviews [e.g. INDEX-TB Guidelines ] or tailThe relational model of KTA focuses on linkage exchange, collaboration and shared learning among different stakeholders. This model is apparent where policy makers, clinicians and researchers work together to develop policy documents  consistently linked systematic reviews to the relevant statements in the document, similar results have been reported by related global literature. Limited or unsystematic focus on systematic reviews for guideline development is not unusual. A global survey found that few guidelines published in 2017 and 2018 based their recommendations on systematic reviews (32%) or systematic overviews (2%); notable exceptions were those prepared by the WHO and two high-income guideline producers , are to highlight or flag the local region-specific  considerations such as benefits, cost, context-specific considerations, while developing international guidelines for global public health related problems. Collaboration between international guidelines development organizations and regional or national institutions (e.g. with remits for India) could enhance transferability of evidence and provide contextual insights for transferring or adapting international guidelines to the Indian context.Additionally, given the lack of qualitative systematic reviews informing the policy documents analysed, we recommend that policy development groups broaden their interest in systematic reviews to consider qualitative research that includes global evidence relevant to issues that are priorities for India. For instance, there are many systematic reviews of qualitative research which, together, offer in-depth understanding of contextual factors that influence women’s choice and use of contraception (India is increasingly contributing to, and explicitly making use of, global efforts to systematically review health research to make policy decisions in health, mainly in the areas of perinatal health and communicable diseases. Over the years, India has successfully used systematic review evidence for some of its high-priority NHPs and has documented the same in national guidelines. Some documents supporting implementation of policy decisions also cite systematic reviews, although more to highlight the need for action and justify policy decisions than to guide implementation. The visibility of systematic review evidence and understanding about the use of evidence in NHPs will shape the future of guideline development and public health policy making in India.