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Purpose: The preplanned exploratory analysis of the BERIL-1 trial presented here aimed to identify biomarkers of response to the combination of buparlisib and paclitaxel.Patients and Methods: BERIL-1 was a multicenter, randomized, double-blind, placebo-controlled phase II study. Patients with recurrent or metastatic squamous cell carcinoma of the head and neck (SCCHN) progressing on/after one previous platinum-based chemotherapy regimen in the recurrent or metastatic setting were treated with either buparlisib plus paclitaxel or placebo plus paclitaxel. Archival tumor tissue and ctDNA samples were analyzed for molecular alterations and immune infiltration using next-generation sequencing or immunohistochemistry.Results: Biomarker analyses were performed in randomized patients (n = 158) with available biomarker data. The most frequently (>5%) mutated genes were TP53, FAT1, TET2, KMT2D, PIK3CA, NOTCH1, NFE2L2, NOTCH2, CCND1, and CDKN2A. Patients with SCCHN tumors (from various primary sites) having HPV-negative status (HR = 0.51), TP53 alterations (HR = 0.55) or low mutational load (HR = 0.57) derived overall survival (OS) benefit with the combination of buparlisib and paclitaxel. OS benefit with this combination was also increased in patients with presence of intratumoral TILs ≥10% (HR = 0.51), stromal TILs ≥15% (HR = 0.53), intratumoral CD8-positive cells ≥5% (HR = 0.45), stromal CD8-positive cells ≥10% (HR = 0.47), or CD8-positive cells in invasive margins >25% (HR = 0.37). A trend for improved progression-free survival with the combination of buparlisib and paclitaxel was also observed in these patients.Conclusions: The BERIL-1 biomarker analyses showed that patients with TP53 alterations, HPV-negative status, low mutational load, or high infiltration of TILs or CD8-positive cells derived survival benefit with the combination of buparlisib and paclitaxel. Clin Cancer Res; 24(11); 2505–16. ©2018 AACR.

Supplementary methods and results Supplementary Table 1: Mutational profiles of patients with HPV-positive vs HPV-negative tumors Supplementary Figure 1: Interaction between immune markers and other biomarkers

Purpose: The preplanned exploratory analysis of the BERIL-1 trial presented here aimed to identify biomarkers of response to the combination of buparlisib and paclitaxel. Patients and Methods: BERIL-1 was a multicenter, randomized, double-blind, placebo-controlled phase II study. Patients with recurrent or metastatic squamous cell carcinoma of the head and neck (SCCHN) progressing on/after one previous platinum-based chemotherapy regimen in the recurrent or metastatic setting were treated with either buparlisib plus paclitaxel or placebo plus paclitaxel. Archival tumor tissue and ctDNA samples were analyzed for molecular alterations and immune infiltration using next-generation sequencing or immunohistochemistry. Results: Biomarker analyses were performed in randomized patients (n = 158) with available biomarker data. The most frequently (>5%) mutated genes were TP53, FAT1, TET2, KMT2D, PIK3CA, NOTCH1, NFE2L2, NOTCH2, CCND1, and CDKN2A. Patients with SCCHN tumors (from various primary sites) having HPV-negative status (HR = 0.51), TP53 alterations (HR = 0.55) or low mutational load (HR = 0.57) derived overall survival (OS) benefit with the combination of buparlisib and paclitaxel. OS benefit with this combination was also increased in patients with presence of intratumoral TILs ≥10% (HR = 0.51), stromal TILs ≥15% (HR = 0.53), intratumoral CD8-positive cells ≥5% (HR = 0.45), stromal CD8-positive cells ≥10% (HR = 0.47), or CD8-positive cells in invasive margins >25% (HR = 0.37). A trend for improved progression-free survival with the combination of buparlisib and paclitaxel was also observed in these patients. Conclusions: The BERIL-1 biomarker analyses showed that patients with TP53 alterations, HPV-negative status, low mutational load, or high infiltration of TILs or CD8-positive cells derived survival benefit with the combination of buparlisib and paclitaxel. Clin Cancer Res; 24(11); 2505–16. ©2018 AACR.

Summary Background Phosphatidylinositol 3-kinase (PI3K) pathway activation in squamous cell carcinoma of the head and neck contributes to treatment resistance and disease progression. Buparlisib, a pan-PI3K inhibitor, has shown preclinical antitumour activity and objective responses in patients with epithelial malignancies. We assessed whether the addition of buparlisib to paclitaxel improves clinical outcomes compared with paclitaxel and placebo in patients with recurrent or metastatic squamous cell carcinoma of the head and neck. Methods In this multicentre, randomised, double-blind, placebo-controlled phase 2 study (BERIL-1), we recruited patients aged 18 years and older with histologically or cytologically confirmed recurrent and metastatic squamous cell carcinoma of the head and neck after disease progression on or after one previous platinum-based chemotherapy regimen in the metastatic setting. Eligible patients were enrolled from 58 centres across 18 countries and randomly assigned (1:1) to receive second-line oral buparlisib (100 mg once daily) or placebo, plus intravenous paclitaxel (80 mg/m 2 on days 1, 8, 15, and 22) in 28 day treatment cycles. Randomisation was done via a central patient screening and randomisation system with an interactive (voice and web) response system and stratification by number of previous lines of therapy in the recurrent and metastatic setting and study site. Patients and investigators (including local radiologists) were masked to treatment assignment from randomisation until the final overall survival analysis. The primary endpoint was progression-free survival by local investigator assessment per Response Evaluation Criteria In Solid Tumors (version 1.1) in all randomly assigned patients. Efficacy analyses were done on the intention-to-treat population, whereas safety was analysed in all patients who received at least one dose of study drug and had at least one post-baseline safety assessment according to the treatment they received. This trial is registered with ClinicalTrials.gov, number NCT01852292, and is ongoing but no longer enrolling patients. Findings Between Nov 5, 2013, and May 5, 2015, 158 patients were enrolled and randomly assigned to receive either buparlisib plus paclitaxel (n=79) or placebo plus paclitaxel (n=79). Median progression-free survival was 4·6 months (95% CI 3·5–5·3) in the buparlisib group and 3·5 months (2·2–3·7) in the placebo group (hazard ratio 0·65 [95% CI 0·45–0·95], nominal one-sided p=0·011). Grade 3–4 adverse events were reported in 62 (82%) of 76 patients in the buparlisib group and 56 (72%) of 78 patients in the placebo group. The most common grade 3–4 adverse events (occurring in ≥10% of patients in the buparlisib group vs the placebo group) were hyperglycaemia (17 [22%] of 76 vs two [3%] of 78), anaemia (14 [18%] vs nine [12%]), neutropenia (13 [17%] vs four [5%]), and fatigue (six [8%] vs eight [10%]). Serious adverse events (regardless of relation to study treatment) were reported for 43 (57%) of 76 patients in the buparlisib group and 37 (47%) of 78 in the placebo group. On-treatment deaths occurred in 15 (20%) of 76 patients in the buparlisib group and 17 (22%) of 78 patients in the placebo group; most were caused by disease progression and none were judged to be related to study treatment. Interpretation On the basis of the improved clinical efficacy with a manageable safety profile, the results of this randomised phase 2 study suggest that buparlisib in combination with paclitaxel could be an effective second-line treatment for patients with platinum-pretreated recurrent or metastatic squamous cell carcinoma of the head and neck. Further phase 3 studies are warranted to confirm this phase 2 finding. Funding Novartis Pharmaceuticals Corporation.

Cystinosis is a rare autosomal recessive storage disorder characterized by defective lysosomal efflux of cystine due to mutations in the CTNS gene encoding the lysosomal cystine transporter, cystinosin. Lysosomal cystine accumulation leads to crystal formation and functional impairment of multiple organs. Moreover, cystinosis is the most common inherited cause of renal Fanconi syndrome in children. Oral cysteamine therapy delays disease progression by reducing intracellular cystine levels. However, because cysteamine does not correct all complications of cystinosis, including Fanconi syndrome, we hypothesized that cystinosin could have novel roles in addition to transporting cystine out of the lysosome. By coimmunoprecipitation experiments and mass spectrometry, we found cystinosin interacts with almost all components of vacuolar H(+)-ATPase and the Ragulator complex and with the small GTPases Ras-related GTP-binding protein A (RagA) and RagC. Furthermore, the mammalian target of rapamycin complex 1 (mTORC1) pathway was downregulated in proximal tubular cell lines derived from Ctns(-/-) mice. Decrease of lysosomal cystine levels by cysteamine did not rescue mTORC1 activation in these cells, suggesting that the downregulation of mTORC1 is due to the absence of cystinosin rather than to the accumulation of cystine. Our results show a dual role for cystinosin as a cystine transporter and as a component of the mTORC1 pathway, and provide an explanation for the appearance of Fanconi syndrome in cystinosis. Furthermore, this study highlights the need to develop new treatments not dependent on lysosomal cystine depletion alone for this devastating disease.

The mechanism of NADH oxidation varies between living organisms, and is by far the most complex oxidizingsystem found in mitochondria. In human mitochondria, a unique, but huge structure, with more than 45 subunits, knownas complex I, copes with NADH oxidation. This review compiles our present knowledge on the organization of thiscomplex and the putative role of a small subset of its subunits. This review also describes the major progress that has beenmade in understanding the molecular bases of respiratory chain complex I deficiency in humans, with mutations identifiedin both the mitochondrial and the nuclear genes encoding complex I subunits. Finally, the puzzling questions raised by thevarying clinical presentations of patients with complex I deficiency are discussed in light of our limited knowledge oncomplex I function in mammalian cells. Key Words: Mitochondria, Respiratory Chain, Complex I deficiency, NADH. MITOCHONDRIAL OXIDATION OF REDUCEDNICOTINAMIDE ADENINE DINUCLEOTIDES:FROM THE MANY DEHYDROGENASES OF PLANTSTO THE UNIQUE MAMMALIAN COMPLEX I

6045Background: A phase II, placebo-controlled study of buparlisib (BUP) and paclitaxel (PAC) in patients (pts) with platinum-pretreated recurrent/metastatic HNSCC (NCT01852292) met its primary end...

e20522Background: Phosphatidylinositol 3-kinase (PI3K) pathway activation may contribute to primary and secondary resistance to platinum (Pt)-based chemotherapy (CT) in sqNSCLC. The pan-PI3K inhibi...

TPS618Background: The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway is frequently activated in hormone receptor-positive (HR+) BC and is associated with resistance to endocrine therapy. The combination of the PI3Kα-specific inhibitor alpelisib (BYL719) and fulvestrant showed promising activity in a phase I study of HR+/human epidermal growth factor 2-negative (HER2–) BC, with the strongest treatment benefit in patients (pts) with PIK3CA-mutant tumors (Janku et al. SABCS 2014, Abstract PD5-5). Further data are required to confirm these observations. Methods: In the phase III, randomized, double-blind SOLAR-1 study (NCT02437318), men and postmenopausal women with HR+/HER2– advanced BC are randomized (1:1) to alpelisib or placebo (300 mg once daily) + fulvestrant (500 mg intramuscularly on Days 1 and 15 of Cycle 1 [28 days], and Day 1 of Cycles ≥ 2) until disease progression or discontinuation for other reason. Pts are assigned to 2 cohorts (PIK3CAmutant vs non-mutant ...

Respiratory chain complex I (NADH:ubiquinone oxidoreductase) deficiency is one of the most frequent causes of mitochondrial disease in humans. The activity of this complex can be confidently measured in most tissue samples, but not in cultured skin fibroblasts or circulating lymphocytes. Highly contaminating non-mitochondrial NADH-quinone oxidoreductase activity in fibroblasts and the limited access of substrates to complex I in lymphocytes hinder its measurement in permeabilized cells. Complex I assay in these cells requires the isolation of mitochondria, which in turn necessitates large quantities of cells and is not feasible when studying circulating lymphocytes. Here we report a simple method to measure complex I activity in a minute amount of either cell type. The procedure strongly reduces contaminating NADH:quinone oxidoreductase activity and permits measuring high rates of rotenone-sensitive complex I activity thanks to effective cell permeabilization.

Background. Cystinosis is caused by mutations in CTNS that encodes cystinosin, the lysosomal cystine transporter. The most severe and frequent form is characterized by a proximal tubulopathy that appears around 6 to 12 months of age. In the absence of treatment, end-stage renal disease is reached by 10 years. Ctns(-/-) mice of a mixed 129Sv x C57BL/6 genetic background show elevated renal cystine levels; however, proximal tubulopathy or end-stage renal disease is not observed. Methods. As renal phenotype can be influenced by genetic background, we generated congenic C57BL/6 and FVB/N Ctns(-/-) mice and assayed renal lesions and function by histological and biochemical studies. Results. C57BL/6 Ctns(-/-) mice showed significantly higher renal cystine levels than the FVB/N strain. Moreover, C57BL/6 mice presented with pronounced histological lesions of the proximal tubules as well as a tubulopathy and progressively developed chronic renal failure. In contrast, renal dysfunction was not observed in the FVB/N strain. Conclusions. Thus, the C57BL/6 strain represents the first Ctns(-/-) mouse model to show clear renal defects. In addition to highlighting the influence of genetic background on phenotype, the C57BL/6 Ctns(-/-) mice represent a useful model for further understanding cystinosin function in the kidney and, specifically, in the proximal tubules.

Cystinosis is an inherited disorder due to mutations in the CTNS gene which encodes cystinosin, a lysosomal transmembrane protein involved in cystine export to the cytosol. Both accumulation of cystine in the lysosome and decreased cystine in the cytosol may participate in the pathogenic mechanism underlying the disease. We observed that cystinotic cell lines have moderate decrease of glutathione content during exponential growth phase. This resulted in increased solicitation of oxidative defences of the cell denoted by concurrent superoxide dismutase induction, although without major oxidative insult under our experimental conditions. Finally, decreased glutathione content in cystinotic cell lines could be counterbalanced by a series of exogenous precursors of cysteine, denoting that lysosomal cystine export is a natural source of cellular cysteine in the studied cell lines.

Leigh syndrome is a subacute necrotising encephalomyopathy frequently ascribed to mitochondrial respiratory chain deficiency. This condition is genetically heterogeneous, as mutations in both mitochondrial (mt) and nuclear genes have been reported. Here, we report the G13513A transition in the ND5 mtDNA gene in three unrelated children with complex I deficiency and a peculiar MRI aspect distinct from typical Leigh syndrome. Brain MRI consistently showed a specific involvement of the substantia nigra and medulla oblongata sparing the basal ganglia. Variable degrees of heteroplasmy were found in all tissues tested and a high percentage of mutant mtDNA was observed in muscle. The asymptomatic mothers presented low levels of mutant mtDNA in blood leucocytes. This mutation, which affects an evolutionary conserved amino acid (D393N), has been previously reported in adult patients with MELAS or LHON/MELAS syndromes, emphasising the clinical heterogeneity of mitochondrial DNA mutations. Since the G13513A mutation was found in 21% of our patients with Leigh syndrome and complex I deficiency (3/14), it appears that this mutation represents a frequent cause of Leigh-like syndrome, which should be systematically tested for molecular diagnosis in affected children and for genetic counselling in their maternal relatives.

Starting from a cohort of 50 NADH-oxidoreductase (complex I) deficient patients, we carried out the systematic sequence analysis of all mitochondrially encoded complex I subunits (ND1 to ND6 and ND4L) in affected tissues. This approach yielded the unexpectedly high rate of 20% mutation identification in our series. Recurrent heteroplasmic mutations included two hitherto unreported (T10158C and T14487C) and three previously reported mutations (T10191C, T12706C and A13514G) in children with Leigh or Leigh-like encephalopathy. The recurrent mutations consistently involved T-->C transitions (p