31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Dystonia-deafness syndrome caused by ACTB p.Arg183Trp heterozygosity shows striatal dopaminergic dysfunction and response to pallidal stimulation

Background Dystonia-deafness syndrome is a well-known clinical entity, with sensorineural deafness typically manifesting earlier than dystonia. ACTB p.Arg183Trp heterozygosity has been reported in six patients to cause combined infant-onset deafness and dystonia manifesting in adolescence or young adulthood. Three of these have received beneficial pallidal stimulation. Brain imaging to assess striatal function has not been reported previously, however. Nor has a comprehensive hypothesis been presented for how the pleiotropic manifestations of this specific beta-actin gene mutation originate developmentally. Case presentation A 19-year-old girl with congenital mild dysmorphic facial features, cochlear implants for infant-onset deafness, and mild cognitive and emotional disability, presented with an adolescent-onset, severe generalized dystonia. Brain MRI and multiple single gene sequencing were inconclusive. Due to life-threatening dystonia, we implanted a neurostimulation device, targeting the postero-ventral internal pallidum bilaterally. The Burke-Fahn-Marsden Dystonia Rating Scale motor/disability scores improved from 87/25 to 21/13 at 2.5 months postoperatively, 26/14 at 3 years, and 30/14 at 4 years. Subsequent whole exome sequencing identified heterozygosity for the ACTB p.Arg183Trp variant. Brain imaging included 123I-ioflupane single photon emission computed tomography (Dopamine Transporter-SPECT), SPECT with 123I-epidepride (binds to dopamine type 2-receptors) and 18 Fluoro-Deoxy-Glucose (FDG)–PET. Both Epidepride-SPECT and FDG-PET showed reduced tracer uptake in the striatum bilaterally, particularly in the putamen. DaT-SPECT was slightly abnormal. Conclusions In this patient with dystonia-deafness syndrome caused by ACTB p.Arg183Trp heterozygosity, unprecedented brain imaging findings strongly indicate striatal neuronal/dopaminergic dysfunction as the underlying cause of the dystonia. Pallidal stimulation provided a substantial improvement of the severe generalized dystonia, which is largely sustained at 4-year follow-up, and we advise this treatment to be considered in such patients. We hypothesize that the pleiotropic manifestations of the dystonia-deafness syndrome caused by this mutation derive from diverse developmental functions of beta-actin in neural crest migration and proliferation (facial dysmorphogenesis), hair cell stereocilia function (infant-onset deafness), and altered synaptic activity patterns associated with pubertal changes in striatal function (adolescent-onset dystonia). The temporal differences in developmental onset are likely due to varying degrees of susceptibility and of compensatory upregulation of other actin variants in the affected structures

STUB1 mutations in autosomal recessive ataxias – evidence for mutation-specific clinical heterogeneity

Background A subset of hereditary cerebellar ataxias is inherited as autosomal recessive traits (ARCAs). Classification of recessive ataxias due to phenotypic differences in the cerebellum and cerebellar structures is constantly evolving due to new identified disease genes. Recently, reports have linked mutations in genes involved in ubiquitination (RNF216, OTUD4, STUB1) to ARCA with hypogonadism. Methods and results With a combination of homozygozity mapping and exome sequencing, we identified three mutations in STUB1 in two families with ARCA and cognitive impairment; a homozygous missense variant (c.194A > G, p.Asn65Ser) that segregated in three affected siblings, and a missense change (c.82G > A, p.Glu28Lys) which was inherited in trans with a nonsense mutation (c.430A > T, p.Lys144Ter) in another patient. STUB1 encodes CHIP (C-terminus of Heat shock protein 70 – Interacting Protein), a dual function protein with a role in ubiquitination as a co-chaperone with heat shock proteins, and as an E3 ligase. We show that the p.Asn65Ser substitution impairs CHIP’s ability to ubiquitinate HSC70 in vitro, despite being able to self-ubiquitinate. These results are consistent with previous studies highlighting this as a critical residue for the interaction between CHIP and its co-chaperones. Furthermore, we show that the levels of CHIP are strongly reduced in vivo in patients’ fibroblasts compared to controls. Conclusions These results suggest that STUB1 mutations might cause disease by impacting not only the E3 ligase function, but also its protein interaction properties and protein amount. Whether the clinical heterogeneity seen in STUB1 ARCA can be related to the location of the mutations remains to be understood, but interestingly, all siblings with the p.Asn65Ser substitution showed a marked appearance of accelerated aging not previously described in STUB1 related ARCA, none display hormonal aberrations/clinical hypogonadism while some affected family members had diabetes, alopecia, uveitis and ulcerative colitis, further refining the spectrum of STUB1 related disease

STUB1 mutations in autosomal recessive ataxias - evidence for mutation-specific clinical heterogeneity

Background: A subset of hereditary cerebellar ataxias is inherited as autosomal recessive traits (ARCAs). Classification of recessive ataxias due to phenotypic differences in the cerebellum and cerebellar structures is constantly evolving due to new identified disease genes. Recently, reports have linked mutations in genes involved in ubiquitination (RNF216, OTUD4, STUB1) to ARCA with hypogonadism. Methods and results: With a combination of homozygozity mapping and exome sequencing, we identified three mutations in STUB1 in two families with ARCA and cognitive impairment; a homozygous missense variant (c.194A > G, p.Asn65Ser) that segregated in three affected siblings, and a missense change (c.82G > A, p.Glu28Lys) which was inherited in trans with a nonsense mutation (c.430A > T, p.Lys144Ter) in another patient. STUB1 encodes CHIP (C-terminus of Heat shock protein 70 – Interacting Protein), a dual function protein with a role in ubiquitination as a co-chaperone with heat shock proteins, and as an E3 ligase. We show that the p.Asn65Ser substitution impairs CHIP’s ability to ubiquitinate HSC70 in vitro, despite being able to self-ubiquitinate. These results are consistent with previous studies highlighting this as a critical residue for the interaction between CHIP and its co-chaperones. Furthermore, we show that the levels of CHIP are strongly reduced in vivo in patients’ fibroblasts compared to controls. Conclusions: These results suggest that STUB1 mutations might cause disease by impacting not only the E3 ligase function, but also its protein interaction properties and protein amount. Whether the clinical heterogeneity seen in STUB1 ARCA can be related to the location of the mutations remains to be understood, but interestingly, all siblings with the p.Asn65Ser substitution showed a marked appearance of accelerated aging not previously described in STUB1 related ARCA, none display hormonal aberrations/clinical hypogonadism while some affected family members had diabetes, alopecia, uveitis and ulcerative colitis, further refining the spectrum of STUB1 related disease

Effects of the Adulteration Technique on the Near-Infrared Detection of Melamine in Milk Powder

The United States Pharmacopeial Convention has led an international collaborative project to develop a toolbox of screening methods and reference standards for the detection of milk powder adulteration. During the development of adulterated milk powder reference standards, blending methods used to combine melamine and milk had unanticipated strong effects on the near-infrared (NIR) spectrum of melamine. The prominent absorbance band at 1468 nm of melamine was retained when it was dry-blended with skim milk powder but disappeared in wet-blended mixtures, where spray-dried milk powder samples were prepared from solution. Analyses using polarized light microscopy, Raman spectroscopy, dielectric relaxation spectroscopy, X-ray diffraction, and mass spectrometry indicated that wet blending promoted reversible and early Maillard reactions with lactose that are responsible for differences in melamine NIR spectra between wet- and dry-blended samples. Targeted detection estimates based solely on dry-blended reference standards are likely to overestimate NIR detection capabilities in wet-blended samples as a result of previously overlooked matrix effects arising from changes in melamine hydrogen-bonding status, covalent complexation with lactose, and the lower but more homogeneous melamine local concentration distribution produced in wet-blended samples. Techniques used to incorporate potential adulterants can determine the suitability of milk reference standards for use with rapid detection methods