Immune monitoring and TCR sequencing of CD4 T cells in a long term responsive patient with metastasized pancreatic ductal carcinoma treated with individualized, neoepitope-derived multipeptide vaccines: a case report.

BACKGROUND: Cancer vaccines can effectively establish clinically relevant tumor immunity. Novel sequencing approaches rapidly identify the mutational fingerprint of tumors, thus allowing to generate personalized tumor vaccines within a few weeks from diagnosis. Here, we report the case of a 62-year-old patient receiving a four-peptide-vaccine targeting the two sole mutations of his pancreatic tumor, identified via exome sequencing. METHODS: Vaccination started during chemotherapy in second complete remission and continued monthly thereafter. We tracked IFN-γ+ T cell responses against vaccine peptides in peripheral blood after 12, 17 and 34 vaccinations by analyzing T-cell receptor (TCR) repertoire diversity and epitope-binding regions of peptide-reactive T-cell lines and clones. By restricting analysis to sorted IFN-γ-producing T cells we could assure epitope-specificity, functionality, and TH1 polarization. RESULTS: A peptide-specific T-cell response against three of the four vaccine peptides could be detected sequentially. Molecular TCR analysis revealed a broad vaccine-reactive TCR repertoire with clones of discernible specificity. Four identical or convergent TCR sequences could be identified at more than one time-point, indicating timely persistence of vaccine-reactive T cells. One dominant TCR expressing a dual TCRVα chain could be found in three T-cell clones. The observed T-cell responses possibly contributed to clinical outcome: The patient is alive 6 years after initial diagnosis and in complete remission for 4 years now. CONCLUSIONS: Therapeutic vaccination with a neoantigen-derived four-peptide vaccine resulted in a diverse and long-lasting immune response against these targets which was associated with prolonged clinical remission. These data warrant confirmation in a larger proof-of concept clinical trial.

Loss-of-function variants in HIVEP2 are a cause of intellectual disability.

Intellectual disability (ID) affects 2-3% of the population. In the past, many genetic causes of ID remained unidentified due to its vast heterogeneity. Recently, whole exome sequencing (WES) studies have shown that de novo variants underlie a significant portion of sporadic cases of ID. Applying WES to patients with ID or global developmental delay at different centers, we identified three individuals with distinct de novo variants in HIVEP2 (human immunodeficiency virus type I enhancer binding protein), which belongs to a family of zinc-finger-containing transcriptional proteins involved in growth and development. Two of the variants were nonsense changes, and one was a 1 bp deletion resulting in a premature stop codon that was reported previously without clinical detail. In silico prediction programs suggest loss-of-function in the mutated allele leading to haploinsufficiency as a putative mechanism in all three individuals. All three patients presented with moderate-to-severe ID, minimal structural brain anomalies, hypotonia, and mild dysmorphic features. Growth parameters were in the normal range except for borderline microcephaly at birth in one patient. Two of the patients exhibited behavioral anomalies including hyperactivity and aggression. Published functional data suggest a neurodevelopmental role for HIVEP2, and several of the genes regulated by HIVEP2 are implicated in brain development, for example, SSTR-2, c-Myc, and genes of the NF-κB pathway. In addition, HIVEP2-knockout mice exhibit several working memory deficits, increased anxiety, and hyperactivity. On the basis of the genotype-phenotype correlation and existing functional data, we propose HIVEP2 as a causative ID gene.

Germline PTPN11 and somatic PIK3CA variant in a boy with megalencephaly-capillary malformation syndrome (MCAP)--pure coincidence?

Megalencephaly-capillary malformation (MCAP) syndrome is an overgrowth syndrome that is diagnosed by clinical criteria. Recently, somatic and germline variants in genes that are involved in the PI3K-AKT pathway (AKT3, PIK3R2 and PIK3CA) have been described to be associated with MCAP and/or other related megalencephaly syndromes. We performed trio-exome sequencing in a 6-year-old boy and his healthy parents. Clinical features were macrocephaly, cutis marmorata, angiomata, asymmetric overgrowth, developmental delay, discrete midline facial nevus flammeus, toe syndactyly and postaxial polydactyly--thus, clearly an MCAP phenotype. Exome sequencing revealed a pathogenic de novo germline variant in the PTPN11 gene (c.1529A>G; p.(Gln510Arg)), which has so far been associated with Noonan, as well as LEOPARD syndrome. Whole-exome sequencing (>100 × coverage) did not reveal any alteration in the known megalencephaly genes. However, ultra-deep sequencing results from saliva (>1000 × coverage) revealed a 22% mosaic variant in PIK3CA (c.2740G>A; p.(Gly914Arg)). To our knowledge, this report is the first description of a PTPN11 germline variant in an MCAP patient. Data from experimental studies show a complex interaction of SHP2 (gene product of PTPN11) and the PI3K-AKT pathway. We hypothesize that certain PTPN11 germline variants might drive toward additional second-hit alterations.

Further delineation of the SATB2 phenotype.

SATB2 is an evolutionarily highly conserved chromatin remodeling gene located on chromosome 2q33.1. Vertebrate animal models have shown that Satb2 has a crucial role in craniofacial patterning and osteoblast differentiation, as well as in determining the fates of neuronal projections in the developing neocortex. In humans, chromosomal translocations and deletions of 2q33.1 leading to SATB2 haploinsufficiency are associated with cleft palate (CP), facial dysmorphism and intellectual disability (ID). A single patient carrying a nonsense mutation in SATB2 has been described to date. In this study, we performed trio-exome sequencing in a 3-year-old girl with CP and severely delayed speech development, and her unaffected parents. Previously, the girl had undergone conventional and molecular karyotyping (microarray analysis), as well as targeted analysis for different diseases associated with developmental delay, including Angelman syndrome, Rett syndrome and Fragile X syndrome. No diagnosis could be established. Exome sequencing revealed a de novo nonsense mutation in the SATB2 gene (c.715C>T; p.R239*). The identification of a second patient carrying a de novo nonsense mutation in SATB2 confirms that this gene is essential for normal craniofacial patterning and cognitive development. Based on our data and the literature published so far, we propose a new clinically recognizable syndrome - the SATB2-associated syndrome (SAS). SAS is likely to be underdiagnosed and should be considered in children with ID, severe speech delay, cleft or high-arched palate and abnormal dentition with crowded and irregularly shaped teeth.

ß-Catenin signaling increases during melanoma progression and promotes tumor cell survival and chemoresistance.

Beta-catenin plays an important role in embryogenesis and carcinogenesis by controlling either cadherin-mediated cell adhesion or transcriptional activation of target gene expression. In many types of cancers nuclear translocation of beta-catenin has been observed. Our data indicate that during melanoma progression an increased dependency on the transcriptional function of beta-catenin takes place. Blockade of beta-catenin in metastatic melanoma cell lines efficiently induces apoptosis, inhibits proliferation, migration and invasion in monolayer and 3-dimensional skin reconstructs and decreases chemoresistance. In addition, subcutaneous melanoma growth in SCID mice was almost completely inhibited by an inducible beta-catenin knockdown. In contrast, the survival of benign melanocytes and primary melanoma cell lines was less affected by beta-catenin depletion. However, enhanced expression of beta-catenin in primary melanoma cell lines increased invasive capacity in vitro and tumor growth in the SCID mouse model. These data suggest that beta-catenin is an essential survival factor for metastatic melanoma cells, whereas it is dispensable for the survival of benign melanocytes and primary, non-invasive melanoma cells. Furthermore, beta-catenin increases tumorigenicity of primary melanoma cell lines. The differential requirements for beta-catenin signaling in aggressive melanoma versus benign melanocytic cells make beta-catenin a possible new target in melanoma therapy.

No evidence of viral genomes in whole-transcriptome sequencing of three melanoma metastases.

Several viruses are known to cause cancer, such as human herpes virus 8 in Kaposi sarcoma and human papilloma viruses in cervical cancer. Recently, Merkel cell polyoma virus (MCPyV) has been described in 80% of Merkel cell carcinomas (MCC). Similarly to MCC and Kaposi sarcoma, melanoma incidence is increased in immunosuppressed patients. We asked whether infection by known or yet unknown viruses may play a role in melanoma development as well. To detect viral sequences expressed in melanoma cells, we analysed three melanoma metastases by whole-transcriptome sequencing and digital transcriptome subtraction. None of the samples investigated harboured viral sequences. In contrast, artificial viral sequences and MCPyV transcripts used as a positive control for the bioinformatics analysis were detected. This renders it less likely that viruses are frequently involved in melanoma induction. A larger number of melanoma transcriptome sequencings are required to rule out viruses as a relevant pathogen.

Suppression of casein kinase 1alpha in melanoma cells induces a switch in beta-catenin signaling to promote metastasis.

Casein kinase 1 alpha (CK1alpha) is a multifunctional Ser/Thr kinase that phosphorylates several substrates. Among those is beta-catenin, an important player in cell adhesion and Wnt signaling. Phosphorylation of beta-catenin by CK1alpha at Ser45 is the priming reaction for the proteasomal degradation of beta-catenin. Interestingly, aside from this role in beta-catenin degradation, very little is known about the expression and functional role of CK1alpha in tumor cells. Here, we show that CK1alpha expression in different tumor types is either strongly suppressed or completely lost during tumor progression and that CK1alpha is a key factor determining beta-catenin stability and transcriptional activity in tumor cells. CK1alpha reexpression in metastatic melanoma cells reduces growth in vitro and metastasis formation in vivo, and induces cell cycle arrest and apoptosis, whereas suppression of CK1alpha in primary melanoma cells induces invasive tumor growth. Inactivation of CK1alpha promotes tumor progression by regulating a switch in beta-catenin-mediated signaling. These results show that melanoma cells developed an efficient new mechanism to activate the beta-catenin signaling pathway and define CK1alpha as a novel tumor suppressor.

Serum components and activated Ha-ras antagonize expression of perivenous marker genes stimulated by beta-catenin signaling in mouse hepatocytes.

Hepatocytes of the periportal and perivenous zones of the liver lobule show marked differences in the contents and activities of many enzymes and other proteins. Previous studies from our and other groups have pointed towards an important role of beta-catenin-dependent signaling in the regulation of expression of genes encoding proteins with preferential perivenous localization, whereas, in contrast, signaling through Ras-dependent pathway(s) may induce a 'periportal' phenotype. We have now conducted a series of experiments to further investigate this hypothesis. In transgenic mice with scattered expression of an activated Ha-ras (Ha-ras(G12V)) mutant in liver, expression of the perivenous markers glutamine synthetase and two cytochrome P450 isoforms was completely abolished in those hepatocytes demonstrating constitutively activated extracellular signal-regulated kinase activity, even though they were located directly adjacent to central veins. Similarly, incubation of primary hepatocytes or hepatoma cells with increasing amounts of serum caused a concentration-dependent attenuation of expression of perivenous marker mRNAs, whereas the expression of periportal markers was increased. The inhibitory effect of high amounts of serum on the expression of perivenous markers was also observed if their expression was stimulated by activation of beta-catenin signaling, and comparable inhibitory effects were seen in cells stably transfected with a T-cell factor/lymphoid-enhancing factor-driven luciferase reporter. Epidermal growth factor could partly mimic serum effects in hepatoma cells, and its effect could be blocked by an inhibitor of extracellular signal-regulated kinase activity. These data suggest that activation of the Ras/mitogen-activated protein kinase (extracellular signal-regulated kinase) pathway favors periportal gene expression while simultaneously antagonizing a perivenous phenotype of hepatocytes.