The kinetics of T and B cell immune recovery after bone marrow transplantation (BMT) is affected by many pre- and post-transplant factors. Because of the profoundly depleted baseline T and B cell immunity in recombination activating gene 2 (RAG-2)-deficient severe combined immunodeficiency (SCID) patients, some of these factors are eliminated, and the immune recovery after BMT can then be clearly assessed. This process was followed in ten SCID patients in parallel to their associated transplant-related complications. Early peripheral presence of T and B cells was observed in 8 and 4 patients, respectively. The latter correlated with pre-transplant conditioning therapy. Cells from these patients carried mainly signal joint DNA episomes, indicative of newly derived B and T cells. They were present before the normalization of the T cell receptor (TCR) and the B cell receptor (BCR) repertoire. Early presentation of the ordered TCR gene rearrangements after BMT occurred simultaneously, but this pattern was heterogeneous over time, suggesting different and individual thymic recovery processes. Our findings early after transplant could suggest the long-term patients' clinical outcome. Early peripheral presence of newly produced B and T lymphocytes from their production and maturation sites after BMT suggests donor stem cell origin rather than peripheral expansion, and is indicative of successful outcome. Peripheral detection of TCR excision circles and kappa-deleting recombination excision circles in RAG-2-deficient SCID post-BMT are early markers of T and B cell reconstitution, and can be used to monitor outcome and tailor specific therapy for patients undergoing BMT. Severe combined immunodeficiency (SCID) is characterized by significantly low levels of T and B cells and profound defective immune function. Bone marrow transplantation (BMT) is the life-saving and life-sustaining treatment procedure for such patients in order to restore their T and B cell immunity The thymus and the bone marrow are the primary anatomic sites for T and B cell neogenesis from undifferentiated hematopoietic progenitor cells. Within these organs, hematopoietic progenitor cells that have been committed to the T and B cell lineage undergo rapid proliferation and differentiation to mature cells. During this process, a diverse receptor repertoire is formed, and the resulting cells are able to respond to a wide array of internally and externally processed antigens A major challenge in the field of BMT is the overcoming the difficulty of monitoring the efficacy of the procedure, especially since the prognosis is affected by many pre- and post-transplant parameters In the present study, we examined the early release of newly formed T and B cells from the thymus and the bone marrow in the peripheral blood of 10 RAG-2-deficient SCID patients who received a transplantation of hematopoietic stem cells from healthy donors. This experimental population provides a unique opportunity to examine immune reconstitution and to identify clinical factors that influence the rapidity and extent of T and B cell neogenesis in vivo.Ten consecutive patients diagnosed at the Sheba Medical Center  as having RAG-2-deficient SCID were enrolled in this study which was approved by the Institutional Review Board. Parents provided signed informed consent to obtain blood from their children. Diagnosis was based on clinical findings, family history, and immunological evaluation, and confirmed by genetic testing. Patients who lacked histocompatible siblings or closely matched related donors (MRD) received match unrelated donor (MUD) or mismatch related donor (MMRD) BMTs. The minimum follow-up after BMT was 6 months. Lymphocytes in all patients were tested for human leukocyte antigen (HLA) A, B, C and DR typing using standard serological methods, or DNA hybridization with sequence-specific oligonucleotide probes in order to find a suitable donor.2/day) was administered to patients who received bone marrow from cytomegalovirus (CMV)-seropositive donors or to CMV-seropositive patients who received stem cell transplantation from a seronegative donor. All blood products were CMV safe and irradiated. Pre-transplant conditioning therapy consisting of various drugs was given to some patients, and the severity of GVHD was graded as described previously 9/l for 3 consecutive days.The patients were treated in positive pressure with heap filter units from the time of diagnosis until discharge. Trimethoprim/sulfametaxozole was used for Pneumocystis jiroveci prophylaxis. Intravenous immunoglobulins  were infused to maintain IgG levels above 6 g/l. Acyclovir  of polymorphic markers. Multiplex tetranucleotide repeat PCR was performed on 2 ng of genomic DNA using the AmpFlSTR SGM Plus kit (Applied Biosystems), which amplifies 10 tetranucleotide repeat loci (plus the X–Y homologous gene amelogenin) labeled in three different colors . Amplified PCR products were subjected to capillary electrophoresis in an ABI Prism 3100 (Applied Biosystems) automated DNA sequencer under the conditions recommended by the manufacturer. The GeneScan 3.7 Analysis software (Applied Biosystems) was used for the analysis of the PCR products.Neutrophil engraftment was considered to have occurred on the first of 3 consecutive days in which the absolute neutrophil count exceeded 0.5×10Cell surface markers of peripheral blood mononuclear cells (PBMCs), using immunofluorescent staining and flow cytometry  with antibodies purchased from Coulter Diagnostics, and lymphocyte proliferation in response to phytohemagglutinin, using tritiated thymidine incorporation, were determined as previously described 8 Briefly, the immunoglobulin heavy chain (IGH) and TCR-γ genes were amplified by PCR using fluorescence-labeled Vh primers or Vγ primers, respectively, according to the standardized Biomed 2 protocol. Fluorescence-labeled PCR products (1 µl of each) were added to a mixture of 8.5 µl deionized formamide and 0.5 µl GeneScan 500TM Rox internal lane standard , and separated using the 3100 Genetic Analyzer (Applied Biosystem). Results were analyzed using the GeneMapper software (Applied Biosystem).TCR and BCR gene rearrangement analyses were performed on genomic DNA obtained from patient's PBMCs without specific cell enrichment, as previously described.3 to 106 copies of the signal joint TREC plasmid. A triplicate was used for each dilution. The obtained cycle threshold (Ct) values of the standard curve were the same in all experiments ±0.5 cycle: 20.8 cycles for 106 copies, 23.6 cycles for 105 copies, 27.7 cycles for 104 copies, and 30.1 cycles for 103 copies. TREC copies were >400 in 40 samples in which immunodeficiency was ruled out. The number of TRECs in a given sample was automatically calculated by comparing the obtained Ct value of a patient's sample to the standard curve using an absolute quantification algorithm.The amount of TRECs was determined by real-time quantitative (RQ)-PCR. Reactions were performed using 0.25 µg genomic DNA extracted from the patient's PBMCs. PCR reactions contained TaqMan universal PCR master mix , specific primers (900 nM) and FAM-TAMRA probes (250 nM) as previously described The amount of signal and coding joint of KRECs was determined by RQ-PCR as described for TRECs above. In order to calculate the change in KREC copies for each patient, the obtained Ct was compared to the Ct of his/her KREC levels before undergoing BMT using the ΛΛCt relative quantification analysis. Amplification of RNAseP  served as a quality control to verify similar amounts of genomic DNA that were used in the assays for both the TREC and KREC analyses. Age-matched normal individuals were used as controls. Each experiment was performed in triplicate, and the threshold line for Ct determination was positioned at the same level.The amount of copies of various TCR gene rearrangements in a given sample was relatively quantified using RQ-PCR methodology as described above. The primers and probes are listed in Patients were diagnosed based on clinical findings supplemented by a basic immune workup and confirmed by genetic analysis. A total of 10 consecutive RAG-deficient SCID patients  were included in this study . Their aThe mean age at transplantation was 6 months (range 3–9 months) . Six patPre-transplant TREC and KREC levels were undetectable in all patients as expected in RAG-2 SCID (data not shown). TREC and KREC copies were quantified in order to assess the neogenesis of newly produced T and B lymphocytes after BMT  and 2. EEarly peripheral presence of newly produced B lymphocytes in the various patients was detected after BMT. KRECs were detected early in 4 of the 10 patients (#7–10) , and allAlthough it is typically not expressed on the cell surface of TCR α/β cells, the γ-chain gene remains rearranged and can be used as a marker for a proper diverse rearrangement of the TCR genes. Therefore, we performed PCR analysis of TCRv γ gene rearrangements in peripheral blood obtained from the patients before and after BMT. Analysis of 4 V γ rearranged TCR genes before BMT revealed different abnormal patterns: monoclonal in 3 patients, oligoclonal in 1 patient, and undetectable in 6 patients. These patterns correlated with the clinical phenotypes of the disease: the 4 patients who displayed the Omenn phenotype (patients #1 and #7–9) had mono- or oligoclonal patterns, while the SCID patients with no Omenn features had an undetectable repertoire. A representative repertoire is given for each pattern in Each patient underwent PCR analysis of 2 distinct IgH BCR gene rearrangements in peripheral blood obtained from them before and after BMT. As expected, the BCR repertoire upon diagnosis of SCID was completely abnormal (data not shown). Between 2–4 months after BMT, they displayed different patterns of the BCR repertoire in agreement with their clinical outcome. Polyclonal, oligoclonal and monoclonal BCR repertoire patterns were found in 7, 1 and 2 patients, respectively, with a good outcome (#6–10) comparing to 0, 3 and 7 patients, respectively, with a poor outcome (#1–5) . InteresThe TCR is created through a sequential order of recombination events between the different TCR genes (TCRD>TCRG>TCRB>TCRA). This sequential order also occurs within each individual gene and can therefore represent different stages of T cell maturation. In order to test if thymus recovery after BMT includes the simultaneous appearance of cells representing these different maturation stages, we measured 4 different TCR gene rearrangements in the peripheral blood of 5 of our RAG-2 SCID study patients over time . VariousRAG-2-deficient patients have the typical T-B-SCID-variant. The undetectable TREC and KREC levels pre-transplantation common to all the patients in our cohort is clearly indicative of absent or dysfunctional T and B cells. We demonstrated that high proportions of T and B cells containing signal joints appeared early after BMT, suggesting cell neogenesis rather than cell proliferation. Their emergence was found to be closely related to the patients' clinical outcome as well as to their having undergone pre-transplant conditioning.+ T cells have been reached, and further characterization of these cells almost always reveals marked functional impairment and a markedly skewed TCR repertoire + cells in peripheral blood T cells begin to engraft quickly after hematopoietic stem cell transplantation, and substantial numbers of phenotypically mature T cells can be detected in peripheral blood within 3 months after transplantation. However, from 6 to 9 months are often required before normal numbers of circulating CD3The different ordered TCR gene rearrangements that occur during thymic maturation of T cells represent different stages of T cell development B cell immune reconstitution and their early peripheral presence after BMT has not been investigated in depth. Even the decision when to discontinue gamma globulin treatment after BMT is not certain, and many parameters, including the duration of therapy, trough levels of serum IgM, IgG, or both, and/or the ability to make antigen-specific antibody after immunization are being considered. The majority of patients lacking donor B cells will require immunoglobulin replacement for life The heterogeneity of our study population in terms of age at the time of transplant, the type of the donors and the conditioning protocols are possible limitations of this work. A recent report showed that the outcome after BMT for SCID is irrespective of donor choice, the conditioning regimen used, and the underlying genetic diagnosis. The overall improved survival is related to a reduced rate of infections which can be due to early diagnosis To summarize, a unique group of patients with profoundly depleted baseline T and B cell immunity was used to track the kinetics of T and B cell immune recovery after BMT. We showed that the early peripheral presence of newly produced B and T lymphocytes from their production and maturation sites indicated a donor stem cell origin. Early release of newly produced B and T lymphocytes was closely associated with disease outcome. Determination of immune reconstitution involves T and B cell recovery and ability to maintain immune tolerance. These capabilities will help to ensure successful engraftment, prevent infection and other BMT-related complications, and provide tolerance to self-antigens. TREC and KREC quantification are important assays for monitoring T and B cell neogenesis and homeostasis after BMT, and, as such, can serve as tools to monitor outcome and to tailor individualized therapy.


EYS revealed its contribution to disease. In view of the high carrier frequency for retinal disease gene mutations in the general population, we considered the overall variant load in each patient to assess if a mutation was causative or reflected accidental carriership in patients with mutations in several genes or with single recessive alleles. For example, truncating mutations in RP1, a gene implicated in both recessive and dominant RP, were causative in biallelic constellations, unrelated to disease when heterozygous on a biallelic mutation background of another gene, or even non-pathogenic if close to the C-terminus. Patients with mutations in several loci were common, but without evidence for di- or oligogenic inheritance. Although the number of targeted genes was low compared to previous studies, the mutation detection rate was highest (70%) which likely results from completeness and depth of coverage, and quantitative data analysis. CNV analysis should routinely be applied in targeted NGS, and mutations in non-coding exons give reason to systematically include 5′-UTRs in disease gene or exome panels. Consideration of all variants is indispensable because even truncating mutations may be misleading.Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are major causes of blindness. They result from mutations in many genes which has long hampered comprehensive genetic analysis. Recently, targeted next-generation sequencing (NGS) has proven useful to overcome this limitation. To uncover “hidden mutations” such as copy number variations (CNVs) and mutations in non-coding regions, we extended the use of NGS data by quantitative readout for the exons of 55 RP and LCA genes in 126 patients, and by including non-coding 5′ exons. We detected several causative CNVs which were key to the diagnosis in hitherto unsolved constellations, e.g. hemizygous point mutations in consanguineous families, and CNVs complemented apparently monoallelic recessive alleles. Mutations of non-coding exon 1 of  CEP290 previously reported to be present in 20% of patients with Leber congenital amaurosis (LCA) and RPGR in male RP patients Retinal dystrophies result from degeneration of photoreceptor and retinal pigment epithelium cells. With a prevalence of ∼1 in 3,000, they represent the major cause of hereditary blindness in developed countries NGS of 55 genes involved in RP and LCA  and more complete (15-fold for more than 99% of target sequences). 37% of the samples sequenced on the 454 platform were mutation-negative (29 of 79 samples), compared to only 18% sequenced on the MiSeq (7 of 38 samples). CNV analysis was only possible with high-coverage NGS as obtained with the MiSeq system.RP1 and EYS in arRP, and RPGRIP1, GUCY2D and TULP1 in LCA) were more prevalent, mutations in many rare genes account for the majority of patients, confirming that these phenotypes are genetically highly heterogeneous and only comprehensively accessible by highly parallel sequencing of all known disease genes. CEP290, previously reported as the predominant LCA gene, was not a major contributor to this phenotype in our cohort, and its hot spot mutation, c.2991+1655A>G, was not found at all. This may partially be due to the ethnic background of LCA patients in our cohort with 43% of patients originating from the Arabian peninsula. In contrast to other large studies USH2A mutations contributed only to a small proportion of arRP. Causative mutations were found in 28 different genes that encode proteins from diverse pathways and cellular compartments. Mutations in ciliary genes were most prevalent . More specifically, causative mutations were detected in 38/53 patients (72%) with autosomal recessive (ar) and in 12/14 (86%) with autosomal dominant (ad) RP . Three prevalent , indicatVirtually any gene may be captured and subjected to NGS aimed not only at qualitative, but also quantitative readout. This utilization of NGS data enables CNV detection and can favourably complement MLPA (multiplex ligation-dependent probe amplification), where the application depends on the availability of commercial kits that currently cover only a fraction of known RD genes. We identified four alleles with pathogenic CNVs comprising one to multiple exons. Below, we describe exemplary constellations with CNVs contributing to retinal disease.EYS account for 5–18% of arRP cases depending on the population EYS mutations were found in 9.4% of arRP patients (five families). One patient was compound heterozygous for a truncating mutation in the coding region and a deletion of non-coding exon 1 at least , predicting an elongated protein with 118 unrelated residues (p.*300Trpext*118). Subsequent quantitative analysis revealed a heterozygous deletion of exon 4 in trans to the no-stop mutation which was thereby recognized as hemizygous  RP11 families often display incomplete penetrance, and dominant inheritance may not be obvious from the family history. In five patients, we identified heterozygous PRPF31 mutations, including deletions of multiple (patient 116) or even all coding exons (patient 113)  of the Usher protein interactome AHI1 and PDZD7Given the multitude of genes implicated in RP and LCA, it is not surprising that NGS, providing a “full picture” of the mutational load, identifies constellations with mutations in several genes. In view of a recent study of genome sequences from 46 control individuals from various regions of the world indicating that one in 4–5 individuals from the general population may be a carrier of null mutations in a gene for inherited retinal degeneration RP1, the most prevalent arRP gene in our cohort, was frequently found together with mutations in other RD genes. The observed constellations resulted in different deductions regarding the pathogenicity of the respective RP1 allele:However, we identified patients with causative biallelic mutations in recessive RP genes who additionally carried heterozygous truncating mutations in secondary loci. RP1 mutations are mostly truncating and may cause adRP RP1 mutations were observed in our adRP patients, but RP1 was the most prevalent arRP gene, with clearly causative biallelic mutations in several cases  and c.3157delT (p.Tyr1053Thrfs*4), and additionally carried a nonsense mutation in CDH23 which has previously been described in recessive deafblindness  RP1 and CDH23, both encoding proteins of the photoreceptor’s connecting cilium, was compatible with both RP1 alleles acting recessively  was found in LCA patient 124, on a homozygous TULP1 nonsense mutation background  demonstrate that even truncating mutations must be assessed with caution and in the context of the full variant load of known disease genes in order to avoid false interpretations: If a monoallelic truncation is found in addition to clearly causative biallelic mutations in another gene, it may either represent accidental carriership for a pathogenic allele (unrelated to disease in that patient) or a non-pathogenic variant in a non-essential gene region .These three scenarios with in-frame deletion-insertion mutation in TULP1 (p.Asp124_132delinsAla) was identified in two independent simplex RP patients (patients 33 and 82), and an SAG nonsense mutation was found in patient 29. Patient 33 in addition carried two CRX missense variants, p.Arg41Gln and p.Tyr142Cys, that we consider likely benign . The sporadic occurrence of RP suggests autosomal recessive inheritance in all three patients, making a dominant-negative effect of the TULP1 and the SAG mutation unlikely. The three DNA samples with monoallelic mutations in TULP1 and SAG mutations were initially sequenced on the GS FLX system; subsequent analysis on the MiSeq platform did not identify additional mutant alleles, in particular no CNVs.Monoallelic mutations in recessive disease genes represent a challenge for interpretation regarding their causality in the patient, especially if there are no biallelic mutations in another gene for the trait that would qualify such mutations as incidental findings . While single non-synonymous variants in recessive disease genes may often represent rare non-pathogenic variants see , the natTULP1 and SAG mutations with the causative mutation in another arRP gene not known at the time of study design, or the “missing alleles” escaped detection by exonic sequencing because they are deep intronic (as exemplified by the LCA mutation c.2991+1655A>GCEP290 or the only known RP23 mutation in the OFD1 gene EYS exon 1 in this study).Patients 33, 82 and 29 are therefore either accidental carriers of the EYS suggests that such exons should be included in upcoming disease gene panels. Mutation-negative cases in our study will in part be due to mutations in RP and LCA genes that were identified after the design of our gene panel . For example, subsequent exome sequencing for patient 15 identified a homozygous nonsense mutation in IMPG2 (data not shown). Updating the panel accordingly will identify the causative mutations in additional patients.As discussed above, mutations in known retinal dystrophy genes may escape detection because of their localization – about 15% of disease-causing mutations localize outside coding exonic sequences RP2 and RPGR have been reported to account for 8.5% of cases with RP of apparently autosomal dominant transmission and for 15% of males with simplex retinal degenerative disease RP2 is uncomplicated, the mutational hot spot exon of RPGR, ORF15, is not accessible by our NGS approach due to its highly repetitive sequence. Because about 2/3 of RPGR mutations reside in RPGRORF15RPGRORF15. However, there was no excess of male mutation-negative RP patients: 41% of RP patients without mutations were male which corresponds to their percentage (40%) in the RP cohort (excluding the three proven X-RP patients).Mutations in the X-linked RP genes PRPF31 detected by quantitative analysis of MiSeq reads resolved the diagnosis in two patients with seemingly recessive RP, and a CNV in EYS represented the “missing allele” in one patient.The rate of mutation-negative samples sequenced on the Illumina MiSeq system was only half compared to the Roche GS FLX platform (18% versus 37%). In contrast to 454 sequencing (GS FLX), analysis of homopolymer stretches is not problematic in Solexa sequencing (MiSeq). Because very few mutations identified by supplementary Sanger sequencing  or with the Miseq were found in such sequence motifs, the higher detection rate on the MiSeq was mainly due to a better coverage in terms of completeness and depth . As detailed above, CNVs in Finally, lack of mutations may result from unclear genetic diagnosis: If an older patient is for the first time seen by an ophthalmologist at a late stage of his disease, it may be impossible to assess if the initial disease was RP or CRD; CRD genes are not comprehensively covered by our current gene panel and the causative mutation could therefore be missed.This is the largest NGS study on retinal dystrophies to date. Compared to other NGS studies on this disease group, we obtained a significantly higher diagnostic yield – which is remarkable because the number of analyzed disease genes (55) in this study was much smaller than in similar studies RP1 truncations unrelated to disease in certain constellations. “Missing alleles” in seemingly accidental carriers of recessive RD gene mutations were partly large CNVs and mutations affecting non-coding 5′ exons, demonstrating that both UTR inclusion and quantitative analysis should be part of a comprehensive NGS approach. Because such mutations may also be deep intronic variants with impact on splicing, genomic sequencing, where necessary followed by RNA analysis, may complement primary exonic sequencing in the future. Careful consideration of all variants led to revision of the assumed mode of inheritance, e.g. in case of PRPF31 mutations in simplex RP patients.In conclusion, the identification of mutations in 28 RD genes in our cohort, with most alterations previously undescribed, clearly demonstrates that this disease group is accessible only by massively parallel multi-gene sequencing. Although our NGS study was rather conservative and confined to only 55 genes, we detected the causative mutations in the majority from a large cohort of RP and LCA patients. Regular updating of such panels and inclusion of genes for related disorders (e.g. cone-rod dystrophies) is needed to maximize the mutation detection rate. CNV detection from high-coverage NGS data was a major benefit from switching to a high-capacity NGS platform. Therefore, we currently favor NGS of an RD gene panel over exome sequencing where RD gene coverage is reduced due to distribution of reads across some 20,000 genes. Both, oligogenic heterozygosity and monoallelic constellations were observed and may require segregation analysis and careful evaluation of clinical data. Importantly, NGS readout should implicate the overall variant load in order to avoid interpretation pitfalls – as exemplified by the identification of As indicated by several exceptional findings in our study, scientific gain of knowledge will strongly benefit from the recent advent of NGS in routine diagnostics and the “byproducts” of such unprecedented large-scale analyses – not only for RD, but for many other genetically heterogeneous conditions.All samples in this study were obtained with written informed consent accompanying the patients’ samples. All clinical investigations have been conducted according to the principles expressed in the Declaration of Helsinki. The study was approved by the institutional review board of the Ethics Committee of the University Hospital of Cologne.A total of 126 patients  were included in this study. Genomic DNA was isolated from EDTA blood following standard protocols. The diagnoses of all patients were established by medical history, family history and detailed clinical evaluation of vision. Ophthalmological examination included stereoscopic funduscopy, standard ERG, perimetry, measurement of dark adaptation, and determination of best-corrected visual acuity in most patients.RPGR exon ORF15 was excluded from panel design. Because the USH2A gene was not included in the panel design, all coding exons of the gene were analyzed either by conventional Sanger sequencing or by a complementary USH2A-including NGS gene panel in arRP patients without mutations in the RP genes covered by our panel.Genomic coordinates of coding and non-coding exons in all isoforms were identified in the RefSeq database (hg19) using the University of California Santa Cruz (USCS) table browser Samples from 79 patients were subjected to NGS on the Roche GS FLX platform . In the second part of the study, 38 samples were sequenced on the Illumina MiSeq system  with only the latter allowing for CNV detection due to high and uniform coverage. Samples from nine patients were analysed on both systems . Between eight (GS FLX) and 20 (MiSeq) samples were pooled and sequenced in a multiplexing procedure. Multiple DNAs were enriched using the NimbleGen SeqCap EZ choice sequence capture approach and sequenced by Roche 454 GS FLX pyrosequencing or by Illumina MiSeq sequencing-by-synthesis technology according to the manufacturerś protocols. In brief, 0.5–1 µg of genomic DNA per sample was sheared using the Covaris S2 AFA system  and ligated to barcoded adaptors for multiplexing. Pre-capture amplified samples were pooled and hybridized to the customized in-solution capture library for 72 hours, subsequently eluted and post-capture amplified by ligation-mediated (LM-) PCR. This amplified enriched DNA was used as input for emulsion PCR (emPCR) and subsequent massively parallel sequencing on one full PTP of a Roche 454 GS FLX platform or as input for direct cluster generation and sequencing on the Illumina MiSeq system (2×150 bp paired-end reads). Uncovered regions of LCA genes (n = 16) in negative samples from LCA patients designated as having “LCA” were sequenced by conventional Sanger sequencing for completeness, whereas in RP samples, gaps of uncovered exons of arRP genes (n = 31) samples were only eliminated by Sanger sequencing in search of a second mutation in an incompletely covered arRP gene).http://picard.sourceforge.net). Finally, GATK in silico prediction software tools: SIFT Demultiplexed reads from the GS FLX platform or paired end reads (2×150 bp) from the Illumina MiSeq instrument were mapped against the hg19 human reference genome using SMALT  with the recommended standard settings. The mapped reads were preprocessed with SAMtools Sequence variants of interest identified by high-throughput sequencing were verified by Sanger sequencing following PCR amplification of the respective coding exons and adjacent intronic sequences by standard protocols. Purified PCR fragments were sequenced using Big Dye Terminator Cycle sequencing and analyzed on an 3500 Genetic Analyzer sequencer . Where applicable, DNA from affected and unaffected family members was analyzed for segregation analysis of putatively causative sequence variants. The resulting sequence data were compared to the reference sequence of the RefSeq database ftp://hgdownload.cse.ucsc.edu/goldenPath/hg19/database/refGene.txt.gz). Potential CNVs were visualized and recalculated with the CNV mode of SeqNext using standard settings and the analysis mode “all vs. all.” Thereby, the normalized relative coverage of every target ROI (region of interest) of a patient sample  was calculated against the normalized average relative target coverage of several control samples (RPC C.) to obtain the ratio relative coverage (ratio RPC). Deletions were reported if the ratio RPC fell below 75%. CNVs that fulfilled these criteria were validated by multiplex ligation dependent probe amplification (MLPA) for the affected gene. For the EYS gene, the SALSA MLPA probemix P-328-A1 EYS, for the CRX gene the SALSA MLPA probemix P221-B1 LCA and for the PRPF31 gene the SALSA MLPA KIT P235-B1 Retinitis Pigmentosa was used . Only CNVs that could be confirmed by MLPA were considered real. MLPA results were visualized with the MLPA module of the SeqPilot software . The ratio RPA (relative peak area) was calculated as the RPA of the patient versus controls.Very high coverage was reproducibly achievable by sequencing with the Illumina MiSeq system and enabled copy number variation (CNV) analysis for most of the analyzed genes. Potential copy number alterations (CNA) were initially identified by VarScan CEP290 gene mutation in all LCA patients prior to NGS analysis, the region of interest in intron 26 was amplified by PCR. Genotyping for the presence of the mutation was performed by pyrosequencing using QIAGEN Pyro Gold chemistry according to the manufactureŕs instructions and subsequent analysis on a PSQ 96MA system .For exclusion of the common c.2991+1655A>G mutation in the In the family of patient 110 afflicted with LCA, we performed genome-wide homozygosity mapping using the Affymetrix GeneChip Human Mapping 10K Array, version 2.0 . GRR File S1File S1 contains the following files.Figure S1.CNVs  of PRPF31 detected by analysis of NGS data. A heterozygous deletion of all 14 PRPF31 exons was identified in patient 113. In patient 116, exons 1–5 were deleted on one gene copy . The dashed line and red arrows indicate lower coverage for heterozygously deleted regions compared to one control sample. Figure S2. Validation of CNVs predicted from NGS data by MLPA. Only confirmed CNVs were considered true CNVs. A. Heterozygous deletion of exon 1 in the EYS gene in patient 57 and his father. B. Heterozygous deletion of exon 4 in the CRX gene in patient 110 and his father. C. Heterozygous deletion of exons 1–14 in the PRPF31 gene in patient 113 and of exons 1–5 in patient 116. RPA: Relative peak area of the patient result file (green) and of the control result files (blue) with standard deviation (error bar). The ratio RPA was calculated as the RPA of the patient versus controls. Deletions are indicated if the ratio RPA falls below 75%. Table S1. Genes analyzed in this study. A. arRP, adRP and LCA genes that were captured and subjected to NGS in this study. B. Functional categorization of genes with causative mutations. Table S2. Additional variants classified as “likely pathogenic”. Classification as pathogenic by at least three out of five bioinformatic prediction programs and a minor allele frequency below 3% in unresolved patients. Although a contribution of these variants to the phenotype cannot be excluded, they were not considered causative. In many cases, they represented monoallelic variants in recessive genes which would not sufficiently explain the phenotype. References S1. References for  and Table S2.(ZIP)Click here for additional data file.


The importance of CD4 T cells in orchestrating the immune system and their role in inducing effective T cell-mediated therapies for the treatment of patients with select established malignancies are undisputable. Through a complex and balanced array of direct and indirect mechanisms of cellular activation and regulation, this functionally diverse family of lymphocytes can potentially promote tumor eradication, long-term tumor immunity, and aid in establishing and/or rebalancing immune cell homeostasis through interaction with other immune cell populations within the highly dynamic tumor environment. However, recent studies have uncovered additional functions and roles for CD4 T cells, some of which are independent of other lymphocytes, that can not only influence and contribute to tumor immunity but paradoxically promote tumor growth and progression. Here, we review the recent advances in our understanding of the various CD4 T cell lineages and their signature cytokines in disease progression and/or regression. We discuss their direct and indirect mechanistic interplay among themselves and with other responding cells of the antitumor response, their potential roles and abilities for “plasticity” and memory cell generation within the hostile tumor environment, and their potentials in cancer treatment and immunotherapy. Cancer cells express antigens that differentiate them from their non-transformed counterparts , most notably dendritic cells (DCs), encounter and capture tumor antigens that are released from either viable or dying tumor cells. This results in the activation and up-regulation of co-stimulatory molecules that facilitate and/or promote the migration of these cells to secondary lymphoid organs such as regional draining lymph nodes  and T-bet transcription factors during immune activation of naïve T cells . ROR-γt is a critical regulator of Th17 cell differentiation and induces IL-17A, IL-17F, IL-26, and CCR6 expression while downregulating IFN-γ production in human naïve T cells . This cell lineage represents a third effector arm of CD4-mediated immune response and complements, in part, the functions of the Th1 and Th2 cell lineages. In addition to IL-17A and IL-17F, human Th17 cells also produce other cytokines such as IL-21, IL-22, and IL-26. In addition, the chemokine receptor CCR6 [which binds to the chemokine CC ligand 20 (CCL20) that is present in many malignant pleural effusions of lung cancer patients] is highly expressed on Th17 cells thus further facilitating their recruitment to sites of tumor growth and inflammation  promoter , are responsible for providing B cell help and supporting B cell expansion and differentiation  transcription factor may be crucial in driving the development of cytotoxic CD4 T cells in vivo , constitutively expressing FoxP3 and the activation marker CD25, originate in the thymus by high affinity interaction of the T cell receptor (TCR) with Ag expressed on the thymic stroma  exert suppression mostly through soluble factors and their suppressive function is not strictly associated with a high level of FoxP3 expression. Moreover, human TReg cell subpopulations have also been further divided into two subsets based on their expression of the “resting” CD45RA (a marker of naïve or antigen-inexperienced cells) or “activated” CD45RO (a marker for memory or antigen-experienced T cells) cell surface markers  express the CC-chemokine receptor CCR7 and L-selectin CD62L following activation. Expression of these receptors enable the TCM subgroup to recirculate through secondary lymphoid organs such as lymph nodes. Such circulation is beneficial since DCs from diverse tissue sites continuously bring antigen to the draining lymph nodes, thereby increasing the effective area of memory cell immunosurveillance for progressing tumor growth due to metastases and/or occult cell outgrowth. Alternatively, effector memory T cells (TEM) lack expression of CCR7 or CD62L and thus have a propensity to migrate to peripheral tissues in response to localized inflammatory stimuli and bolster the process of immune surveillance at such sites. Moreover, expression of other surface chemokine receptors such as CCR5 have been associated with polarized Th1 memory T cell subsets  among responding CD8 T cells promoted the generation of terminally differentiated short-lived effector cells whereas lower levels facilitated long-lived, self renewing memory T cell development  that is associated with the development of memory T cells  suggesting a role for this transcription factor in the regulation of polarized CD4 T cell functions  were associated with a higher level of IFN-γ production during the recall response in a corresponding cell subpopulation  in the development of Th1 cell differentiation and memory phenotype under various stimulating conditions ex vivo  have been implicated in promoting inflammation responsible for immunopathology in both cancer and several autoimmune disorders. Studies in various murine tumor models have suggested that Th17 cells may be associated with tumor initiation and growth in the context of chronic inflammation (Kawakami et al., Alternatively, pre-clinical murine tumor studies have correlated the presence of intratumoral Th17 cells with reduced tumor growth and effective antitumor immunity (Muranski et al., in vivo (Muranski et al., Investigations on the association between Th17 cells and promising clinical outcomes in cancer patients, have suggested a link and close interplay between the Th17 and Th1 cell lineages (Kryczek et al., + TReg cell subsets have been associated with the prevention of immunopathology during excessive and/or unwanted inflammation and prevention of autoimmune disease. However, in the context of cancer, such cells have been associated with facilitating the suppression of the antitumor response through various tolerance induction and tissue homeostatic mechanisms. However, the role and prognostic value of TReg cells in cancer has recently been disputed (Wilke et al., + TReg cell expression has been associated with both a poor prognosis and diminished survival rates (Woo et al., +FoxP3+ T cells in malignant ascites of patients with ovarian carcinomas correlated with advanced tumor staging and reduced survival. Alternatively, in colorectal cancer, several investigators did not find any differences between patients with high or low TReg cell infiltration (Loddenkemper et al., + cells suggesting no major immunosuppressive role of TReg cells in colorectal cancer (Salama et al., The immunoregulatory roles of CD4in vivo significantly reduced human TReg cell trafficking to ovarian carcinomas (Curiel et al., in vitro migration assays (Mizukami et al., + TReg cell trafficking (Kleinewietfeld et al., + TReg cell subpopulations may become enriched within tumors, through a variety of different chemokine receptor/ligand interactions. Furthermore, cytokines and chemokines produced, in part, by either tumor cells, tumor infiltrating lymphocytes, and/or APCs within the tumor milieu may preferentially support such TReg cell expansion, retention, survival, and in some cases, their “further” differentiation and/or change in phenotype (Campbell and Koch, + TReg cell subsets is to maintain and/or restore local immune cell homeostasis during polarized Th1, Th2, and Th17 cell-mediated immune responses. This led to the identification and observation that human Th cell subsets and TReg cells appear to undergo functional specialization in parallel resulting in the development of functionally distinct TReg cell subsets capable of co-localizing with and effectively regulating different types of Th cell responses in vivo (Hall et al., + TReg cells can down-regulate FoxP3 expression and their associated regulatory properties and in some cases acquire an effector cell phenotype that expresses IFN-γ and/or IL-17 (Gavin et al., +FoxP3+ TReg cells remains controversial (Rubtsov et al., Select chemokines and their corresponding receptors have been shown to play a role in the recruitment of specific T cell subsets into tumors and sites of inflammation (Sallusto et al., + TReg cells may possess other underappreciated anti-cancer functions. For example, such cells may have the ability to limit the extent and potential of inflammatory responses to induce tumor development and carcinogenesis. Using a murine herpes viral model, investigators observed that TReg cell-mediated down-modulation of inflammatory responses in secondary lymphoid tissues can actually optimize ensuing immune responses to local infection by more effectively redirecting it to sites of initial infection (Lund et al., + TReg cells may inhibit tumor-promoting inflammatory responses induced by local microbes, which may help explain their presence and favorable association with outcomes to these cancers (Haas et al., Alternatively, CD4The immune system has the capacity to either obstruct tumor development and deter established tumors, or to promote carcinogenesis and tumor progression. Here, we reviewed how CD4 T cells and their various functionally distinct subpopulations contribute to the antitumor immune response and potentially influence this process. Several key principles emerge. Distinct effector and memory CD4 T cell subsets have important roles in the antitumor response. Key among these roles is the provision of orchestrating and/or regulating other immune cells that result in promoting tumor eradication, long-term tumor immunity, and establishing or rebalancing immune cell homeostasis within the tumor environment. Such roles are mediated, in part, by the production of signature cytokines by specific CD4 T cell lineages and through direct cytotoxic effects on tumor cells. The efficacy of such CD4 effector T cell responses may not only depend on memory cell generation, phenotype, and their functional capacity to interact with other immune cell populations, but also in some cases, on their ability to evolve through “cellular plasticity” within the hostile tumor environment. Conceivably, this trait of “cellular Darwinism” by the various CD4 T cell lineages could endow them with considerable flexibility to procure effective tumor immunity. Alternatively, it appears that such CD4 T cell subsets and their signature cytokines can also contribute and facilitate tumor-promoting activities. This may be due, in part, on the abilities of select CD4 T cell lineages and their subpopulations to modulate effector cell population dynamics and thus affect the balance between effective and ineffective antitumor responses. Achieving an “appropriate cellular balance” appears highly context-dependent. Further studies on the biological activities and mechanisms of how the various polarized CD4 effector and/or memory T cell subsets influence and/or facilitate the immune response as a whole in patients with different types of cancers should further enhance the development of more effective cancer treatment strategies.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The extension and the transmurality of the myocardial infarction are of high predictive value for clinical outcome. The aim of the study was to characterize the ability of longitudinal, circumferential and radial strain measured by 2-dimensional speckle tracking echocardiography (2D-STE) to predict the extent of necrosis in myocardial segments following acute myocardial infarction and to separate transmural necrotic segments from non-transmural necrotic segments in a full 18-segment porcine model.2D-STE strain was assessed in long- and short-axis following myocardial infarction in ten open-chest anesthetized pigs. Strain was defined according to systolic peak values. In segments displaying both negative and positive peaks, only the peak with the highest absolute value was utilized. Necrosis was measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining and expressed as percent of each myocardial segment.p < 0.001), but was stronger for longitudinal strain (r2 = 0.52) than circumferential strain (r2 = 0.38) and radial strain (r2 = 0.23). The area under the receiver operator characteristic curve (AUC) for separating transmural necrotic segments (>50% necrosis) from predominantly viable segments (0–50% necrosis) was significantly larger for longitudinal strain  when compared with circumferential strain  and radial strain , indicating a stronger ability of longitudinal strain to identify segments with transmural necrosis.Significant correlations were found between the extension of necrosis and all measured parameters of myocardial deformation (Peak strain values derived from 2D-STE correlate well with the extent of necrosis in myocardial segments following acute myocardial infarction. Longitudinal strain most accurately reflects myocardial segmental viability in this setting. Two-dimensional speckle tracking echocardiography 2D-STE) is an increasingly recognized technique that measures myocardial deformation by automatic tracking of interference patterns from conventional gray scale B-mode images during the cardiac cycle. In contrast to tissue Doppler derived strain, 2D-STE has the advantage of being relatively independent of insonation angle, and can therefore assess motion in three dimensions ,2. 2D-STD-STE is The extent of the necrotic myocardium following myocardial infarction (MI) has prognostic as well as therapeutic implications . Traditi2 was kept between 4 and 6 kPa. Continous anesthesia with midazolam 0.3 mg/Kg/hour , fentanyl 0.02 mg/kg/hour  and pentobarbital 4 mg/Kg/hour (Nycomed Pharma) was established through a central venous catheter in the left jugular vein. The pigs received amiodarone as arrhythmia prophylaxis and 2500 IU of heparin to prevent from clotting of vascular catheters. Blood pressure was measured in the descending aorta with a polyethylene catheter through the right femoral artery and the bladder was drained through a cystostoma. The pigs received 10 ml/Kg/hour glucose enriched sodium chloride (1.25 g glucose/l sodium chloride) as basal fluid replacement. The heart was exposed through a median sternotomy. The pericardium was opened and a flow probe was placed snugly around the pulmonary trunk for continuous measurements of cardiac output. After baseline measurements of basic hemodynamics, MI was induced by placing a small metal clamp on the left anterior descending coronary artery (LAD) 6 cm distal to the division of the left main coronary artery. The position of the LAD clamp was determined from pilot experiments, which indicated that a substantial number of pigs developed ventricular fibrillation during ischemia when the LAD was clamped within a shorter distance from the division of the left main coronary artery. After one hour of ischemia, the clamp was removed and the infarcted area was reperfused for three hours.The experimental protocol was approved by the local steering committee of the Norwegian Animal Experiments Authority. Animal care was done in accordance with guidelines from the U.S. National Institute of Health . Ten pigs fasted overnight, but with free access to water were premedicated with im. ketamine  and atropine  and transferred to the operating room. After iv. bolus injections of fentanyl  and pentobarbital (Nycomed Pharma), the pigs were tracheostomized and ventilated on a respirator . Tidal volume was controlled by repeated blood gas analysis, and pCOAll images were obtained after opening of the pericardium and the recordings were performed at baseline and following 3 hours of reperfusion. The thoracic cavity was filled with 38°C of saline, in order to provide air-free insonation. The left ventricle was visualized by basal, middle and apical short axis images as well as long axis images in 2-, 3- and 4-chamber views Figure . A PhiliAfter three hours of reperfusion, the pigs received bolus injections of pentobarbital. The aorta was clamped and the hearts were perfused with 3 L of saline through a cannulla in the ascending aorta to wash out the blood from the myocardium, which would otherwise interfere with the tetrazolium staining . After sTo estimate intra- and interobserver variability, the full 18-segment model was re-analyzed for longitudinal, circumferential and radial strain in five randomly chosen pigs.Variability was assessed by intraclass correlation coefficients. Intraclass correlation coefficients were analyzed with SPSS  using two-way random single measures with absolute agreement according to intra class correlation coefficient 2.1 by Shrout and Fleiss . Intra-ot tests. The relationship between necrosis and strain was assessed by linear regression. Comparison of the number of segments exhibiting positive strain values between longitudinal and short-axis derived parameters of myocardial deformation were analyzed with Fisher’s exact test. Comparisons of strain values obtained from segments at baseline, and in viable, subendocardial and transmural necrotic segments following MI were analyzed with one way analysis of variance and the Bonferroni posthoc test. The optimal cut off values for longitudinal, circumferential and radial strain were determined from the receiver operator characteristic (ROC) curve by choosing values that combined the highest sensitivity with the highest possible specificity. Diagnostic accuracy was defined as true negative and true positive segments divided by all segments. Comparison of ROC curves was performed with the Medcalc 11.1 statistical program  according to the method described by DeLong et al.[Data are presented as mean ± standard deviation, unless otherwise noted. Differences in hemodynamic variables between baseline and following ischemia and reperfusion were analyzed with paired samples ng et al.. All othBasic hemodynamic data, ejection fraction, end diastolic volume and end systolic volume at baseline and following ischemia and three hours of reperfusion are summarized in Table Representative cross sections of the heart following staining with Evan’s blue and TTC are demonstrated in Figure 2 = 0.52), followed by circumferential strain (r2 = 0.38) and radial strain (r2 = 0.23).Highly significant correlations were found between necrosis and longitudinal, circumferential and radial strain respectively Table . The strVPeak strain values obtained at baseline and following MI from long- and short-axis images are displayed in Figure (t) –L0)/L0, in which L0 represents the end diastolic distance between to pixels and L(t) the longest distance between these two pixels during systole. Accordingly negative strain values indicate shortening of the myocardium, which is normally seen in the circumferential and longitudinal projections. In contrast, there is a thickening of the myocardial wall in the radial direction, which results in positive radial strain under normal conditions. The positive peaks displayed by the transmural necrotic segments in Figure Representative strain curves from viable (gray) and transmural necrotic myocardial segments (black) are displayed in Figure As illustrated in Figure p < 0.05) and radial strain (p < 0.01). Longitudinal strain also had the highest diagnostic accuracy to separate transmural necrotic segments from subendocardial necrotic segments, but the area under the ROC curve failed to reach statistical significance when compared with circumferential strain and radial strain (data not shown).Diagnostic accuracies for these myocardial deformation parameters to discriminate between transmural necrocis and predominantly viable segments were assessed by ROC curves. The corresponding cut off values, sensitivities, specificities and diagnostic accuracies are summarized in Figure Results from inter- and intraobserver variability analysis are summarized in Table The present study is to our knowledge the first that has compared circumferential strain, radial strain and longitudinal strain with respect to the extension of necrosis in acute ischemia in a complete model, covering all segments of the left ventricle. Our results indicate that peak longitudinal strain better separates transmural necrotic segments from predominantly viable segments compared with circumferential strain and radial strain. Chan et al. have previously examined the diagnostic accuracy of circumferential strain and longitudinal strain in patients with chronic ischemic ventricular dysfunction. In that study it was demonstrated that circumferential strain was able to differentiate transmural and subendocardial necrotic segments as well as transmural necrotic and normal myocardial segments . FurtherIn contrast to the study by Chan et al., the present study has utilized peak positive or negative systolic strain values in acute ischemia, which resulted in positive longitudinal strain values in a substantial number of the transmural necrotic segments, indicating systolic stretching of the myocardium. Positive peak longitudinal strain has previously been demonstrated in necrotic myocardium from dogs subjected to occlusion and reperfusion  and was In contrast to the present study, Sjøli et al. utilized peak strain in acute ischemia to demonstrate that circumferential strain separated transmural from subendocardial necrosis better than longitudinal strain on a segmental level. It is noticeable that the mean longitudinal strain values from transmural necrotic segments in the study by Sjøli’s group are considerably lower than those of the present study. As illustrated in Figure Another theoretical assumption for finding differences between humans and pigs could be that the porcine left ventricle has different myocardial fiber orientation compared to humans. However, studies of human and porcine myocardial architecture have reported that they are essentially similar -27. FurtPositive longitudinal strain and negative circumferential strain reported from an identical myocardial segment might seem as a contradiction to the incompressibility of the myocardial tissue. However, as previously demonstrated segments with acute transmural necrosis exhibited biphasic strain curves in all three dimensions, indicating tissue stretch in the first part of systole and a contraction like deformation in opposite direction in the end of systole. Thus, peak positive and negative systolic strain in the three dimensions was measured at different time points during ejection time period. The abnormal systolic longitudinal lengthening (positive strain) seems as an accurate indicator of transmural necrotic segments in the acute phase, while circumferential or radial strain to a significant lesser extent develop abnormal systolic lengthening and radial thinning despite transmural necrosis.The relatively high number of transmural necrotic segments exhibiting positive peak longitudinal strain values when compared with circumferential strain and radial strain may reflect differences in wall stress sustained by the myocardium in long and short axis. According to the law of Laplace, local wall stress increases with increasing radius of the curvature. Longitudinal fibres, might therefore be exposed to higher wall stress compared to the wall stress generated by the relatively smaller radii of the short-axis curvatures, which are relevant for circumferential and radial strain . Thus, dThe pig as a research animal is a limitation to this study. The porcine heart’s poor tolerance to ischemia restricted us to induce relatively small infarctions, which resulted in the vast majority of the transmural necrotic segments being located in the apex. Although our results indicate that peak longitudinal strain better separates transmural necrotic segments from predominantly viable segments than circumferential strain and radial strain in acute ischemia, more research is needed to establish whether this is true also for larger infarctions and in the human heart. On the other hand, 2D-STE might be of more benefit to those patients suffering from ischemia with small infarctions, than patients with large infarctions and obvious signs of extensive myocardial ischemia, such as ST-elevation.The 18-segment model in the present study with six apical segments also makes it difficult to directly compare our results with other studies which have employed 16-segment models. Since the 18-segment model consists of two more apical segments than the 16-segment model, it may be argued that the two extra apical segments are decisive for our results. To examine whether our results would change with four segments in the apex, a modified 16-segment model was created by averaging strain values from both septal and lateral segments. Measurements of necrosis was then recalculated for the two 120° apical septal and apical lateral segments, yielding a modified 16-segment apical model with 4 segments in the apex (2x60° + 2x120°). When ROC curve analysis was performed on this modified 16-segment model, the area under the curve for longitudinal strain was still found to be significantly larger than for circumferential strain and radial strain, for separating transmural necrotic segments from predominantly viable segments.We conclude that 2D-STE provides reliable and reproducible estimates of the extent of segmental necrosis. By measuring peak positive or negative strains during ejection time, our data suggest that longitudinal strain provides the most accurate estimate of the degree of transmurality in an acute ischemia-reperfusion model.AUC: Area under the curve; LAD: Left anterior descending coronary artery; MI: Myocardial infarction; ROC: Receiver operator characteristic; 2D-STE: Two-dimensional speckle tracking echocardiography; TTC: 2,3,5-triphenyltetrazolium chloride.The author(s) declare that they have no competing interests.The authors contributions were as follows: EA, ES and RB designed the study. EA and ES performed the experiments. AR obtained all echocardiographic images. EA and AR performed the echocardiographic analysis. EA carried out statistics and wrote the first manuscript draft. AR, RB and ES were all involved in the revision of the first manuscript draft. All authors read and approved the final manuscript.


Circulating tumor cell (CTC) enumeration promises to be an important predictor of clinical outcome for a range of cancers. Established CTC enumeration methods primarily rely on affinity capture of cell surface antigens, and have been criticized for underestimation of CTC numbers due to antigenic bias. Emerging CTC capture strategies typically distinguish these cells based on their assumed biomechanical characteristics, which are often validated using cultured cancer cells. In this study, we developed a software tool to investigate the morphological properties of CTCs from patients with castrate resistant prostate cancer and cultured prostate cancer cells in order to establish whether the latter is an appropriate model for the former. We isolated both CTCs and cultured cancer cells from whole blood using the CellSearch® system and examined various cytomorphological characteristics. In contrast with cultured cancer cells, CTCs enriched by CellSearch® system were found to have significantly smaller size, larger nuclear-cytoplasmic ratio, and more elongated shape. These CTCs were also found to exhibit significantly more variability than cultured cancer cells in nuclear-cytoplasmic ratio and shape profile. Circulating tumor cells (CTCs) have been implicated as potential seeds of cancer metastasis and are therefore of great importance in research, disease management, and drug development In addition to their expression of tumor antigens, it is broadly accepted that CTCs have distinct biomechanical characteristics, including larger size than leukocytes, greater nuclear to cytoplasmic (N:C) ratio, as well as distinct nuclear morphology We developed a software tool to analyze the cytomorphological properties of cancer cells. We employed this tool to examine both patient CTC and model cancer cell line morphology, following CellSearch® enrichment. These results will provide important data to aid in CTC identification based on combined antigen and biomechanical criteria http://research.ubc.ca/ethics/clinical-research-ethics-board). The CRPC patients included in this study ranged in age, from 53–83 years, and PSA levels, from 21.1-2200 μg/L  were obtained with written informed consent and collected using protocols approved by the UBC Clinical Ethics Review board (200 μg/L . Blood sCTCs isolation and enumeration were performed using the CellSearch® system as previously described 2. PC3 (ATCC: CRL-1435) cells was cultured similarly, but using DMEM  medium instead. Cultured cancer cells were spiked into 7.5 ml of blood from a healthy donor into CellSave® Vacutainer tubes and processed within 48 hours identically as the patient specimen.Human prostate cancer cell lines including LNCaP (ATCC: CRL-1740), DU145 (ATCC: HTB-81), and C4-2 (ATCC: CRL-1595) were propagated in culture using RPMI-1640 medium  with 10% fetal bovine serum at 37°C with 5% COTo study the morphology of CTCs and cultured cancer cells, we exported images of individual cells from the CellSearch® system and analyzed them using a software program we developed using LabView . To calcTo analyse the eccentricity of cell shape, an ellipse was fitted to the outline of the cell. The overview of estimating the best-fit ellipse using LabView software is shown in To calibrate size measurements from the CellSearch® images, we separately measured the size of the cultured prostate cancer cells in suspension using the CEDEX XS imaged-based cell analyzer . Grown cultured cancer cells are trypsinized and re-suspended in the culture medium. Cell counts were evaluated using a 1∶1 dilution of cell suspension in trypan blue . A 10 μl of cell suspension is loaded on the Smart Slide , and then read to measure the cell diameter. The conversion factor from pixels to micrometers can be determined using the following equation,The size of CTCs from patient samples was estimated by products of the conversion factor and area of CTCs measured from CellSearch® images.N) to cytoplasmic (AC) area, where AC is considered as the area of the cell excluding AN.The nuclear cytoplasmic ratio is defined as the ratio of nuclear area  size estimates. Furthermore, our estimate of cell diameter is consistent with the small mean cell volume reported by Ligthart and colleagues +CK+CD45− prostate cancer CTCs Analyzing images of cells processed using the CellSearch® system and calibrated against standard microscopy, we found significant size differences between prostate cancer CTCs and cultured prostate cancer cells . SpecifiWe also considered the possibility of our patient selection criteria (CTC count >40) may have biased for a greater number of smaller CTCs. While the selected patients were chemotherapy-negative, they would have participated in a range of therapeutic interventions and would represent patients in the late stages of the disease. Due to these or other unique physiological burdens within our patient cohort, caution should be exercised in generalizing these results to all CRPC CTCs. However, we observed no correlation between CTC cell size and cell count  that wou+ CTCs are consistently smaller than cultured cancer cells is potentially important for emerging label-free CTC enrichment strategies. Firstly, enrichment of CTCs based on size alone may have limited efficacy for the capture of the smaller CTCs found in CRPC patients because they will not be as clearly discriminable from patient leukocytes. While larger cancer cells are typically used to demonstrate the efficacy of these techniques, such as HELA (>20 µm), LNCaP (18 µm), MCF-7 (>15 µm), MDA-231 (15 µm) Similarly, our observation that prostate cancer EpCAMThe use of cultured cancer cells spiked into blood from healthy donors may model the separation of these cells from hematological cells that differ in cell size but the common pre-treatment of these cells with trypsin, to dissociate them from tissue culture flasks, or sample processing using the CellSearch affinity capture strategy may also dramatically influence the cell shape. Through comparison of trypsinized cultured cells and CRPC CTCs, following CellSearch® CTC enrichment, we evaluated whether these cultured cells are appropriate models for patient-derived CTCs. In contrast to cultured cells, that were generally round in shape, CTCs exhibited significant shape variability with many cells having a more elongated shaped . We quanThe nuclear cytoplasmic ratio (N/C) is defined as the ratio of the apparent nuclear area and the apparent cell area with the nucleus subtracted. Compared to cultured cancer cells, CTCs are expected to have larger N/C because of their smaller cell size and likely larger nuclear size due to possible chromosomal abnormalities. We found the median N/C for all cultured cancer cells to be 1.12 while average median N/C ratio from the CTC of patients, enriched by CellSearch® system to be 1.43. This observation further underscores the potential efficacy of deformability-based CTC enrichment, as the nucleoplasm contributes 10-fold more to cell stiffness than does the cytoplasm One concern associated with measuring cell size using the CellSearch® system is whether storing cell samples in the CellSave® tubes modifies the size of the cell and nucleus. To investigate, we compared cultured cancer cells spiked into blood from healthy donors processed immediately with the same cells processed after 48 hours of storage. The cell diameter was found to decrease by ∼6%, while the nuclear diameter was found to decrease by ∼10% from 0 to 48 hours . This reIn conclusion, CTCs isolated from castrate resistant prostate cancer patients, using the CellSearch® system, were smaller in size, more elongated in shape, and had greater N/C when compared to cultured cancer cells. CTCs also showed significantly greater variability in shape and N/C. While the system only captures EpCAM-high cells, CTC images from CellSearch® enumeration are widely available and this analytical strategy could be applied to identify characteristic morphological features of CellSearch®-enriched CTCs. The morphological differences between cultured cell lines and CTC need to be considered in the design and testing of devices that isolate CTC in a label-free fashion based on cytomorphological criteria.Figure S1Screen-shot of the LabView® program developed to analyze images obtained from the CellSearch® system. The program acquires the images for each CTC candidate. A selected image (highlighted in yellow) is analyzed to measure the area in pixels. An ellipse is fitted to this image and overlaid on top of the original image for checking. Parameters for intermediate image processing steps, as well as statistics for the whole collection are also displayed.(DOCX)Click here for additional data file.Figure S2Rejected images of cell fragments from CTC identification. These images of cell fragments commonly appeared during image analysis and were not included in the CTC count or cell size measurements. Typical CTC fragments include a nucleus partly covered by cytokeratin, or a nucleus completely separated from cytokeratin. These fragments likely originated from CTCs undergoing apoptosis.(DOCX)Click here for additional data file.Figure S3Cell size versus CTC count. There appeared to be no correlation between CTC cell size and cell count for CTCs identified by CellSearch from patients with metastatic castrate resistant prostate cancer. The cell size ranged from 6.9 µm to 8.95 µm; while the CTC count varied from 11 to 106.(DOCX)Click here for additional data file.Table S1Patient information summary. All patients were diagnosed with metastatic castration resistant prostate cancer (mCRPC). There was no significant correlation between PSA level and size of CTCs.(DOCX)Click here for additional data file.


In spite of intense research efforts, pancreatic ductal adenocarcinoma remains one of the most deadly malignancies in the world. We and others have previously identified a subpopulation of pancreatic cancer stem cells within the tumor as a critical therapeutic target and additionally shown that the tumor stroma represents not only a restrictive barrier for successful drug delivery, but also serves as a paracrine niche for cancer stem cells. Therefore, we embarked on a large-scale investigation on the effects of combining chemotherapy, hedgehog pathway inhibition, and mTOR inhibition in a preclinical mouse model of pancreatic cancer.Prospective and randomized testing in a set of almost 200 subcutaneous and orthotopic implanted whole-tissue primary human tumor xenografts.The combined targeting of highly chemoresistant cancer stem cells as well as their more differentiated progenies, together with abrogation of the tumor microenvironment by targeting the stroma and enhancing tissue penetration of the chemotherapeutic agent translated into significantly prolonged survival in preclinical models of human pancreatic cancer. Most pronounced therapeutic effects were observed in gemcitabine-resistant patient-derived tumors. Intriguingly, the proposed triple therapy approach could be further enhanced by using a PEGylated formulation of gemcitabine, which significantly increased its bioavailability and tissue penetration, resulting in a further improved overall outcome.This multimodal therapeutic strategy should be further explored in the clinical setting as its success may eventually improve the poor prognosis of patients with pancreatic ductal adenocarcinoma. Pancreatic ductal adenocarcinoma (hereafter referred to as “pancreatic cancer” or PDAC) is the fourth most frequent cause of cancer-related death world-wide Hedgehog and mTOR signaling, and simultaneous targeting of these two pathways, in combination with Gemcitabine, represents a novel treatment strategy for epithelial cancers such as pancreatic cancer With increasing evidence supporting the existence of cancer stem cells, a new horizon is emerging in the development of therapeutic strategies against pancreatic cancer. Cancer stem cells represent a subpopulation of cells distinguishable from the bulk of the tumor based on their exclusive ability to drive tumorigenesis and metastasis. These cells also play a crucial and driving role in disease relapse in vivo evaluation of the combination therapy was performed in clinically most relevant models of patient-derived pancreatic cancer whole-tissue xenografts . SIBI was administered for only 3 weeks to reduce potentially deleterious effects. Gem was given for a total time period of 60 days in accordance with common clinical practice  practice Fig. 1C.in vitro studies that cancer stem cells can indeed be eliminated by the addition of an inhibitor of the mTOR pathways &Since we have shown in comprehensive in vivo effects of combination therapy on cancer stem cell populations, we explanted and digested representative tumors of each group after completion of the 3 weeks of triple therapy, and analyzed by flow cytometry the expression of surface markers previously linked to a cancer stem cell phenotype +CD133+CD44+ cells in Gem-treated tumors was regularly 2-3-fold higher as compared to untreated tumors [data not shown and In order to evaluate the s), the addition of a hedgehog pathway inhibitor markedly decreased the stroma content , an observation that is well in line with previous published reports s), and was statistically significant as compared to tumors treated with Gem alone . Interestingly, we observed similar effects in orthotopic tumors .More detailed histological investigation of the tumors showed that the different treatment regimens also modified the cellular composition of the tumor. While the primary tumor-derived xenografts used for this study displayed a reasonably high amount of stroma (35–70%) in the groups treated with Gem only  cancer PolyEthyleneGlycol-bound Gem (PEG-Gem) as the extended in vivo circulation time and higher tissue penetration of PEG-Gem may generate superior effects as compared to standard Gem. First, we evaluated the in vitro effects of PEG-Gem as compared to Gem on freshly isolated primary human pancreatic cancer cells. For this purpose, four matching primary cell cultures generated from in vivo-expanded pancreatic cancer tissues were treated for 48 hours with either standard Gem or PEG-Gem and were subsequently analyzed by flow cytometry for the induction of apoptosis or cell death, as well as for their cancer stem cell content. Regarding the percentage of apoptotic and dead cells, no differences could be observed between treatment groups . In addition, we did not observe differences between standard Gem and PEG-Gem treatment regarding the content of CD133+ cells in vitro  and, subsequently, a decrease in the survival of these xenograft-bearing animals. As this might be related to the limited bioavailability of the chemotherapeutic agent as an essential part of this combination therapy, we further advanced our treatment strategy by modifying the chemotherapy. Specifically, Vandana et al. have recently shown that modifying gemcitabine via PEGylation leads to enhanced bioavailability in the circulation as compared to native gemcitabine. Although they have also shown better in vivo setting by showing that PEG-Gem treatment significantly delayed the time of tumor progression by 72 days. This enhanced treatment response is certainly impressive as it represents more than half of the study period. As expected, however, tumors ultimately progressed resulting in virtually no difference in median survival of the PEG-Gem treated mice compared to mice treated with traditional Gem alone. While these data confirm that PEGylation of Gem does indeed improve drug availability and delivery, respectively, by enhancing the circulation time and tissue penetration, as expected, PEG-Gem alone is clearly not sufficient to overcome the chemoresistance of cancer stem cells. Therefore, we next investigated the effects of replacing regular Gem with PEG-Gem in our multimodal approach for targeting pancreatic tumors, which had originally responded to Gem+SIBI+Rapa treatment, but eventually relapsed under this specific treatment regimen. Intriguingly, using the PEG-Gem+SIBI+Rapa combination we not only observed virtually complete tumor regression, but most importantly we obtained 100% survival throughout the 125d study period in this highly therapy-resistant tumor. While this observation does not exclude later re-growth as seen in PDAC-Pax22, these data are very promising and are consistent with the notion that further improving the formulation of the combined drugs is mandatory for extending in vitro findings to the much more complex in vivo setting.Indeed, we were then able to validate and extend this concept to the in vivo, as can be seen by reduced tumor growth in combination with Gem and thus significantly enhanced survival time. Furthermore, SIBI can be safely administered in vivo, as we saw no adverse effects on total body weight or white blood cell counts. Importantly, this is well in line with previous observations using other smoothened inhibitors in vivo, only slightly, but non-significantly decreased white blood cell counts and had no effect on the body weight of the animals as compared to respective controls. It is important to note, however, that the healthy and relatively young mice used for this study are likely more capable of compensating for putative adverse effects on the normal stem cell compartments during triple therapy treatment. Therefore, it will be important to further validate the safety of this treatment regimen in human patients in order to ultimately apply it to the mostly aged and moribund patients suffering from pancreatic cancer.The utilized new smoothened inhibitor SIBI-C1  was also highly effective In conclusion, here we provide compelling evidence for the efficacy of a multimodal therapy targeting differentiated cells as well as cancer stem cells in pancreatic cancer, resulting in long-term survival in mice. Thus, these data confirm and expand previous findings from our laboratory in a very large cohort of animals with patient-derived pancreatic cancer xenografts After patients' informed consent had been obtained, excess tissues from resected pancreatic carcinomas was xenografted at Johns Hopkins Medical Institutions  and Hospital de Madrid - Centro Integral Oncológico Clara Campal , respectively, under the indicated Institutional Review Board-approved protocols 3 pieces of primary, in vivo expanded pancreatic cancer tissue pieces were either orthotopically or subcutaneously implanted into the pancreas of 6–8 weeks old female nude mice (Harlan Europe) as described previously 2)/2. Survival was defined as the time point when tumors reached 1 cm3 and mice had to be removed from the study. White blood cell counts were performed with an Abacus Junior Vet hematology analyzer .All animal experiments were conducted in accordance with institutional guidelines and were approved by the Institutional Animal Care and Use Committee of the CNIO (Protocol PA34/2012– “Xenotransplant model for human pancreatic cancer”). Animals were housed and maintained in laminar flow cabinets under specific pathogen-free conditions. Briefly, 8 mmin vitro and in vivo tumor models of glioblastoma in vitro experiments, SIBI-C1 was dissolved in DMSO and used at a concentration of 10 µM.Gemcitabine was purchased from Lilly , dissolved in sterile water and administered twice a week (125 mg/kg i.p.) for 60 days. Rapa  was orally administered via the drinking water as described previously 2 solution in the presence of 2 mM TEA (PEG-NHS/Gemcitabine/TEA molar ratio = 1∶4∶20). The reaction mixture was then kept on constant magnetic stirring overnight at room temperature. The reaction mixture was later subjected to dialysis using a dialysis membrane (MWCO: molecular weight cut-off = 3.5 kDa) against distilled water to remove free and unreacted gemcitabine. Subsequently, the dialyzed solution was freeze-dried using a lyophilizer  at a temperature of −48°C and 0.05 mbar to obtain the powdered form of the conjugate. The characterization of the PEGylated gemcitabine was performed as described previously PEGylated gemcitabine was synthesized by Sahoo and colleagues as described previously and with modifications Table S1 in File S2.Total RNAs from human primary pancreatic cancer cells and spheres were extracted with TRIzol kit (Life Technologies Inc.) according to the manufacturer’s instructions. One microgram of total RNA was used for cDNA synthesis with SuperScript II reverse transcriptase (Life Technologies Inc.) and random hexamers. Quantitative real-time PCR was performed using SYBR Green PCR master mix (Qiagen), according to the manufacturer’s instructions. The list of utilized primers is depicted in Table S2 in File S2.Total protein extracts were obtained using M-PER Mammalian Protein Extraction Reagent (Thermo Scientific) supplemented with phosphatase and protease inhibitors. Pellets were incubated during 1 h in lysis buffer at 4°C, and centrifuged at 13,000 rpm during 10 min at 4°C. Total protein concentration was measured with BCA Protein Assay kit (Pierce) and 25–100 mg protein were separated by SDS/PAGE and transferred to PVDF membranes. Upon antibody incubation, membranes were visualized by enhanced chemoluminescence (Amersham). GAPDH was used as a loading control. A complete list of used antibodies is included in To characterize pancreatic cancer stem cells, the following antibodies were used: anti-CD133/1-APC , anti-CD44-PE anti-EpCAM FITC  or appropriate isotype-matched control antibodies. Samples were analyzed by flow cytometry, using a FACSCanto II (BD), and data were analyzed with FloJo 9.4.4 . Apoptosis and cell death analyses were performed using DAPI and an Annexin V fluorescein isothiocyanate (FITC) staining kit (BD).Formalin-fixed, paraffin-embedded tumor sections were stained with a CK19 antibody , and then visualized with a rabbit anti-mouse and anti-rabbit HRP-conjugated antibody (both Epitomics). Nuclear counterstaining was performed using Hematoxylin. The stroma quantification was performed by two independent investigators, one an experienced pathologist (E.G.).in vitro studies, tumors were enzymatically digested with collagenase and pancreatic cancer adherent cell and sphere cultures were generated and expanded as previously described 5 cells per well were seeded in 6-well plates, treated with either standard gemcitabine or PEG-Gem at a concentration of 100 ng/mL after 24 h, and analyzed on day 3 by flow cytometry to detect apoptosis and cancer stem cell content.For Results for continuous variables are expressed as means ± standard error of the mean (SEM) unless stated otherwise. Overall comparison of continuous variables was performed with the Kruskal-Wallis test followed by post hoc pairwise comparison using the Mann-Whitney U test. Survival was compared using a Log Rank test. P values <0.05 were considered statistically significant. All analyses were performed with SPSS 19 .File S1Figure S1A: Effect of SIBI-C1 on Hedgehog pathway gene expression. Fold increase mRNA expression levels of SHH and GLI2 in gemcitabine resistant primary cancer cells . Figure S1B: Effect of combination therapy on tumor composition. Representative histological pictures showing stroma content in the respective treatment groups in gemcitabine resistant subcutaneously implanted tumors , .(TIF)Click here for additional data file.File S2Table S1: Utilized qRT-PCR primers. Table S2: Utilized antibodies.(TIF)Click here for additional data file.