Disruption of TUFT1, a Desmosome-Associated Protein, Causes Skin Fragility, Woolly Hair, and Palmoplantar Keratoderma.

Desmosomes are dynamic complex protein structures involved in cellular adhesion. Disruption of these structures by loss-of-function variants in desmosomal genes leads to a variety of skin- and heart-related phenotypes. In this study, we report TUFT1 as a desmosome-associated protein, implicated in epidermal integrity. In two siblings with mild skin fragility, woolly hair, and mild palmoplantar keratoderma but without a cardiac phenotype, we identified a homozygous splice-site variant in the TUFT1 gene, leading to aberrant mRNA splicing and loss of TUFT1 protein. Patients' skin and keratinocytes showed acantholysis, perinuclear retraction of intermediate filaments, and reduced mechanical stress resistance. Immunolabeling and transfection studies showed that TUFT1 is positioned within the desmosome and that its location is dependent on the presence of the desmoplakin carboxy-terminal tail. A Tuft1-knockout mouse model mimicked the patients' phenotypes. Altogether, this study reveals TUFT1 as a desmosome-associated protein, whose absence causes skin fragility, woolly hair, and palmoplantar keratoderma.

PD-L1 expression in urothelial bladder cancer varies more among specimen types than between companion assays.

Urothelial bladder cancer (UBC) patients ineligible to platinum-based chemotherapy can be treated with immune-checkpoint inhibitors (ICI) in Programmed Death Ligand 1 (PD-L1) positive cases. Although concordance exists between different PD-L1 assays, little is known on PD-L1 expression variability in matched UBC samples. We compared PD-L1 expression in whole slides of matched transurethral resections (TURBT), radical cystectomies (RC), and lymph node metastasis (LN). Immunohistochemistry using the VENTANA PD-L1 (SP263) assay was performed on 115 patients and scored positive if expression occurred in ≥25% immune cells (IC), ≥25% tumour cells (TC), or both. PD-L1 was positive in 42.7% TURBT, 39.8% RC, and 27.3% LN specimens. Concordance was moderate (κ=0.52; P<0.001) between TURBT and RC, and fair between LN and TURBT (κ=0.31; P=0.048) or RC (κ=0.25; P=0.075). Comparison with the VENTANA PD-L1 (SP142) assay which had been performed previously on the same cohort showed moderate to substantial inter-assay agreement (κ=0.42-0.66). Although TC staining is not part of the SP142 scoring algorithm, discordant PD-L1 assay outcome could be attributed to SP263 TC≥25% staining in only 41% of cases. These results show that PD-L1 expression variability between matched specimens is higher than that between individual assays. Optimal specimen determination for PD-L1 testing needs to be addressed in future studies.

Napabucasin overcomes cisplatin resistance in ovarian germ cell tumor-derived cell line by inhibiting cancer stemness.

BACKGROUND: Cisplatin resistance of ovarian yolk sac tumors (oYST) is a clinical challenge due to dismal patient prognosis, even though the disease is extremely rare. We investigated potential association between cisplatin resistance and cancer stem cell (CSC) markers in chemoresistant oYST cells and targeting strategies to overcome resistance in oYST. METHODS: Chemoresistant cells were derived from chemosensitive human oYST cells by cultivation in cisplatin in vitro. Derivative cells were characterized by chemoresistance, functional assays, flow cytometry, gene expression and protein arrays focused on CSC markers. RNAseq, methylation and microRNA profiling were performed. Quail chorioallantoic membranes (CAM) with implanted oYST cells were used to analyze the micro-tumor extent and interconnection with the CAM. Tumorigenicity in vivo was determined on immunodeficient mouse model. Chemoresistant cells were treated by inhibitors intefering with the CSC properties to examine the chemosensitization to cisplatin. RESULTS: Long-term cisplatin exposure resulted in seven-fold higher IC50 value in resistant cells, cross-resistance to oxaliplatin and carboplatin, and increased migratory capacity, invasiveness and tumorigenicity, associated with hypomethylation of differentially methylated genes/promotors. Resistant cells exhibited increased expression of prominin-1 (CD133), ATP binding cassette subfamily G member 2 (ABCG2), aldehyde dehydrogenase 3 isoform A1 (ALDH3A1), correlating with reduced gene and promoter methylation, as well as increased expression of ALDH1A3 and higher overall ALDH enzymatic activity, rendering them cross-resistant to DEAB, disulfiram and napabucasin. Salinomycin and tunicamycin were significantly more toxic to resistant cells. Pretreatment with napabucasin resensitized the cells to cisplatin and reduced their tumorigenicity in vivo. CONCLUSIONS: The novel chemoresistant cells represent unique model of refractory oYST. CSC markers are associated with cisplatin resistance being possible targets in chemorefractory oYST.

Switches of SOX17 and SOX2 expression in the development of squamous metaplasia and squamous intraepithelial lesions of the uterine cervix.

AIMS: The dynamics and topographical distribution of SOX17 and SOX2 expression was studied in the transformation zone (TZ) of the uterine cervix. This TZ is a dynamic area where switches from glandular into squamous epithelium can be recognized, new squamocolumnar junctions are formed, and premalignant lesions originate. SOX17 and SOX2 show mutually exclusive expression patterns in the normal uterine cervix, with SOX2 being exclusively found in squamous epithelium, while SOX17 is detected in endocervical columnar cells and reserve cells. METHODS AND RESULTS: Normal cervices and squamous intraepithelial lesions (SIL) were studied with immunohistochemistry, methylation of SOX17, human papilloma virus (HPV) genotyping, and in situ hybridization. In the TZ squamous metaplasia originating from these reserve cells can still show SOX17 expression, while also remnants of SOX17-positive immature metaplasia can be recognized in the normal squamous epithelium. SOX17 expression is gradually lost during maturation, resulting in the exclusive expression of SOX2 in the majority of (SIL). This loss of SOX17 expression is independent of methylation of the CpG island in its promotor region. HPV can be detected in SOX17-positive immature metaplastic regions in the immediate vicinity of SOX2-positive SIL, suggesting that switches in SOX17 and 2 expression can occur upon HPV infection. CONCLUSIONS: This switch in expression, and the strong association between the distribution of reserve cells and squamous areas within the columnar epithelium in the TZ, suggests that reserve cell proliferations, next to basal cells in the squamous epithelium, are potential targets for the formation of squamous lesions upon viral infection.

SOX17 expression and its down-regulation by promoter methylation in cervical adenocarcinoma in situ and adenocarcinoma.

AIMS: SOX17 expression has not been studied in glandular lesions of the uterine cervix like adenocarcinoma in situ (AIS) and invasive adenocarcinomas (AdC), whereas SOX17 promoter CpG island methylation has been reported. Therefore, the aim of this study was to relate the topographical distribution of SOX17 expression and SOX17 methylation status to each other, and to SOX2 expression, human papillomavirus (HPV) type, and physical status of the virus. METHODS AND RESULTS: Immunohistochemistry was used in 45 cases to assess expression of SOX17 and SOX2. SOX17 promoter methylation was determined in 25 cases by means of bisulphite conversion and methylation-specific polymerase chain reaction. SOX17 and SOX2 showed a mutually exclusive expression pattern in normal epithelium, with a sharp delineation in the squamocolumnar junction. SOX17 was found in endocervical columnar and reserve cells, whereas SOX2 was exclusively found in squamous epithelium. In both glandular lesions and cases with coexisting glandular and squamous intraepithelial components, a complex combination of SOX17 and SOX2 expression patterns was seen and mutually exclusive expression was lost. Frequently, gain of expression of SOX2 was found and expression of SOX17 was lost. Methylation of the CpG island in the SOX17 promoter was shown to be strongly associated with loss of expression of SOX17 (P = 0.0016). CONCLUSIONS: In this study, we show for the first time a direct correlation between the topographical distribution of SOX17 expression and the methylation status of its gene promoter. This explains the heterogeneity of SOX17 expression in the glandular lesions of the cervix. No correlation was found between HPV type and physical status of the virus on the one hand and methylation status on the other.

Interobserver Agreement in Vascular Invasion Scoring and the Added Value of Immunohistochemistry for Vascular Markers to Predict Disease Relapse in Stage I Testicular Nonseminomas.

Vascular invasion has been identified as an informative risk factor for relapse in stage I testicular nonseminomas, used to tailor treatment. We investigated interobserver agreement in vascular invasion reporting and studied the potential additional value of immunohistochemistry for vascular markers for predicting relapse. Patients (n=52) with stage I testicular nonseminomas undergoing surveillance (1993-2006) were included (median follow-up of 66 mo). Two formalin-fixed paraffin-embedded blocks with >1 cm tissue and tumor/normal parenchyma interface were stained with hematoxylin and eosin and CD31, FVIII, and D2-40. Slides were assessed by 3 independent testicular germ cell tumor-dedicated pathologists, and agreement was assessed using Cohen κ statistic. Sensitivity, specificity, and accuracy of vascular invasion scoring in predicting relapse were calculated. Agreement among testicular germ cell tumor-dedicated pathologists was moderate (κ=0.49 to 0.54), as was performance in predicting disease relapse (particularly, specificity of 86%). Immunohistochemistry increased overall sensitivity (71%), but decreased specificity (71%) in predicting relapse. All patients (n=8) with both blood and lymphatic vascular invasion developed a relapse. In multivariable analysis (including age, tumor size, rete testis invasion, and serum tumor markers), only vascular invasion had an independent impact in predicting relapse. Assessment of vascular invasion by testicular germ cell tumor-dedicated pathologists is good and is clinically meaningful, predicting disease relapse. Immunohistochemistry for vascular markers improves sensitivity of detecting disease relapse and allows for the identification of high-risk patients with both blood and lymphatic vascular invasion simultaneously, potentially of interest for tailored chemotherapy.

Olfactomedin 4 (OLFM4) expression is associated with nodal metastases in esophageal adenocarcinoma.

To date no informative biomarkers exist to accurately predict presence of lymph node metastases (LNM) in esophageal adenocarcinoma (EAC). We studied the discriminative value of Olfactomedin 4 (OLFM4), an intestinal stem cell marker, in EAC. Patients who had undergone esophagectomy as single treatment modality for both advanced (pT2-4) and early (pT1b) adenocarcinoma of the esophagus or gastro-esophageal junction were selected for this study from an institutional database (Erasmus MC University Medical Center, Rotterdam, The Netherlands). Surgical resection specimens of 196 advanced and 44 early EAC were examined. OLFM4 expression was studied by immunohistochemistry and categorized as low (<30%) or high (> = 30%) expression. Low OLFM4 was associated with poor differentiation grade in both advanced (60% vs. 34.8%, p = 0.001) and early EAC (39.1% vs. 9.5%, p = 0.023). LNM were present in 161 (82.1%) of advanced and 9 (20.5%) of early EAC respectively. Low OLFM4 was independently associated with the presence of LNM in advanced EAC in multivariable analysis (OR 2.7; 95% CI, 1.16-6.41; p = 0.022), but not in early EAC (OR 2.1; 95% CI, 0.46-9.84; p = 0.338). However, the difference in association with LNM between advanced (OR 2.7; 95% CI, 1.18-6.34; p = 0.019) and early (OR 2.3; 95% CI, 0.47-11.13; p = 0.302) EAC was non-significant (p = 0.844), suggesting that the lack of significance in early EAC is due to the small number of patients in this group. OLFM4 was not of significance for the disease free and overall survival. Overall, low expression of intestinal stem cell marker OLFM4 was associated with the presence of LNM. Our study suggests that OLFM4 could be an informative marker with the potential to improve preoperative assessment in patients with EAC. Further studies are needed to confirm the value of OLFM4 as a biomarker for LNM.

Molecular Alterations in Dog Pheochromocytomas and Paragangliomas.

8658860258318000Recently, genetic alterations in the genes encoding succinate dehydrogenase subunit B and D (SDHB and SDHD) were identified in pet dogs that presented with spontaneously arising pheochromocytomas (PCC) and paragangliomas (PGL; together PPGL), suggesting dogs might be an interesting comparative model for the study of human PPGL. To study whether canine PPGL resembled human PPGL, we investigated a series of 50 canine PPGLs by immunohistochemistry to determine the expression of synaptophysin (SYP), tyrosine hydroxylase (TH) and succinate dehydrogenase subunit A (SDHA) and B (SDHB). In parallel, 25 canine PPGLs were screened for mutations in SDHB and SDHD by Sanger sequencing. To detect large chromosomal alterations, single nucleotide polymorphism (SNP) arrays were performed for 11 PPGLs, including cases for which fresh frozen tissue was available. The immunohistochemical markers stained positive in the majority of canine PPGLs. Genetic screening of the canine tumors revealed the previously described variants in four cases; SDHB p.Arg38Gln (n = 1) and SDHD p.Lys122Arg (n = 3). Furthermore, the SNP arrays revealed large chromosomal alterations of which the loss of chromosome 5, partly homologous to human chromosome 1p and chromosome 11, was the most frequent finding (100% of the six cases with chromosomal alterations). In conclusion, canine and human PPGLs show similar genomic alterations, suggestive of common interspecies PPGL-related pathways.

Molecular heterogeneity and early metastatic clone selection in testicular germ cell cancer development.

BACKGROUND: Testicular germ cell cancer (TGCC), being the most frequent malignancy in young Caucasian males, is initiated from an embryonic germ cell. This study determines intratumour heterogeneity to unravel tumour progression from initiation until metastasis. METHODS: In total, 42 purified samples of four treatment-resistant nonseminomatous (NS) TGCC were investigated, including the precursor germ cell neoplasia in situ (GCNIS) and metastatic specimens, using whole-genome and targeted sequencing. Their evolution was reconstructed. RESULTS: Intratumour molecular heterogeneity did not correspond to the supposed primary tumour histological evolution. Metastases after systemic treatment could be derived from cancer stem cells not identified in the primary cancer. GCNIS mostly lacked the molecular marks of the primary NS and comprised dominant clones that failed to progress. A BRCA-like mutational signature was observed without evidence for direct involvement of BRCA1 and BRCA2 genes. CONCLUSIONS: Our data strongly support the hypothesis that NS is initiated by whole-genome duplication, followed by chromosome copy number alterations in the cancer stem cell population, and accumulation of low numbers of somatic mutations, even in therapy-resistant cases. These observations of heterogeneity at all stages of tumourigenesis should be considered when treating patients with GCNIS-only disease, or with clinically overt NS.

The MicroRNA-371 Family as Plasma Biomarkers for Monitoring Undifferentiated and Potentially Malignant Human Pluripotent Stem Cells in Teratoma Assays.

Predicting developmental potency and risk of posttransplantation tumor formation by human pluripotent stem cells (hPSCs) and their derivatives largely rely on classical histological analysis of teratomas. Here, we investigated whether an assay based on microRNAs (miRNA) in blood plasma is able to detect potentially malignant elements. Several hPSCs and human malignant germ cell tumor (hGCT) lines were investigated in vitro and in vivo after mouse xenografting. The multiple conventional hPSC lines generated mature teratomas, while xenografts from induced hPSCs (hiPSCs) with reactivated reprogramming transgenes and hGCT lines contained undifferentiated and potentially malignant components. The presence of these elements was reflected in the mRNA and miRNA profiles of the xenografts with OCT3/4 mRNA and the miR-371 and miR-302 families readily detectable. miR-371 family members were also identified in mouse plasma faithfully reporting undifferentiated elements in the xenografts. This study demonstrated that undifferentiated and potentially malignant cells could be detected in vivo.

Correction: c-MET receptor as potential biomarker and target molecule for malignant testicular germ cell tumors.

[This corrects the article DOI: 10.18632/oncotarget.25867.].

c-MET receptor as potential biomarker and target molecule for malignant testicular germ cell tumors.

Type II testicular germ cell tumors (TGCTs) represent the most frequent malignancy in Caucasian males (20-40 years). Even if diagnosed with disseminated disease, >80% of patients are cured; however, a small percentage of cases progress and result in death. It is commonly accepted that these cancers arise from a disturbed testicular embryonic niche that leads to the block of gonocyte differentiation. The subsequent development of the invasive seminomas and non-seminomas is due to a combination of genetic, epigenetic and microenvironment-based alterations (genvironment). Hepatocyte growth factor (HGF) is present in the testicular microenvironment, together with its receptor c-MET, from early embryonic development to an adult stage. In addition, c-MET is a well-known proto-oncogene involved in the onset and progression of various human cancers. Herein, we have investigated the expression and availability of HGF and c-MET in TCam-2, NCCIT and NT2D1 cells, which are type II (T)GCT representative cell lines, and the effect of c-MET activation/repression on the regulation of cancerous biological processes. We found that NT2D1 cells increase their proliferation, polarized migration, and invasion in response to HGF administration. NCCIT cells respond to HGF stimulation only partially, whereas TCam-2 cells do not respond to HGF, at least according to the investigated parameters. Interestingly, the immunohistochemical study of c-MET distribution in TGCTs confirm its presence in both seminoma and non-seminoma lesions with different patterns. Notably, we found the highest c-MET immunoreactivity in the epithelial elements of the various components of TGCTs: teratoma, yolk sac tumor and choriocarcinoma.

Concordance of PD-L1 expression in matched urothelial bladder cancer specimens.

AIMS: Programmed death ligand 1 (PD-L1) expression has predictive value for response to immune-checkpoint inhibitor treatment in urothelial cancer patients. The consistency of PD-L1 expression among different specimen types, however, is unknown. The aim of this study is to compare PD-L1 expression in matched transurethral resections of the bladder (TURB), cystectomy specimens and lymph node metastases of urothelial cancer patients. METHODS AND RESULTS: We performed PD-L1 (SP142) immunohistochemistry on whole tissue slides of 115 urothelial carcinoma patients who had undergone TURB, followed by radical cystectomy and/or pelvic lymph node dissection. The PD-L1 assay was positive if PD-L1 expression in immune cells occupied ≥5% of the tumour area. PD-L1 was positive in 15 of 97 (15.5%) TURB, 17 of 98 (17.3%) cystectomies and nine of 49 (18.4%) lymph node metastases. Agreement of PD-L1 assay outcome between cystectomy and TURB (kappa = 0.34; P = 0.002) and cystectomy and lymph node metastasis (kappa = 0.35; P = 0.034) was fair; there was no agreement between TURB and lymph node metastasis (kappa = 0.045; P = 0.82). Discordance of PD-L1 outcome in matched TURB and cystectomy specimens occurred more frequently after neoadjuvant therapy (53.3% versus 25.4%; P = 0.03), and was not associated with other clinicopathological parameters. CONCLUSIONS: Urothelial bladder cancer patients showed fair agreement of PD-L1 assay outcome in cystectomies and matched TURB or lymph node specimens. PD-L1 expression was discordant more often after neoadjuvant therapy. Therefore, immune-checkpoint inhibitor studies should take into account specimen type and neoadjuvant therapy in assessing the predictive value of PD-L1 expression.

Prognostic value of intra-tumoral CD8+ /FoxP3+ lymphocyte ratio in patients with resected colorectal cancer liver metastasis.

BACKGROUND AND OBJECTIVES: Patients with isolated colorectal-cancer-liver-metastases (CRCLM) frequently undergo metastatectomy. Tumor-infiltrating-lymphocytes (TILs) have prognostic potential in the setting of primary colorectal cancer, however, their role in CRCLM is less studied. We aimed to study the spatial distribution and prognostic role of tumor-infiltrating CD8+ cytotoxic T-cells and FoxP3+ regulatory T-cells at the metastatic site of CRCLM patients. METHODS: TILs were isolated from fresh metastatic tissues of 47 patients with CRCLM. Archived paraffin-embedded tissue, from the same patients, was retrieved. CD8+ and FoxP3+ cells, both in the intra-tumoral and the peri-tumoral compartments, were measured by immunohistochemistry on full tissue sections. Proportions of cytotoxic T-cells (CD8+ ) and regulatory T-cells (CD4+ CD25+ FoxP3+ ), within CD45+ TILs, were measured by flow-cytometry. RESULTS: By immunohistochemistry, individual densities of intra-tumoral or peri-tumoral CD8+ and FoxP3+ cells were not prognostic of survival. However, the intra-tumoral, but not the peri-tumoral, CD8+ /FoxP3+ ratio was an independent predictor of survival (HR 0.43, 95%CI 0.19-0.95, P = 0.032). By flow cytometry, the intra-tumoral CD8+ /regulatory T-cell ratio was also an independent predictor of survival (HR 0.45, 95%CI 0.20-0.99, P = 0.044). CONCLUSIONS: The ratio of cytotoxic (CD8+ ) to regulatory (FoxP3+ ) T-cells, in the intra-tumoral compartment, but not in the peri-tumoral compartment, can predict survival after resection of CRCLM.

Characterization of Endothelial Cells Associated with Hematopoietic Niche Formation in Humans Identifies IL-33 As an Anabolic Factor.

Bone marrow formation requires an orchestrated interplay between osteogenesis, angiogenesis, and hematopoiesis that is thought to be mediated by endothelial cells. The nature of the endothelial cells and the molecular mechanisms underlying these events remain unclear in humans. Here, we identify a subset of endoglin-expressing endothelial cells enriched in human bone marrow during fetal ontogeny and upon regeneration after chemotherapeutic injury. Comprehensive transcriptional characterization by massive parallel RNA sequencing of these cells reveals a phenotypic and molecular similarity to murine type H endothelium and activation of angiocrine factors implicated in hematopoiesis, osteogenesis, and angiogenesis. Interleukin-33 (IL-33) was significantly overexpressed in these endothelial cells and promoted the expansion of distinct subsets of hematopoietic precursor cells, endothelial cells, as well as osteogenic differentiation. The identification and molecular characterization of these human regeneration-associated endothelial cells is thus anticipated to instruct the discovery of angiocrine factors driving bone marrow formation and recovery after injury.

Biallelic and monoallelic ESR2 variants associated with 46,XY disorders of sex development.

PURPOSE: Disorders or differences of sex development (DSDs) are rare congenital conditions characterized by atypical sex development. Despite advances in genomic technologies, the molecular cause remains unknown in 50% of cases. METHODS: Homozygosity mapping and whole-exome sequencing revealed an ESR2 variant in an individual with syndromic 46,XY DSD. Additional cases with 46,XY DSD underwent whole-exome sequencing and targeted next-generation sequencing of ESR2. Functional characterization of the identified variants included luciferase assays and protein structure analysis. Gonadal ESR2 expression was assessed in human embryonic data sets and immunostaining of estrogen receptor-ß (ER-ß) was performed in an 8-week-old human male embryo. RESULTS: We identified a homozygous ESR2 variant, c.541_543del p.(Asn181del), located in the highly conserved DNA-binding domain of ER-ß, in an individual with syndromic 46,XY DSD. Two additional heterozygous missense variants, c.251G>T p.(Gly84Val) and c.1277T>G p.(Leu426Arg), located in the N-terminus and the ligand-binding domain of ER-ß, were found in unrelated, nonsyndromic 46,XY DSD cases. Significantly increased transcriptional activation and an impact on protein conformation were shown for the p.(Asn181del) and p.(Leu426Arg) variants. Testicular ESR2 expression was previously documented and ER-ß immunostaining was positive in the developing intestine and eyes. CONCLUSION: Our study supports a role for ESR2 as a novel candidate gene for 46,XY DSD.

VASA mRNA (DDX4) detection is more specific than immunohistochemistry using poly- or monoclonal antibodies for germ cells in the male urogenital tract.

VASA, also known as DDX4, is reported to be specifically expressed in cells belonging to the germ cell lineage, both in males and females. Therefore, it could be an informative protein biomarker to be applied on semen to differentiate between obstructive and nonobstructive azoospermia (OA and NOA, respectively). In addition, it could be of value to predict sperm retrieval based on testicular sperm extraction. Immunocytochemistry of proven OA semen using both polyclonal and monoclonal antibodies against VASA showed positive staining of both cells and cell sized particles. This is spite of being the absolute negative controls, completely lacking germ lineage derived cells and material. In order to identify the source of the VASA-positive material, a detailed screen of different anatomical parts of the whole male urogenital tract was performed of multiple cases using immunohistochemistry.The polyclonal antibody stained, besides the expected germ cells in the testis, epithelium of the bladder and the seminal vesicles. The monoclonal antibody only stained the latter. To investigate whether the immunohistochemical staining is associated with the presence of the corresponding VASA mRNA, samples of seminal vesicles, bladder, testis, and semen (with and without germ cells) were investigated using the specific quantitative reverse transcription-polymerase chain reaction (qRT-PCR) on 42 samples. A positive result was detected in testis and semen containing germ cells (n = 10 and 8), being negative in semen without germ cells (n = 11), bladder (n = 3), and seminal vesicles (n = 10).Two commercially available VASA antibodies (mono- and polyclonal) are not specific. In contrast, VASA-mRNA evaluation, using qRT-PCR, is specific for the presence of germ cells, therefore, is an interesting molecular biomarker for germ cell detection in semen.

Gene Expression Differences between Ductal Carcinoma in Situ with and without Progression to Invasive Breast Cancer.

To understand the molecular alterations driving the progression of ductal carcinoma in situ (DCIS), we compared patients with pure DCIS and patients with DCIS and synchronous invasive breast cancer (IBC). Twelve patients with extensive pure DCIS were included as a representation of indolent lesions with limited invasive capacity. These cases were matched with 12 patients with a limited DCIS component and IBC, representing lesions with a high invasive potential. Matching included age and surrogate DCIS subtypes. Gene expression profiling was performed on DCIS cells to identify transcriptional differences between these two groups. The identified genes were validated by immunohistochemistry. Nine genes showed significantly different expression. Most of these genes were highly expressed in DCIS samples with IBC, including PLAU (P = 0.002), COL1A1 (P = 0.006), KRT81 (P = 0.009), S100A7 (P = 0.015), SCGB1D2 (P = 0.023), KRT18 (P = 0.029), and NOTCH3 (P = 0.044), whereas EGFR and CXCL14 showed a higher expression in cases with pure DCIS (P = 0.015 and P = 0.028, respectively). This difference was only significant for SCGB1D2 (P = 0.009). Hierarchical clustering revealed distinct clustering of patients with and without invasion. Patients with pure DCIS have a different gene expression pattern as compared to patients with DCIS and synchronous IBC. These genes may pinpoint to driver pathway(s) that play an important role in DCIS progression.

Loss-of-Function Variants in MYLK Cause Recessive Megacystis Microcolon Intestinal Hypoperistalsis Syndrome.

Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is a congenital disorder characterized by loss of smooth muscle contraction in the bladder and intestine. To date, three genes are known to be involved in MMIHS pathogenesis: ACTG2, MYH11, and LMOD1. However, for approximately 10% of affected individuals, the genetic cause of the disease is unknown, suggesting that other loci are most likely involved. Here, we report on three MMIHS-affected subjects from two consanguineous families with no variants in the known MMIHS-associated genes. By performing homozygosity mapping and whole-exome sequencing, we found homozygous variants in myosin light chain kinase (MYLK) in both families. We identified a 7 bp duplication (c.3838_3844dupGAAAGCG [p.Glu1282_Glyfs∗51]) in one family and a putative splice-site variant (c.3985+5C>A) in the other. Expression studies and splicing assays indicated that both variants affect normal MYLK expression. Because MYLK encodes an important kinase required for myosin activation and subsequent interaction with actin filaments, it is likely that in its absence, contraction of smooth muscle cells is impaired. The existence of a conditional-Mylk-knockout mouse model with severe gut dysmotility and abnormal function of the bladder supports the involvement of this gene in MMIHS pathogenesis. In aggregate, our findings implicate MYLK as a gene involved in the recessive form of MMIHS, confirming that this disease of the visceral organs is heterogeneous with a myopathic origin.

Tumor cell expression of immune inhibitory molecules and tumor-infiltrating lymphocyte count predict cancer-specific survival in pancreatic and ampullary cancer.

Understanding the mechanisms of immune resistance in pancreatic and ampullary cancers is crucial for the development of suitable biomarkers and effective immunotherapeutics. Our aim was to examine the expression of the immune inhibiting molecules PD-L1, Galectin-9, HVEM, IDO and HLA-G, as well as CD8+ and FoxP3+ tumor infiltrating lymphocytes (TIL), in pancreatic and ampullary cancers, and to relate their individual, as well as their combined expression, to cancer survival. Tumor tissue from 224 patients with resected pancreatic (n = 148) and ampullary (n = 76) cancer was used to construct tissue-microarrays. Expression of immune inhibitory molecules and TIL was examined by immunohistochemistry. We show that immune inhibitory molecules are prevalently expressed. Moreover, high tumor expression of PD-L1 (p = 0.002), Gal-9 (p = 0.003), HVEM (p = 0.001), IDO (p = 0.049), HLA-G (p = 0.004) and high CD8/FoxP3 TIL ratio (p = 0.006) were associated with improved cancer-specific survival. All immune biomarkers, with the exception of IDO, were individually predictive of cancer-specific survival when adjusted for clinicopathologic characteristics. For every additional immune biomarker present survival was almost two-fold prolonged (HR 0.57 95%CI 0.47-0.69, p < 0.0001). When patients with pancreatic and ampullary cancer were analyzed separately the results were similar. We conclude that pancreas and ampullary cancers are rich in expression of immune-inhibitory molecules. These molecules can be targets for future immunotherapeutics, as well as form powerful immunological biomarkers. We propose that such immune biomarker panels be included in future prospective immunotherapy trials.