Longitudinal Multi-omics Analyses Identify Responses of Megakaryocytes, Erythroid Cells, and Plasmablasts as Hallmarks of Severe COVID-19.

Temporal resolution of cellular features associated with a severe COVID-19 disease trajectory is needed for understanding skewed immune responses and defining predictors of outcome. Here, we performed a longitudinal multi-omics study using a two-center cohort of 14 patients. We analyzed the bulk transcriptome, bulk DNA methylome, and single-cell transcriptome (>358,000 cells, including BCR profiles) of peripheral blood samples harvested from up to 5 time points. Validation was performed in two independent cohorts of COVID-19 patients. Severe COVID-19 was characterized by an increase of proliferating, metabolically hyperactive plasmablasts. Coinciding with critical illness, we also identified an expansion of interferon-activated circulating megakaryocytes and increased erythropoiesis with features of hypoxic signaling. Megakaryocyte- and erythroid-cell-derived co-expression modules were predictive of fatal disease outcome. The study demonstrates broad cellular effects of SARS-CoV-2 infection beyond adaptive immune cells and provides an entry point toward developing biomarkers and targeted treatments of patients with COVID-19.

Loss of copy numbers of retrotransposons (HERVK) on chromosome 7p11.2 impacts EGFR (Epidermal Growth Factor Receptor)-induced phenotypes for platinum sensitivity and long-term survival in ovarian cancer-A study from the OVCAD consortium.

We analyzed variations in the epidermal growth factor receptor (EGFR) gene and 5'-upstream region to identify potential molecular predictors of treatment response in primary epithelial ovarian cancer. Tumor tissues collected during debulking surgery from the prospective multicenter OVCAD study were investigated. Copy number variations in the human endogenous retrovirus sequence human endogenous retrovirus K9 (HERVK9) and EGFR Exons 7 and 9, as well as repeat length and loss of heterozygosity of polymorphic CA-SSR I and relative EGFR mRNA expression were determined quantitatively. At least one EGFR variation was observed in 94% of the patients. Among the 30 combinations of variations discovered, enhanced platinum sensitivity (n = 151) was found dominantly with HERVK9 haploidy and Exon 7 tetraploidy, overrepresented among patients with survival ≥120 months (24/29, p = .0212). EGFR overexpression (≥80 percentile) was significantly less likely in the responders (17% vs. 32%, p = .044). Multivariate Cox regression analysis, including age, FIGO stage, and grade, indicated that the patients' subgroup was prognostically significant for CA-SSR I repeat length <18 CA for both alleles (HR 0.276, 95% confidence interval 0.109-0.655, p = .001). Although EGFR variations occur in ovarian cancer, the mRNA levels remain low compared to other EGFR-mutated cancers. Notably, the inherited length of the CA-SSR I repeat, HERVK9 haploidy, and Exon 7 tetraploidy conferred three times higher odds ratio to survive for more than 10 years under therapy. This may add value in guiding therapies if determined during follow-up in circulating tumor cells or circulating tumor DNA and offers HERVK9 as a potential therapeutic target.

EGFR as a stable marker of prostate cancer dissemination to bones.

BACKGROUND: Prostate cancer (PCa) is among the most commonly diagnosed malignancies in men. Although 5-year survival in patients with localised disease reaches nearly 100%, metastatic disease still remains incurable. Therefore, there is a need for markers indicating metastatic dissemination. METHODS: EGFR overexpression (EGFRover) was tracked in 1039 primary tumours, circulating tumour cells from 39 d'Amico high-risk patients and metastatic samples from 21 castration-resistant PCa cases. EGFR status was compared to clinical parameters and multiple molecular factors were assessed using immunohistochemistry and gene ontology analysis. The functional aspect of EGFR was evaluated by plating PC-3 cells on soft and rigid matrices. RESULTS: EGFRover was found in 14% of primary tumours, where it was associated with shorter metastasis-free survival and was an independent indicator of worse overall survival. EGFRover correlated with a pro-migratory and pro-metastatic phenotype of tumour cells as well as rich collagen fibre content. All circulating tumour cells (detected in 13% of cases) were positive for EGFR, independent of their EMT-related phenotype. EGFRover was more prevalent in castration-resistant bone metastases (29% of patients) and supported growth of human PCa cells on rigid matrices mimicking bone stiffness. CONCLUSIONS: EGFRover is a stable, EMT-independent marker of PCa disseminating to rigid organs, preferentially bones.

Somatic aberrations of BRCA1 gene are associated with ALDH1, EGFR, and tumor progression in prostate cancer.

BRCA1 is a pivotal tumor suppressor. Its dysfunction is known to play a role in different tumors. Among others, BRCA1 germline mutations account for higher risk and more aggressive course of prostate cancer (PCa). In addition, somatic BRCA1 gene loss was demonstrated to be a signature of PCa dissemination to lymph nodes and peripheral blood, and indicate worse clinical outcome. In order to substantiate the data for BRCA1 gene loss in PCa and reveal its phenotypical background, BRCA1 gene status was assessed in a large cohort of PCa patients and compared to different molecular factors. BRCA1 gene dosage was assessed in 2398 tumor samples from 1,199 PCa patients using fluorescent in situ hybridization. It was compared to clinico-pathological parameters, patients' outcome as well as selected proteins (Ki-67, apoptosis marker, cytokeratins, vimentin, E- and N-cadherin, ALDH1 and EGFR) examined immunohistochemically. BRCA1 losses were found in 10%, whereas gains appeared in 7% of 603 informative PCa patients. BRCA1 losses correlated to higher T stage (p = 0.027), Gleason score (p = 0.039), shorter time to biochemical recurrence in patients with Gleason score > 7 independently of other factors (multivariate analysis, p = 0.005) as well as expression of proteins regulating stemness and epithelial-mesenchymal transition, that is, ALDH1 (p = 0.021) and EGFR (p = 0.011), respectively. BRCA1 gains correlated to shorter time to metastasis (p = 0.012) and expression of ALDH1 (p = 0.014). These results support the assumption that BRCA1 gene losses contribute to a progressive and stem cell-like phenotype of PCa. Furthermore, they reveal that also BRCA1 gain conceivably representing loss-of-function might mark more invasive tumors.

Exploring prostate cancer genome reveals simultaneous losses of PTEN, FAS and PAPSS2 in patients with PSA recurrence after radical prostatectomy.

The multifocal nature of prostate cancer (PCa) creates a challenge to patients' outcome prediction and their clinical management. An approach that scrutinizes every cancer focus is needed in order to generate a comprehensive evaluation of the disease, and by correlating to patients' clinico-pathological information, specific prognostic biomarker can be identified. Our study utilized the Affymetrix SNP 6.0 Genome-wide assay to investigate forty-three fresh frozen PCa tissue foci from twenty-three patients. With a long clinical follow-up period that ranged from 2.0-9.7 (mean 5.4) years, copy number variation (CNV) data was evaluated for association with patients' PSA status during follow-up. From our results, the loss of unique genes on 10q23.31 and 10q23.2-10q23.31 were identified to be significantly associated to PSA recurrence (p < 0.05). The implication of PTEN and FAS loss (10q23.31) support previous reports due to their critical roles in prostate carcinogenesis. Furthermore, we hypothesize that the PAPSS2 gene (10q23.2-10q23.31) may be functionally relevant in post-operative PSA recurrence because of its reported role in androgen biosynthesis. It is suggestive that the loss of the susceptible region on chromosome 10q, which implicates PTEN, FAS and PAPSS2 may serve as genetic predictors of PSA recurrence after radical prostatectomy.

Frequent genetic alterations in EGFR- and HER2-driven pathways in breast cancer brain metastases.

Current standard systemic therapies for treating breast cancer patients with brain metastases are inefficient. Targeted therapies against human epidermal growth factor receptors are of clinical interest because of their alteration in a subset of breast cancers (BCs). We analyzed copy number, mutation status, and protein expression of epidermal growth factor receptor (EGFR), human epidermal growth factor 2 (HER2), phosphatase and tensin homologue (PTEN), and PI3K catalytic subunit (PIK3CA) in 110 ductal carcinoma in situ, primary tumor, and metastatic BC samples. Alterations in EGFR, HER2, and PTEN, alone or in combination, were found in a significantly larger fraction of breast cancer brain metastases tumor tissue compared with samples from primary tumors with good prognosis, bone relapse, or other distant metastases (all P < 0.05). Primary tumor patients with a subsequent brain relapse showed almost equally high frequencies of especially EGFR and PTEN alteration as the breast cancer brain metastases patients. PIK3CA was not associated with an increased risk of brain metastases. Genetic alterations in both EGFR and PTEN were especially common in triple-negative breast cancer patients and rarely were seen among HER2-positive patients. In conclusion, we identified two independent high-risk primary BC subgroups for developing brain metastases, represented by genetic alterations in either HER2 or EGFR/PTEN-driven pathways. In contrast, none of these pathways was associated with an increased risk of bone metastasis. These findings highlight the importance of both pathways as possible targets in the treatment of brain metastases in breast cancer.

The interplay of HER2/HER3/PI3K and EGFR/HER2/PLC-γ1 signalling in breast cancer cell migration and dissemination.

HER2 signalling by heterodimerisation with EGFR and HER3 in breast cancer is associated with worst outcome of the afflicted patients, which is attributed not only to the aggressiveness of such tumours but also to therapy resistance. Thus, in the present study we investigated the role of EGFR, HER2 and HER3 lateral signalling in cell migration by applying the MDA-MB-468-HER2 (MDA-HER2) breast cancer cell line, representing a valid model system. Knockdown of HER3 expression by siRNA resulted in decreased phosphorylated AKT (pAKT) levels, abrogated epidermal growth factor (EGF)-mediated PLC-γ1 activation and a diminished EGF-induced migratory activity, depicting the interplay of EGF receptor (EGFR)/HER2/PLC-γ1 and HER2/HER3/PI3K signalling in mediating the migration of EGFR/HER2/HER3-expressing breast cancer cells. Since therapy failure usually arises from metastatic cells, we further investigated whether HER3 signalling was active in established breast cancer disseminated tumour cell (DTC) lines as well as in primary DTCs derived from breast cancer patients. EGF treatment of DTC lines resulted solely in increased pAKT S473 levels, whereas in MDA-HER2 cells both pAKT S473 and pAKT T308 levels were increased upon EGF stimulation. Moreover, despite active HER3 molecules, as indicated by pTyr1222 staining, about 90% of analysed breast cancer patient DTCs exhibited very low or even no detectable pAKT S473 levels, suggesting that these cells might have fallen into dormancy. In summary, our data indicate the important role in EGFR, HER2 and HER3 lateral signalling in breast cancer cell migration. Moreover, our data further show that primary tumour cells and DTCs can vary in their HER activation status, which is important to know in the context of cancer therapy.

15-Hydroxyprostaglandin dehydrogenase associates with poor prognosis in breast cancer, induces epithelial-mesenchymal transition, and promotes cell migration in cultured breast cancer cells.

Breast cancer is the most frequent cancer and the leading cause of cancer-related deaths in women worldwide. The prognosis of breast cancer is tightly correlated with the degree of spread beyond the primary tumour. Arachidonic acid (AA) and prostaglandin E(2) (PGE(2)) are known to regulate tumour metastasis enabling epithelial-mesenchymal transition (EMT). However, the detailed role of 15-hydroxyprostaglandin dehydrogenase (HPGD), the key enzyme degrading prostaglandin E(2) , remains unclear in breast cancer. Here, we show that HPGD mRNA is overexpressed in a subset of clinical breast cancers compared to normal breast tissue samples and that high HPGD mRNA expression associates with poor prognosis. Immunohistochemical staining of primary breast cancer and lymph node metastasis tissue samples confirmed high HPGD protein expression in 20% of the samples, as well as associated HPGD expression with aggressive characteristics, such as increased risk of disease relapse and shorter disease-free survival. Results from cultured cells indicated abundant HPGD expression in highly metastatic breast cancer cells, and impairment of HPGD expression using RNA interference led to a significant decrease in transforming growth factor-ß signalling, in cellular arachidonic acid levels as well as in cell migration. Furthermore, gene expression microarray analysis followed by quantitative RT-PCR validation showed that HPGD silencing decreased aryl hydrocarbon receptor signalling and induced mesenchymal-epithelial transition. In conclusion, our results indicate that HPGD is highly expressed in metastatic and aggressive breast cancer and promotes EMT and migration in breast cancer cells.

Genome-wide investigation of multifocal and unifocal prostate cancer-are they genetically different?

Prostate cancer is widely observed to be biologically heterogeneous. Its heterogeneity is manifested histologically as multifocal prostate cancer, which is observed more frequently than unifocal prostate cancer. The clinical and prognostic significance of either focal cancer type is not fully established. To investigate prostate cancer heterogeneity, the genetic profiles of multifocal and unifocal prostate cancers were compared. Here, we report observations deduced from tumor-tumor comparison of copy number alteration data of both focal categories. Forty-one fresh frozen prostate cancer foci from 14 multifocal prostate cancers and eight unifocal prostate cancers were subjected to copy number variation analysis with the Affymetrix SNP 6.0 microarray tool. With the investigated cases, tumors obtained from a single prostate exhibited different genetic profiles of variable degrees. Further comparison identified no distinct genetic pattern or signatures specific to multifocal or unifocal prostate cancer. Our findings suggest that samples obtained from multiple sites of a single unifocal prostate cancer show as much genetic heterogeneity and variability as separate tumors obtained from a single multifocal prostate cancer.

Hybrid cells derived from breast epithelial cell/breast cancer cell fusion events show a differential RAF-AKT crosstalk.

BACKGROUND: The biological phenomenon of cell fusion has been linked to several characteristics of tumour progression, including an enhanced metastatogenic capacity and an enhanced drug resistance of hybrid cells. We demonstrated recently that M13SV1-EGFP-Neo breast epithelial cells exhibiting stem cell characteristics spontaneously fused with MDA-MB-435-Hyg breast cancer cells, thereby giving rise to stable M13MDA435 hybrid cells, which are characterised by a unique gene expression profile and migratory behaviour. Here we investigated the involvement of the PLC-ß/γ1, PI3K/AKT and RAS-RAF-ERK signal transduction cascades in the EGF and SDF-1α induced migration of two M13MDA435 hybrid cell clones in comparison to their parental cells. RESULTS: Analysis of the migratory behaviour by using the three-dimensional collagen matrix migration assay showed that M13SV1-EGFP-Neo cells as well as M13MDA435 hybrid cells, but not the breast cancer cell line, responded to EGF stimulation with an increased locomotory activity. By contrast, SDF-1α solely stimulated the migration of M13SV1-EGFP-Neo cells, whereas the migratory activity of the other cell lines was blocked. Analysis of signal transduction cascades revealed a putative differential RAF-AKT crosstalk in M13MDA435-1 and -3 hybrid cell clones. The PI3K inhibitor Ly294002 effectively blocked the EGF induced migration of M13MDA435-3 hybrid cells, whereas the EGF induced locomotion of M13MDA435-1 hybrid cells was markedly increased. Analysis of RAF-1 S259 phosphorylation, being a major mediator of the negative regulation of RAF-1 by AKT, showed decreased pRAF-1 S259 levels in LY294002 treated M13MDA435-1 hybrid cells. By contrast, pRAF-1 S259 levels remained unaltered in the other cell lines. Inhibition of PI3K/AKT signalling by Ly294002 relieves the AKT mediated phosphorylation of RAF-1, thereby restoring MAPK signalling. CONCLUSIONS: Here we show that hybrid cells could evolve exhibiting a differential active RAF-AKT crosstalk. Because PI3K/AKT signalling has been chosen as a target for anti-cancer therapies our data might point to a possible severe side effect of AKT targeted cancer therapies. Inhibition of PI3K/AKT signalling in RAF-AKT crosstalk positive cancer (hybrid) cells could result in a progression of these cells. Thus, not only the receptor (activation) status, but also the activation of signal transduction molecules should be analysed thoroughly prior to therapy.

Influence of whole arm loss of chromosome 16q on gene expression patterns in oestrogen receptor-positive, invasive breast cancer.

A whole chromosome arm loss of 16q belongs to the most frequent and earliest chromosomal alterations in invasive and in situ breast cancers of all common subtypes. Besides E-cadherin, several putative tumour suppressor genes residing on 16q in breast cancer have been investigated. However, the significance of these findings has remained unclear. Thus, other mechanisms leading to gene loss of function (eg haploinsufficiency, or distortion of multiple regulative subnetworks) remain to be tested as a hypothesis. To define the effect on gene expression of whole-arm loss of chromosome 16q in invasive breast cancer, we performed global gene expression analysis on a series of 18 genetically extensively characterized invasive ductal breast carcinomas and verified the results by quantitative real-time PCR (qRT-PCR). The distribution of the differential genes across the genome and their expression status was studied. A second approach by qRT-PCR in an independent series of 30 breast carcinomas helped to narrow down the observed effect. Whole-arm chromosome 16q losses, irrespective of other chromosomal changes, are associated with decreased expression of a number of candidate genes located on 16q (eg CDA08, CGI-128, SNTB2, NQO1, SF3B3, KIAA0174, ATBF1, GABARAPL2, KARS, GCSH, MBTPS1 and ZDHHC7) in breast carcinomas with a low degree of genetic instability. qRT-PCR provided evidence to suggest that the expression of these genes was reduced in a gene dosage-dependent manner. The differential expression of the candidate genes according to the chromosomal 16q-status vanished in genetically advanced breast cancer cases and changed ER status. These results corroborate previous reports about the importance of whole-arm loss of chromosome 16q in breast carcinogenesis and give evidence for the first time that haploinsufficiency, in the sense of a gene dosage effect, might be an important contributing factor in the early steps of breast carcinogenesis.

Inositol 1,4,5-trisphosphate 3-kinase-A is a new cell motility-promoting protein that increases the metastatic potential of tumor cells by two functional activities.

Cellular migration is an essential prerequisite for metastatic dissemination of cancer cells. This study demonstrates that the neuron/testis-specific F-actin-targeted inositol 1,4,5-trisphosphate 3-kinase-A (ITPKA) is ectopically expressed in different human tumor cell lines and during tumor progression in the metastatic tumor model Balb-neuT. High expression of ITPKA increases invasive migration in vitro and metastasis in a xenograft SCID mouse model. Mechanistic studies show that ITPKA promotes migration of tumor cells by two different mechanisms as follows: growth factor independently high levels of ITPKA induce the formation of large cellular protrusions by directly modulating the actin cytoskeleton. The F-actin binding activity of ITPKA stabilizes and bundles actin filaments and thus increases the levels of cellular F-actin. In growth factor-stimulated cells, the catalytically active domain enhances basal ITPKA-induced migration by activating store-operated calcium entry through production of inositol 1,3,4,5-tetrakisphosphate and subsequent inhibition of inositol phosphate 5-phosphatase. These two functional activities of ITPKA stimulating tumor cell migration place the enzyme among the potential targets of anti-metastatic therapy.

Detection and clinical relevance of early disseminated breast cancer cells depend on their cytokeratin expression pattern.

The factors determining the clinical relevance of disseminated tumor cells (DTC) in breast cancer patients are largely unknown. Here we compared the specificity and clinical performance of two antibodies frequently used for DTC detection. Reactivities of antibodies A45-B/B3 (A45) and AE1/AE3 (AE) for selected cytokeratins (CK) were assessed by 2-DE Western Blot analysis. Using these antibodies bone marrow aspirates from 391 breast cancer patients (M(0), pT1-3, pN0-3) were screened for the presence of DTC. To obtain prognostic information, patients were followed up over a median of 83 months for time to relapse and 99 months for time to death. Among the analyzed CK, AE detected CK5, CK7, CK8, and CK19, whereas A45 recognized CK7 and CK18. In total, 24 of 391 patients (6.1%) were DTC-positive for A45, and 41 (10.5%) for AE. Although concordance between the two antibodies was 84.4%, overlap among positive cases was only 3.2%. DTC-positivity with AE and A45 was more frequent in patients of higher nodal status (P=0.019 and P=0.036, respectively). Nearly all patients with A45-positive DTC had hormone receptor-positive tumors (23/24), while detection of AE-positive DTC was more frequent among hormone receptor negative patients (P=0.006). Survival analyses of all patients revealed shorter distant disease-free survival (P=0.039) for patients with A45-positive DTC, whereas the prognostic relevance of AE-positive DTC was restricted to node-positive patients. The clinical utility of immunocytochemical (ICC) DTC detection depends on the anti-CK antibody used, which may reflect the complex CK composition of DTC.

Longitudinal Analysis of Circulating Tumor Cells in Colorectal Cancer Patients by a Cytological and Molecular Approach: Feasibility and Clinical Application.

INTRODUCTION: Liquid biopsies allowing for individualized risk stratification of cancer patients have become of high significance in individualized cancer diagnostics and treatment. The detection of circulating tumor cells (CTC) has proven to be highly relevant in risk prediction, e.g., in colorectal cancer (CRC) patients. In this study, we investigate the clinical relevance of longitudinal CTC detection over a course of follow-up after surgical resection of the tumor and correlate these findings with clinico-pathological characteristics. METHODS: In total, 49 patients with histologically proven colorectal carcinoma were recruited for this prospective study. Blood samples were analyzed for CTC presence by two methods: first by marker-dependent immunofluorescence staining combined with automated microscopy with the NYONE® cell imager and additionally, indirectly, by semi-quantitative Cytokeratin-20 (CK20) RT-qPCR. CTC quantification data were compared and correlated with the clinico-pathological parameters. RESULTS: Detection of CTC over a post-operative time course was feasible with both applied methods. In patients who were pre-operatively negative for CTCs with the NYONE® method or below the cut-off for relative CK20 mRNA expression after analysis by PCR, a statistically significant rise in the immediate post-operative CTC detection could be demonstrated. Further, in the cohort analyzed by PCR, we detected a lower CTC load in patients who were adjuvantly treated with chemotherapy compared to patients in the follow-up subgroup. This finding was contrary to the same patient subset analyzed with the NYONE® for CTC detection. CONCLUSION: Our study investigates the occurrence of CTC in CRC patients after surgical resection of the primary tumor and during postoperative follow-up. The resection of the tumor has an impact on the CTC quantity and the longitudinal CTC analysis supports the significance of CTC as a prognostic biomarker. Future investigations with an even more extended follow-up period and larger patient cohorts will have to validate our results and may help to define an optimal longitudinal sampling scheme for liquid biopsies in the post-operative monitoring of cancer patients to enable tailored therapy concepts for precision medicine.

The Long-Term Prognostic Significance of Circulating Tumor Cells in Ovarian Cancer-A Study of the OVCAD Consortium.

INTRODUCTION: We previously reported the prognostic impact of circulating tumor cells (CTCs) in a multicenter study on minimal residual disease in primary ovarian cancer. With additional follow-up data, we evaluated the combined CTC approach (CTCscombo), in particular for the patients who had survived more than five years. MATERIAL AND METHODS: Blood samples taken at baseline and six months after adjuvant treatment (follow-up) were assessed by quantitative PCR (qPCR) measuring PPIC transcripts and immunofluorescent staining (IF). A positive result with either IF or qPCR was classified as CTCcombo-positive. Further, PPIC was assessed in the primary tumor tissue. RESULTS: The concordance of IF and qPCR was 65% at baseline and 83% after treatment. Results showed that 50.5% of the baseline and 29.5% of the follow-up samples were CTCcombo-positive. CTCscombo after treatment were associated with increased mortality after adjusting for FIGO stage (HR 2.574, 95% CI: 1.227-5.398, p = 0.012), a higher risk of recurrence after adjusting for peritoneal carcinosis (HR 4.068, 95% CI: 1.948-8.498, p < 0.001), and increased mortality after five survived years. DISCUSSION: The two-sided analytical approach revealed CTC subpopulations associated with ovarian cancer progression and may illuminate a potential treatment-related shift in molecular phenotypes. That approach can identify patients who have elevated risk of recurrence and death due to ovarian cancer and who may require risk-adapted treatment strategies.

Low Tumor-to-Stroma Ratio Reflects Protective Role of Stroma against Prostate Cancer Progression.

Tumor-to-stroma ratio (TSR) is a prognostic factor that expresses the relative amounts of tumor and intratumoral stroma. In this study, its clinical and molecular relevance was evaluated in prostate cancer (PCa). The feasibility of automated quantification was tested in digital scans of tissue microarrays containing 128 primary tumors from 72 PCa patients stained immunohistochemically for epithelial cell adhesion molecule (EpCAM), followed by validation in a cohort of 310 primary tumors from 209 PCa patients. In order to investigate the gene expression differences between tumors with low and high TSR, we applied multigene expression analysis (nCounter® PanCancer Progression Panel, NanoString) of 42 tissue samples. TSR scores were categorized into low (<1 TSR) and high (≥1 TSR). In the pilot cohort, 31 patients (43.1%) were categorized as low and 41 (56.9%) as high TSR score, whereas 48 (23.0%) patients from the validation cohort were classified as low TSR and 161 (77.0%) as high. In both cohorts, high TSR appeared to indicate the shorter time to biochemical recurrence in PCa patients (Log-rank test, p = 0.04 and p = 0.01 for the pilot and validation cohort, respectively). Additionally, in the multivariate analysis of the validation cohort, TSR predicted BR independent of other factors, i.e., pT, pN, and age (p = 0.04, HR 2.75, 95%CI 1.07-7.03). Our data revealed that tumors categorized into low and high TSR score show differential expression of various genes; the genes upregulated in tumors with low TSR score were mostly associated with extracellular matrix and cell adhesion regulation. Taken together, this study shows that high stroma content can play a protective role in PCa. Automatic EpCAM-based quantification of TSR might improve prognostication in personalized medicine for PCa.

Discovery of a novel unfolded protein response phenotype of cancer stem/progenitor cells from the bone marrow of breast cancer patients.

Metastases arise from disseminated tumor cells (DTC) that colonize secondary organs. However, DTC survival strategies to start metastatic outgrowth are unclear. The hostile (hypoxic, hypoglycemic) microenvironmental conditions of the bone marrow serve as an ideal model environment for investigation of DTC survival strategies under environmental stress. We investigated the breast cancer DTC cell line BC-M1 established from the bone marrow of a cancer patient by 2-D DIGE and MS analysis. We observed specific overexpression of the unfolded protein response (UPR) proteins Grp78, Grp94, and protein disulfide-isomerase in breast, lung, and prostate cancer DTC cell lines from the bone marrow. The UPR contributes to survival under adverse environmental conditions including chemotherapy. We show in cellular models that Grp78 expression of the UPR is regulated by tyrosine 1248 of ErbB-2. The breast cancer DTC cell lines shared stem/progenitor cell cancer phenotypes (CD44(high)/CD24(low)). Immunocytochemical staining of bone marrow samples from breast cancer patients confirmed in situ high expression of Grp78 and Grp94 in DTC of breast cancer patients, indicating the potential of both proteins as novel markers for DTC detection. Our results suggest the presence of a previously not recognized stress resistant DTC population that combines stem/progenitor attributes with an UPR phenotype.

Selective regain of egfr gene copies in CD44+/CD24-/low breast cancer cellular model MDA-MB-468.

BACKGROUND: Increased transcription of oncogenes like the epidermal growth factor receptor (EGFR) is frequently caused by amplification of the whole gene or at least of regulatory sequences. Aim of this study was to pinpoint mechanistic parameters occurring during egfr copy number gains leading to a stable EGFR overexpression and high sensitivity to extracellular signalling. A deeper understanding of those marker events might improve early diagnosis of cancer in suspect lesions, early detection of cancer progression and the prediction of egfr targeted therapies. METHODS: The basal-like/stemness type breast cancer cell line subpopulation MDA-MB-468 CD44high/CD24-/low, carrying high egfr amplifications, was chosen as a model system in this study. Subclones of the heterogeneous cell line expressing low and high EGF receptor densities were isolated by cell sorting. Genomic profiling was carried out for these by means of SNP array profiling, qPCR and FISH. Cell cycle analysis was performed using the BrdU quenching technique. RESULTS: Low and high EGFR expressing MDA-MB-468 CD44+/CD24-/low subpopulations separated by cell sorting showed intermediate and high copy numbers of egfr, respectively. However, during cell culture an increase solely for egfr gene copy numbers in the intermediate subpopulation occurred. This shift was based on the formation of new cells which regained egfr gene copies. By two parametric cell cycle analysis clonal effects mediated through growth advantage of cells bearing higher egfr gene copy numbers could most likely be excluded for being the driving force. Subsequently, the detection of a fragile site distal to the egfr gene, sustaining uncapped telomere-less chromosomal ends, the ladder-like structure of the intrachromosomal egfr amplification and a broader range of egfr copy numbers support the assumption that dynamic chromosomal rearrangements, like breakage-fusion-bridge-cycles other than proliferation drive the gain of egfr copies. CONCLUSION: Progressive genome modulation in the CD44+/CD24-/low subpopulation of the breast cancer cell line MDA-MB-468 leads to different coexisting subclones. In isolated low-copy cells asymmetric chromosomal segregation leads to new cells with regained solely egfr gene copies. Furthermore, egfr regain resulted in enhanced signal transduction of the MAP-kinase and PI3-kinase pathway. We show here for the first time a dynamic copy number regain in basal-like/stemness cell type breast cancer subpopulations which might explain genetic heterogeneity. Moreover, this process might also be involved in adaptive growth factor receptor intracellular signaling which support survival and migration during cancer development and progression.

Isolation and Enumeration of CTC in Colorectal Cancer Patients: Introduction of a Novel Cell Imaging Approach and Comparison to Cellular and Molecular Detection Techniques.

Circulating tumour cells (CTC) were proven to be prognostically relevant in cancer treatment, e.g., in colorectal cancer (CRC). This study validates a molecular detection technique through using a novel cell imaging approach for CTC detection and enumeration, in comparison to a size-based cellular and correlated the data to clinico-pathological characteristics. Overall, 57 CRC patients were recruited for this prospective study. Blood samples were analysed for CTCs by three methods: (1) Epithelial marker immunofluorescence staining combined with automated microscopy using the NYONE® cell imager; (2) isolation by size using membrane filtration with the ScreenCell® Cyto IS device and immunofluorescence staining; (3) detection by semi-quantitative Cytokeratin-20 RT-qPCR. Enumeration data were compared and correlated with clinic-pathological parameters. CTC were detected by either approach; however, with varying positivity rates: NYONE® 36.4%, ScreenCell® 100%, and PCR 80.5%. All methods revealed a positive correlation of CTC presence and higher tumour burden, which was most striking using the ScreenCell® device. Generally, no intercorrelation of CTC presence emerged amongst the applied techniques. Overall, enumeration of CTC after isolation by size demonstrated to be the most reliable strategy for the detection of CTC in CRC patients. Ongoing studies will have to unravel the prognostic value of this finding, and validate this approach in a larger cohort.

Biological importance of a polymorphic CA sequence within intron 1 of the epidermal growth factor receptor gene (EGFR) in high grade central osteosarcomas.

Expression of EGFR in high grade osteosarcomas has been observed to be correlated with an improved prognosis. Yet, the underlying mechanism remained unclear since amplifications of EGFR have rarely been described. Recently, the length of a polymorphic CA repeat located at a 5'-regulatory sequence in the intron 1 of the EGFR gene (SSR I) has been shown to be associated with its basal transcriptional activity. We therefore determined the allelic length of CA SSR-I in 219 cases of high grade osteosarcoma and correlated the results with EGFR expression in 34 cases, the presence of amplifications within the CA SSR-I repeat in 59 cases, and clinical follow-up. Our results confirm that in osteosarcoma patients short alleles are more frequent than longer ones, 16 CA repeats being the most frequent. The allele composition differed significantly from the one recently described in a healthy control population (P < 0.01). Short alleles tended to be associated with increased expression of EGFR. Amplifications of the EGFR gene were seen in 13.5% of cases. Significant correlations between allele length composition and neoadjuvant chemotherapy response or long term clinical outcome could not be established. While we were able to show that high frequency of EGFR expression in osteosarcomas is associated with predominantly short alleles of EGFR-CA SSR I, persisting shortcomings in the correspondence with clinical data point toward the existence of additional, putatively more important transcription control mechanisms for EGFR in osteosarcomas which might account for the good prognostic value of EGFR expression.