Erratum: Cuceu, C., et al. Independent Mechanisms Lead to Genomic Instability in Hodgkin Lymphoma: Microsatellite or Chromosomal Instability. Cancers 2018, 10, 233.

The authors wish to make the following corrections to this paper [...].

Independent Mechanisms Lead to Genomic Instability in Hodgkin Lymphoma: Microsatellite or Chromosomal Instability †.

Background: Microsatellite and chromosomal instability have been investigated in Hodgkin lymphoma (HL). Materials and Methods: We studied seven HL cell lines (five Nodular Sclerosis (NS) and two Mixed Cellularity (MC)) and patient peripheral blood lymphocytes (100 NS-HL and 23 MC-HL). Microsatellite instability (MSI) was assessed by PCR. Chromosomal instability and telomere dysfunction were investigated by FISH. DNA repair mechanisms were studied by transcriptomic and molecular approaches. Results: In the cell lines, we observed high MSI in L428 (4/5), KMH2, and HDLM2 (3/5), low MSI in L540, L591, and SUP-HD1, and none in L1236. NS-HL cell lines showed telomere shortening, associated with alterations of nuclear shape. Small cells were characterized by telomere loss and deletion, leading to chromosomal fusion, large nucleoplasmic bridges, and breakage/fusion/bridge (B/F/B) cycles, leading to chromosomal instability. The MC-HL cell lines showed substantial heterogeneity of telomere length. Intrachromosmal double strand breaks induced dicentric chromosome formation, high levels of micronucleus formation, and small nucleoplasmic bridges. B/F/B cycles induced complex chromosomal rearrangements. We observed a similar pattern in circulating lymphocytes of NS-HL and MC-HL patients. Transcriptome analysis confirmed the differences in the DNA repair pathways between the NS and MC cell lines. In addition, the NS-HL cell lines were radiosensitive and the MC-cell lines resistant to apoptosis after radiation exposure. Conclusions: In mononuclear NS-HL cells, loss of telomere integrity may present the first step in the ongoing process of chromosomal instability. Here, we identified, MSI as an additional mechanism for genomic instability in HL.

The Transition between Telomerase and ALT Mechanisms in Hodgkin Lymphoma and Its Predictive Value in Clinical Outcomes.

Background: We analyzed telomere maintenance mechanisms (TMMs) in lymph node samples from HL patients treated with standard therapy. The TMMs correlated with clinical outcomes of patients. Materials and Methods: Lymph node biopsies obtained from 38 HL patients and 24 patients with lymphadenitis were included in this study. Seven HL cell lines were used as in vitro models. Telomerase activity (TA) was assessed by TRAP assay and verified through hTERT immunofluorescence expression; alternative telomere lengthening (ALT) was also assessed, along with EBV status. Results: Both TA and ALT mechanisms were present in HL lymph nodes. Our findings were reproduced in HL cell lines. The highest levels of TA were expressed in CD30-/CD15- cells. Small cells were identified with ALT and TA. Hodgkin and Reed Sternberg cells contained high levels of PML bodies, but had very low hTERT expression. There was a significant correlation between overall survival (p < 10-3), event-free survival (p < 10-4), and freedom from progression (p < 10-3) and the presence of an ALT profile in lymph nodes of EBV+ patients. Conclusion: The presence of both types of TMMs in HL lymph nodes and in HL cell lines has not previously been reported. TMMs correlate with the treatment outcome of EBV+ HL patients.

Establishment and Characterization of a Reliable Xenograft Model of Hodgkin Lymphoma Suitable for the Study of Tumor Origin and the Design of New Therapies.

To identify the cells responsible for the initiation and maintenance of Hodgkin lymphoma (HL) cells, we have characterized a subpopulation of HL cells grown in vitro and in vivo with the aim of establishing a reliable and robust animal model for HL. To validate our model, we challenged the tumor cells in vivo by injecting the alkylating histone-deacetylase inhibitor, EDO-S101, a salvage regimen for HL patients, into xenografted mice. Methodology: Blood lymphocytes from 50 HL patients and seven HL cell lines were used. Immunohistochemistry, flow cytometry, and cytogenetics analyses were performed. The in vitro and in vivo effects of EDO-S101 were assessed. Results: We have successfully determined conditions for in vitro amplification and characterization of the HL L428-c subline, containing a higher proportion of CD30-/CD15- cells than the parental L428 cell line. This subline displayed excellent clonogenic potential and reliable reproducibility upon xenografting into immunodeficient NOD-SCID-gamma (-/-)(NSG) mice. Using cell sorting, we demonstrate that CD30-/CD15- subpopulations can gain the phenotype of the L428-c cell line in vitro. Moreover, the human cells recovered from the seventh week after injection of L428-c cells into NSG mice were small cells characterized by a high frequency of CD30-/CD15- cells. Cytogenetic analysis demonstrated that they were diploid and showed high telomere instability and telomerase activity. Accordingly, chromosomal instability emerged, as shown by the formation of dicentric chromosomes, ring chromosomes, and breakage/fusion/bridge cycles. Similarly, high telomerase activity and telomere instability were detected in circulating lymphocytes from HL patients. The beneficial effect of the histone-deacetylase inhibitor EDO-S101 as an anti-tumor drug validated our animal model. Conclusion: Our HL animal model requires only 10³ cells and is characterized by a high survival/toxicity ratio and high reproducibility. Moreover, the cells that engraft in mice are characterized by a high frequency of small CD30-/CD15- cells exhibiting high telomerase activity and telomere dysfunction.

Differential immunohistochemical and biological profile of squamous cell carcinoma of the breast.

BACKGROUND: Pure or metaplastic squamous cell carcinoma (SCC) of the breast is a rare entity with an unclear pathogeny and aggressive clinical behaviour. An attempt was made to characterize its differential immunohistochemical and biological profile. PATIENTS AND METHODS: Twenty-seven cases of SCC (pure or not) of the breast were matched with 27 ductal invasive carcinomas (IDC) for age, tumour size, nodal involvement and year of diagnosis. The expression levels of oestrogen receptor (ER), progesterone receptor (PR), Ki-67, epidermal growth factor receptor (EGFR), HER2, Cyclin Bl, hTERT, cytokeratins (CK) 5/6 and p63 were determined immunohistochemically in both cohorts. The presence of the human papilloma virus (HPV) genome was investigated by polymerase chain reaction (PCR). RESULTS: Pure and metaplastic SCC displayed common profiles typifying a basal origin: they never expressed ER or PR, were HER2-negative in 93% of cases, exhibited positivity for CK5/6 or EGF-R in 75% and 85%, and for p63 in 70% of cases and were highly proliferative. These profiles were markedly different from those of matched controls (p<0.001 for five markers) except for HER2 and hTERT. The HPVgenome was detected in 2 out of 14 cases (14%) of SCC. CONCLUSION: The expression profile of SCC of the breast was markedly different from that of IDC. A typical "basal-like" phenotype was displayed that may explain part of their behaviour and justify specific therapeutic approaches. HPV infection was not a leading oncogenic event in SCC of the breast.

Transmission of Induced Chromosomal Aberrations through Successive Mitotic Divisions in Human Lymphocytes after In Vitro and In Vivo Radiation.

The mechanisms behind the transmission of chromosomal aberrations (CA) remain unclear, despite a large body of work and major technological advances in chromosome identification. We reevaluated the transmission of CA to second- and third-division cells by telomere and centromere (TC) staining followed by M-FISH. We scored CA in lymphocytes of healthy donors after in vitro irradiation and those of cancer patients treated by radiation therapy more than 12 years before. Our data demonstrate, for the first time, that dicentric chromosomes (DCs) decreased by approximately 50% per division. DCs with two centromeres in close proximity were more efficiently transmitted, representing 70% of persistent DCs in ≥M3 cells. Only 1/3 of acentric chromosomes (ACs), ACs with four telomeres, and interstitial ACs, were paired in M2 cells and associated with specific DCs configurations. In lymphocytes of cancer patients, 82% of detected DCs were characterized by these specific configurations. Our findings demonstrate the high stability of DCs with two centromeres in close proximity during cell division. The frequency of telomere deletion increased during cell cycle progression playing an important role in chromosomal instability. These findings could be exploited in the follow-up of exposed populations.

Catalytic subunit of human telomerase reverse transcriptase is an independent predictor of survival in patients undergoing curative resection of hepatic colorectal metastases: a multicenter analysis.

PURPOSE: To determine the role of the catalytic subunit of human telomerase reverse transcriptase (hTERT) in predicting survival after resection of hepatic colorectal metastases (CRM). PATIENTS AND METHODS: Two hundred one patients who underwent curative resection of hepatic CRM between 1990 and 2000 were identified from a multicenter database. The CRM were analyzed for hTERT nucleolar expression by standard immunohistochemical techniques. hTERT expression and known clinicopathologic factors of survival were examined. RESULTS: With a median follow-up of 80 months, 152 patients (75.6%) had died; the 5-year overall survival was 30.7%. On univariate analysis, number of metastases greater than two (P = .0005), extrahepatic disease (P = .0054), disease-free interval less than 12 months (P = .006), carcinoembryonic antigen level greater than 200 ng/mL (P = .0071), and positive hTERT nucleolar staining (P < .0001) were associated with decreased survival. On multivariate analysis, three factors independently predicted survival: number of metastases (relative risk [RR] = 1.74; P = .0011); disease-free interval (RR = 1.70; P = .0035); and positive hTERT nucleolar staining (RR = 2.03; P < .0001). Patients with none or one of these factors had a 5-year survival rate of 48%, whereas those with two or three of these factors had a 5-year survival of 15% (P < .0001). CONCLUSION: hTERT nucleolar expression is associated with worse survival after resection of hepatic CRM. hTERT expression in conjunction with number of hepatic metastases and disease-free interval may permit more accurate prediction of survival after resection of hepatic CRM.

Telomere shortening and telomerase reverse transcriptase expression in preinvasive bronchial lesions.

PURPOSE: Telomerase, a ribonucleoprotein complex whose activity is related to the expression of its catalytic subunit human telomerase reverse transcriptase (hTERT), restores telomere length in tumor cells and enables immortality after p53/Rb inactivation has been achieved. To determine the timing of hTERT derepression during bronchial carcinogenesis and its relationship with telomere shortening and the p53/Rb pathway alterations, we did an immunohistochemical and in situ hybridization study in preinvasive and invasive bronchial lesions. EXPERIMENTAL DESIGN: hTERT, P53, P16, cyclin D1, Bax-to-Bcl2 ratio, and Ki67 immunostainings were done in 106 preneoplastic lesions and in paired lung carcinoma and normal bronchial mucosae. Concomitantly, hTERT mRNA levels and qualitative telomere shortening were assessed by in situ hybridization and fluorescence in situ hybridization, respectively, in a subset of preneoplastic and neoplastic lesions. RESULTS: Telomerase was increasingly expressed from normal epithelium to squamous metaplasia, dysplasia, and carcinoma in situ, and decreased in invasive carcinoma (P < 0.0001), with a direct correlation between protein and mRNA levels of expression (P < 0.0001). hTERT expression was directly correlated with P53, Ki67, and Bcl2-to-Bax ratio, suggesting a coupling between telomerase reactivation, proliferation, and resistance to apoptosis. Telomere signals significantly decreased as early as squamous metaplasia and progressively increased over the spectrum of preneoplastic lesions. CONCLUSIONS: Telomere shortening represents an early genetic abnormality in bronchial carcinogenesis, preceding telomerase expression and p53/Rb inactivation, which predominate in high-grade preinvasive lesions.

Epidermal growth factor receptor, HER-2/neu and related pathways in lung adenocarcinomas with bronchioloalveolar features.

Lung adenocarcinomas with bronchioalveolar features (ABAF), formerly called bronchioloalveolar cancers (BAC), constitute a distinct clinical, radiological and pathological entity among lung malignancies. Epidermal growth factor receptor (EGFR) and to a less extent, HER-2/neu, are known to be overexpressed in non-small lung cancers, but their exact status in ABAF is not well-documented. Stimulation of these two receptors results in the initiation of two major cascades, namely phosphatidylinositol 3-kinase (PI-3K) and Ras-dependent pathways. We have therefore studied the expressions of EGFR, HER-2/neu as well as phosphorylated AKT (pAKT) and phosphorylated extracellular-signal regulated kinase (ERK), which are key molecules in these two pathways, in 15 ABAF patients. EGFR was found to be overexpressed in 9 of 15 patients (60%). HER-2/neu overexpression was detected in 6 of the 14 tumors tested (43%). pAKT and pERK were both found to be positive in 13 of 15 patients (87%). Six of the seven tumors with mucinous pattern were negative for EGFR, while all of the other eight cases were positive (P=0.001). Mucinous tumors were also less likely than non-mucinous tumors to overexpress HER-2/neu (17% versus 63%, respectively). These findings suggest that ABAF, particularly those with non-mucinous histology, commonly harbors EGFR and HER-2/neu overexpression. PI-3K and Ras-dependant pathways that lie downstream are generally activated, even in the absence of EGFR and/or HER-2/neu overexpression. ABAF may be a particularly promising candidate for EGFR-targeted strategies and this possibility merits extensive evaluation in clinical trials.

Global quantification of γH2AX as a triage tool for the rapid estimation of received dose in the event of accidental radiation exposure.

The phosphorylation of the H2AX histone to form γH2AX foci has been shown to be an accurate biomarker of ionizing radiation exposure. It is well established that there is a one-to-one correlation between the number of γH2AX foci and radiation-induced double strand breaks in cellular DNA, which can be translated to the received dose. However, manual counting of foci is time-consuming, and cannot accommodate high throughput analysis required to obtain rapid results for medical triage purposes in the case of large-scale accidental exposure. Furthermore, the accuracy of γH2AX measurements could potentially be compromised by delays between the time of exposure and analysis of results, as well as inter-cellular and inter-individual variability of this biological response. To evaluate more rapid approaches of quantifying γH2AX for use in an emergency situation, and to determine the impact of inter-individual variability, we compared two methods of global γH2AX fluorescence quantification (low magnification immunofluorescence microscopy and flow cytometry) to the well-established γH2AX foci scoring method in human primary fibroblasts. All three approaches were well correlated, indicating that global γH2AX fluorescence measurements are suitable for dose estimation. For rapid triage in an emergency situation, we propose the use of flow cytometry, as it is more highly correlated with foci scoring and because of the speed and ease of the method. Dose response curves (0.25-6Gy) using flow cytometry measurements showed that inter-individual variability in global γH2AX fluorescence is statistically insignificant at 4h post-irradiation. Based on these data, we propose calibration curves that can be applied to populations exposed to moderate radiation doses to estimate individual received doses, independent of individual radiosensitivity, at this specific time point post-irradiation using human fibroblasts and lymphocytes. Furthermore, we define three triage categories that could facilitate immediate and follow-up care in the case of a radiological accident.

Loss of PTEN expression is not uncommon, but lacks prognostic value in stage I NSCLC.

The PTEN gene is a tumor-suppressor gene that is inactivated in several types of human tumors. The loss of PTEN expression has been supported as a prognostic marker. Using immunohistochemical analysis, we retrospectively analyzed PTEN expression in specimens from 53 patients with completely resected stage I non-small cell lung cancer (NSCLC) for whom clinical follow-up data were available. Seven of the 53 specimens showed a total lack of staining for PTEN. No statistically significant relationship could be found between PTEN expression and clinicopathological parameters. Although genetic alterations of the PTEN gene are rare in NSCLC, loss of PTEN protein is not an uncommon event in early-stage NSCLC (13.2%). Here, we also report that the level of PTEN protein expression is not an independent prognostic marker in early-stage NSCLC.

[Epidermal growth factor receptor and chemoprevention of epithelial tumor: rationale and development methods]. / Récepteur du facteur de croissance épidermique et chimioprévention des cancers épithéliaux: rationnel et voies de développement.

Among the many pathways dysregulated during the carcinogenic process, REGF seems to be one of the most promising pathways to target in order to achieve chemopreventive and anticancer effects. Indeed, chemoprevention, the use of natural or synthetic compounds in order to reverse, suppress or prevent the carcinogenic process, aims at the cellular level at regulating the growth and sensitivity to apoptosis of premalignant and malignant clones. REGF activation leads to uncontrolled cellular proliferation, resistance to apoptosis, angiogenesis and invasion. Furthermore, REGF is frequently overexpressed in many epithelial tumors. This review will focus on the rationale and the ongoing research areas related to chemopreventive approaches targeting REGF.

Differential expression of biomarkers in primary non-small cell lung cancer and metastatic sites.

INTRODUCTION: The use of biomarkers to evaluate the presence of a target or to select a specific therapy is increasingly advocated. The correlation of biomarker expression between the primary tumor and its corresponding metastasis has not yet been well documented and analyzed in patients with non-small cell lung cancer (NSCLC). METHODS: The expression of epidermal growth factor receptor (EGFR), excision repair cross-complementing (ERCC1), vascular-endothelial growth factor receptor, and Ki-67 was immunohistochemically analyzed in tumor samples of primary NSCLC and one corresponding metastasis in a population of 49 patients. RESULTS: Sixteen cases (33%) displayed clear discordance in the EGFR status between the primary tumor and the metastasis, with a significant trend toward downregulation of EGFR in the metastasis (p = 0.01). The ERCC1 status was discordant in 20 cases (41%), with a trend toward overexpression in brain and adrenal metastases (p = 0.01 and p = 0.08, respectively). The vascular-endothelial growth factor receptor and Ki-67 statuses were discordant in 13 (27%) and 15 (31%) cases, respectively. No difference in expression was observed between synchronous and metachronous metastasis. CONCLUSION: Biomarker expression is discordant between the primary tumor and its corresponding metastasis in about one third of patients with NSCLC. These findings should be considered in the setting of clinical trials and further explored using frozen material and high-throughput techniques.

Senescence-associated oxidative DNA damage promotes the generation of neoplastic cells.

Studies on human fibroblasts have led to viewing senescence as a barrier against tumorigenesis. Using keratinocytes, we show here that partially transformed and tumorigenic cells systematically and spontaneously emerge from senescent cultures. We show that these emerging cells are generated from senescent cells, which are still competent for replication, by an unusual budding-mitosis mechanism. We further present data implicating reactive oxygen species that accumulate during senescence as a potential mutagenic motor of this post-senescence emergence. We conclude that senescence and its associated oxidative stress could be a tumor-promoting state for epithelial cells, potentially explaining why the incidence of carcinogenesis dramatically increases with advanced age.

The molecular detection of circulating tumor cells in bladder cancer using telomerase activity.

PURPOSE: The detection of circulating tumor cells and micrometastases may have important prognostic and therapeutic implications. We investigated telomerase activity as a molecular marker for detecting bladder carcinoma cells in blood. MATERIALS AND METHODS: Peripheral blood mononuclear cells were isolated from whole blood using Ficoll/Hypaque. Immuno-magnetic beads labeled with an epithelial specific antibody were used to harvest epithelial cells from peripheral blood mononuclear cells. Telomerase activity was detected in this select population using the telomerase-polymerase chain reaction-enzyme-linked immunosorbent assay test based on the telomerase repeat amplification protocol method. The clinical applicability of this technique was explored by evaluating 30 patients with muscle invasive or metastatic bladder carcinoma and 17 healthy volunteers. RESULTS: Telomerase expression was detected in 27 of the 30 patients (90%) with high grade, muscle invasive or metastatic bladder cancer but in none of the 17 healthy controls. CONCLUSIONS: This test is a minimally invasive and specific approach for detecting circulating epithelial cells in patients with bladder cancer. This method may have great value for monitoring cancer progression.

Human telomerase reverse transcriptase (hTERT) and Ki-67 are better predictors of survival than established clinical indicators in patients undergoing curative hepatic resection for colorectal metastases.

BACKGROUND: We evaluated hTERT and Ki-67 expression in patients who underwent curative resection of hepatic colorectal metastases to determine if these markers of cell proliferation correlated better with survival than an established scoring system that is based on clinical predictors. METHODS: Patients operated on between 1993 and 1997 whose survival time was known were analyzed. For each patient, the clinical prognostic score was derived on the basis of primary node status, disease-free interval, number of hepatic tumors, largest tumor, and carcinoembryonic antigen level, and tumor specimens were analyzed for Ki-67 and hTERT with use of standard immunohistochemical techniques. The immunohistochemical analysis was blinded to all patient characteristics. RESULTS: The study included 66 patients. Twenty-six survived less than 2 years after surgery, 19 survived 2-5 years, and 21 survived more than 5 years. Ki-67 positivity and hTERT positivity (labeling indexes greater than or equal to 50%) were observed in 24 patients and 23 patients, respectively. The clinical score did not predict survival, although there was a weak trend toward a lower score in patients with better survival. Both Ki-67 (P =.04) and hTERT (P =.0001) correlated better with survival than did the clinical score. CONCLUSIONS: In patients undergoing curative resection of hepatic colorectal metastases, hTERT and Ki-67 are better predictors of survival than is a score based on clinical features.