Reconstructing tumor history in breast cancer: signatures of mutational processes and response to neoadjuvant chemotherapy⋆.

BACKGROUND: Different endogenous and exogenous mutational processes act over the evolutionary history of a malignant tumor, driven by abnormal DNA editing, mutagens or age-related DNA alterations, among others, to generate the specific mutational landscape of each individual tumor. The signatures of these mutational processes can be identified in large genomic datasets. We investigated the hypothesis that genomic patterns of mutational signatures are associated with the clinical behavior of breast cancer, in particular chemotherapy response and survival, with a particular focus on therapy-resistant disease. PATIENTS AND METHODS: Whole exome sequencing was carried out in 405 pretherapeutic samples from the prospective neoadjuvant multicenter GeparSepto study. We analyzed 11 mutational signatures including biological processes such as APOBEC-mutagenesis, homologous recombination deficiency (HRD), mismatch repair deficiency and also age-related or tobacco-induced alterations. RESULTS: Different subgroups of breast carcinomas were defined mainly by differences in HRD-related and APOBEC-related mutational signatures and significant differences between hormone-receptor (HR)-negative and HR-positive tumors as well as correlations with age, Ki-67 and immunological parameters were observed. We could identify mutational processes that were linked to increased pathological complete response rates to neoadjuvant chemotherapy with high significance. In univariate analyses for HR-positive tumors signatures, S3 (HRD, P < 0.001) and S13 (APOBEC, P = 0.001) as well as exonic mutation rate (P = 0.002) were significantly correlated with increased pathological complete response rates. The signatures S3 (HRD, P = 0.006) and S4 (tobacco, P = 0.011) were prognostic for reduced disease-free survival of patients with chemotherapy-resistant tumors. CONCLUSION: The results of this investigation suggest that the clinical behavior of a tumor, in particular, response to neoadjuvant chemotherapy and disease-free survival of therapy-resistant tumors, could be predicted by the composition of mutational signatures as an indicator of the individual genomic history of a tumor. After additional validations, mutational signatures might be used to identify tumors with an increased response rate to neoadjuvant chemotherapy and to define therapy-resistant subgroups for future therapeutic interventions.

Tumor mutational burden and immune infiltration as independent predictors of response to neoadjuvant immune checkpoint inhibition in early TNBC in GeparNuevo.

BACKGROUND: The predictive value of tumor mutational burden (TMB), alone or in combination with an immune gene expression profile (GEP), for response to neoadjuvant therapy in early triple negative breast cancer (TNBC) is currently not known, either for immune checkpoint blockade (ICB) or conventional chemotherapy. PATIENTS AND METHODS: We obtained both whole exome sequencing and RNA-Seq data from pretreatment samples of 149 TNBC of the recent neoadjuvant ICB trial, GeparNuevo. In a predefined analysis, we assessed the predictive value of TMB and a previously developed immune GEP for pathological complete remission (pCR). RESULTS: Median TMB was 1.52 mut/Mb (range 0.02-7.65) and was significantly higher in patients with pCR (median 1.87 versus 1.39; P = 0.005). In multivariate analysis, odds ratios for pCR per mut/Mb were 2.06 [95% confidence intervals (CI) 1.33-3.20, P = 0.001] among all patients, 1.77 (95% CI 1.00-3.13, P = 0.049) in the durvalumab treatment arm, and 2.82 (95% CI 1.21-6.54, P = 0.016) in the placebo treatment arm, respectively. We also found that both continuous TMB and immune GEP (or tumor infiltrating lymphocytes) independently predicted pCR. When we stratified patients in groups based on the upper tertile of TMB and median GEP, we observed a pCR rate of 82% (95% CI 60% to 95%) in the group with both high TMB and GEP in contrast to only 28% (95% CI 16% to 43%) in the group with both low TMB and GEP. CONCLUSIONS: TMB and immune GEP add independent value for pCR prediction. Our results recommend further analysis of TMB in combination with immune parameters to individually tailor therapies in breast cancer.

A randomised phase II study investigating durvalumab in addition to an anthracycline taxane-based neoadjuvant therapy in early triple-negative breast cancer: clinical results and biomarker analysis of GeparNuevo study.

BACKGROUND: Combining immune-checkpoint inhibitors with chemotherapy yielded an increased response rates in patients with metastatic triple-negative breast cancer (TNBC). Therefore, we evaluated the addition of durvalumab to standard neoadjuvant chemotherapy (NACT) in primary TNBC. PATIENTS AND METHODS: GeparNuevo is a randomised phase II double-blind placebo-controlled study randomising patients with TNBC to durvalumab or placebo given every 4 weeks in addition to nab-paclitaxel followed by standard EC. In the window-phase durvalumab/placebo alone was given 2 weeks before start of nab-paclitaxel. Randomisation was stratified by stromal tumour-infiltrating lymphocyte (sTILs). Patients with primary cT1b-cT4a-d disease, centrally confirmed TNBC and sTILs were included. Primary objective was pathological complete response (pCR) (ypT0 ypN0). RESULTS: A total of 174 patients were randomised, 117 participated in the window-phase. Median age was 49.5 years (range 23-76); 47 patients (27%) were younger than 40 years; 113 (65%) had stage ≥IIA disease, 25 (14%) high sTILs, 138 of 158 (87%) were PD-L1-positive. pCR rate with durvalumab was 53.4% (95% CI 42.5% to 61.4%) versus placebo 44.2% (95% CI 33.5% to 55.3%; unadjusted continuity corrected χ2P = 0.287), corresponding to OR = 1.45 (95% CI 0.80-2.63, unadjusted Wald P = 0.224). Durvalumab effect was seen only in the window cohort (pCR 61.0% versus 41.4%, OR = 2.22, 95% CI 1.06-4.64, P = 0.035; interaction P = 0.048). In both arms, significantly increased pCR (P < 0.01) were observed with higher sTILs. There was a trend for increased pCR rates in PD-L1-positive tumours, which was significant for PD-L1-tumour cell in durvalumab (P = 0.045) and for PD-L1-immune cell in placebo arm (P = 0.040). The most common immune-related adverse events were thyroid dysfunction any grade in 47%. CONCLUSIONS: Our results suggest that the addition of durvalumab to anthracycline-/taxane-based NACT increases pCR rate particularly in patients treated with durvalumab alone before start of chemotherapy. TRIAL REGISTRATION: ClinicalTrials.gov number: NCT02685059.

A gene signature to predict high tumor-infiltrating lymphocytes after neoadjuvant chemotherapy and outcome in patients with triple-negative breast cancer.

Background: In patients with triple-negative breast cancer (TNBC), the extent of tumor-infiltrating lymphocytes (TILs) in the residual disease after neoadjuvant chemotherapy (NACT) is associated with better prognosis. Our objective was to develop a gene signature from pretreatment samples to predict the extent of TILs after NACT and then to test its prognostic value on survival. Patients and methods: Using 99 pretreatment samples, we generated a four-gene signature associated with high post-NACT TILs. Prognostic value of the signature on distant relapse-free survival (DRFS) was first assessed on the training set (n = 99) and then on an independent validation set (n = 115). Results: A four-gene signature combining the expression levels of HLF, CXCL13, SULT1E1, and GBP1 was developed in baseline samples to predict the extent of lymphocytic infiltration after NACT. In a multivariate analysis performed on the training set, this signature was associated with DRFS [hazard ratio (HR): 0.28, for a one-unit increase in the value of the four-gene signature, 95% confidence interval (CI): 0.13-0.63)]. In a multivariate analysis performed on an independent validation set, the four-gene signature was significantly associated with DRFS (HR: 0.17, 95% CI: 0.06-0.43). The four-gene signature added significant prognostic information when compared with the clinicopathologic pretreatment model (likelihood ratio test in the training set P = 0.004 and in the validation set P = 0.002). Conclusions: A four-gene signature predicts high levels of TILs after anthracycline-containing NACT and outcome in patients with TNBC and adds prognostic information to a clinicopathological model at diagnosis.

Human equilibrative nucleoside transporter 1 expression analysed by the clone SP 120 rabbit antibody is not predictive in patients with pancreatic cancer treated with adjuvant gemcitabine - Results from the CONKO-001 trial.

BACKGROUND: High expression of human equilibrative nucleoside transporter 1 (hENT1) is considered to predict survival in patients treated with adjuvant gemcitabine for pancreatic cancer. A standard evaluation system for immunohistochemical analysis (antibody, scoring system) has not yet been established. METHODS: CONKO-001, a prospective randomised phase III study investigated the role of adjuvant gemcitabine (gem) as compared to observation (obs). Tumour samples of 156 patients were analysed by immunohistochemistry with the rabbit monoclonal antibody SP120 (Ventana Medical Systems) for expression of hENT1. Kaplan-Meier analyses for median disease-free survival (DFS) and overall survival (OS) were performed in dependence of hENT1 expression measured analogously to Farrell et al. 2009 and Poplin et al. 2013. RESULTS: For the 88 gem and 68 obs patients, median DFS/OS was 12.9/22.7 months and 6.2/19.1 months. High hENT1 expression was not associated with improved median DFS (Farrell: no hENT1 22.2 months, low hENT1 13.7 months, high hENT1 12.1 months, p=0.248; Poplin: low hENT1 13.2 months versus high hENT1 11.5 months, p=0.5) or median OS (Farrell: no hENT1 21.7 months, low hENT1 24.7 months, high hENT1 19.5, p=0.571; Poplin: low hENT1 24.4 months versus high hENT1 19.7 months, p=0.92;) in the gem group or in the obs group (median DFS Farrell: no hENT1 5.1 months, low hENT1 6.2 months, high hENT1 7.5 months, p=0.375; Poplin: low hENT1 6.2 months versus high hENT1 5.9 months, p=0.83; median OS Farrell: no hENT1 20.2months, low hENT1 17.7 months, high HENT1 19.1 months, p=0.738; Poplin: low hENT1 17.7 months versus high hENT1 20.4 months, p=0.65) measured by the Farrell or Poplin Score. CONCLUSIONS: We cannot confirm a predictive role of hENT1 measured by the clone SP120 rabbit antibody in our study population. Reproducible standard procedures are urgently needed prior to the implementation or exclusion of hENT1 as a predictive biomarker in the treatment of pancreatic cancer. TRIAL REGISTRATION: ISRCTN34802808.

Expression of secreted protein acidic and rich in cysteine (SPARC) in breast cancer and response to neoadjuvant chemotherapy.

BACKGROUND: Secreted protein acidic and rich in cysteine (SPARC) has been suggested as a new biomarker and therapeutic target in breast cancer, as well as other tumor types. PATIENTS AND METHODS: We evaluated the frequency of SPARC expression among different molecular breast cancer subtypes and its role for therapy response after neoadjuvant chemotherapy. In this study, pretherapeutic core biopsies of 667 patients from the neoadjuvant GeparTrio trial were evaluated for SPARC expression by immunohistochemistry using a standardized immunoreactive score (IRS). RESULTS: An increased SPARC expression (IRS ≥6) was observed in 26% of all tumors. In triple-negative tumors, SPARC expression was increased in 37% of tumors, compared with other molecular subtypes (23% HR+/HER2-, 29% HR+/HER2+ and 22% HR-/HER2+; P = 0.038). Increased SPARC expression was associated with an increased pathological complete response (pCR) rate of 27%, compared with 15% in tumors with low SPARC expression (P < 0.001). In the triple-negative subgroup, pCR rates were 47% in tumors with high SPARC expression, compared with 26% in tumors with low SPARC expression (P = 0.032). In multivariable analysis, SPARC was independently predictive in the overall population (P = 0.010) as well as the triple-negative subgroup (P = 0.036). CONCLUSIONS: SPARC is frequently expressed in breast cancer with triple-negative breast cancer revealing the highest expression rate. High SPARC expression of the primary tumor is associated with a higher chance of achieving a pathological complete remission after TAC or TAC-NX chemotherapy. As SPARC is an albumin-binding protein and might mediate intratumoral accumulation of albumin bound drugs, SPARC should be further evaluated as a predictive marker especially for response to albumin-bound drugs like nab-paclitaxel. CLINICAL TRIAL NUMBER: NCT00544765.

α-Smooth muscle actin expression and desmoplastic stromal reaction in pancreatic cancer: results from the CONKO-001 study.

BACKGROUND: Previous investigations in pancreatic cancer suggest a prognostic role for α-smooth muscle actin (α-SMA) expression and stromal density in the peritumoural stroma. The aim of this study was to further validate the impact of α-SMA expression and stromal density in resectable pancreatic cancer patients treated with adjuvant gemcitabine compared with untreated patients. METHODS: CONKO-001 was a prospective randomised phase III study investigating the role of adjuvant gemcitabine as compared with observation. Tissue samples of 162 patients were available for immunohistochemistry on tissue microarrays to evaluate the impact of α-SMA expression and stromal density impact on patient outcome. RESULTS: High α-SMA expression in tumour stroma was associated with worse patient outcome (DFS: P=0.05, OS: P=0.047). A dense stroma reaction was associated with improved disease-free survival (DFS) and overall survival (OS) in the overall study population (DFS: P=0.001, OS: P=0.001). This positive prognostic impact was restricted to patients with no adjuvant treatment (DFS: P<0.001, OS: P<0.001). In multivariable analysis, α-SMA and stromal density expression were independently predictive factors for survival. CONCLUSIONS: Our data confirm the negative prognostic impact of high α-SMA expression in pancreatic cancer patients after curatively intended resection. In contrast to former investigations, we found a positive prognostic impact for a dense stroma. This significant influence was restricted to patients who received no adjuvant therapy.

[Osseous tuberculosis as a rare differential diagnosis of femoral head necrosis]. / Ossäre Tuberkulose als seltene Differenzialdiagnose der Femurkopfnekrose.

BACKGROUND: We report on a 60-year-old immunocompetent German male patient without risk factors, who had been suffering from pain in the right hip for 8 months. DIAGNOSTICS: Radiographs showed destruction of the femoral head with a collapse of the main weight-bearing area, which was interpreted as femoral head necrosis. THERAPY: A cement-free total hip prosthesis was then implanted. The femoral head was sent for routine histological analysis and PCR amplification yielded a positive result for Mycobacterium tuberculosis complex DNA, leading to immediate guideline-based tuberculostatic treatment. CONCLUSION: Tuberculosis should be considered as a differential diagnosis in the case of destruction of the femoral head, especially in immunocompromised patients, patients with a foreign background or destructive osteoarthritis of the hip with an atypical course. Antibiotic treatment is necessary postoperatively. Under this therapy, a good clinical outcome can be expected comparable to that achieved in patients with primary osteoarthritis without infection.

SPARC expression in resected pancreatic cancer patients treated with gemcitabine: results from the CONKO-001 study.

BACKGROUND: Previous investigations in pancreatic cancer suggested a prognostic role for secreted protein acidic and rich in cysteine (SPARC) expression in the peritumoral stroma but not for cytoplasmic SPARC expression. The aim of this study was to evaluate the impact of SPARC expression in pancreatic cancer patients treated with gemcitabine compared with untreated patients. PATIENTS AND METHODS: CONKO-001 was a prospective randomized phase III study investigating the role of adjuvant gemcitabine when compared with observation. Tissue samples of 160 patients were available for SPARC immunohistochemistry on tissue microarrays to evaluate its impact on patient outcome. RESULTS: Strong stromal SPARC expression was associated with worse disease-free survival (DFS) and overall survival (OS) in the overall study population (DFS: P = 0.005, OS: P = 0.033). Its negative prognostic impact was restricted to patients treated with gemcitabine (DFS: P = 0.007, OS: P = 0.006). High cytoplasmic SPARC expression also was associated with worse patient outcome (DFS: P = 0.041, OS: P = 0.011). Again the effect was restricted to patients treated with gemcitabine (DFS: P = 0.002, OS: P = 0.003). In multivariable analysis, SPARC expression was independently predictive of patient outcome. CONCLUSIONS: Our data confirm the prognostic significance of SPARC expression after curatively intended resection. The negative prognostic impact was restricted to patients who received adjuvant treatment with gemcitabine, suggesting SPARC as a predictive marker for response to gemcitabine.

Evaluation of Mucin-1 protein and mRNA expression as prognostic and predictive markers after neoadjuvant chemotherapy for breast cancer.

BACKGROUND: Mucin-1 (MUC1) is a promising antigen for the development of tumor vaccines. We evaluated the frequency of MUC1 expression and its impact on therapy response and survival after neoadjuvant chemotherapy for breast cancer. PATIENTS AND METHODS: Pre-treatment core biopsies of patients from the GeparTrio neoadjuvant trial (NCT 00544765) were evaluated for MUC1 by immunohistochemistry (IHC; N = 691) and quantitative RT-PCR (qRT-PCR; N = 286) from formalin-fixed paraffin-embedded (FFPE) samples. RESULTS: MUC1 protein and mRNA was detectable in the majority of cases and was associated with hormone-receptor-positive status (P < 0.001). High MUC1 protein and mRNA expression were associated with lower probability of pathologic complete response (P = 0.017 and P < 0.001) and with longer patient survival (P = 0.03 and P < 0.001). In multivariable analysis, MUC1 protein and mRNA expression were independently predictive (P = 0.001 and P < 0.001). MUC1 protein and mRNA expression were independently prognostic for overall survival (P = 0.029 and P = 0.015). CONCLUSIONS: MUC1 is frequently expressed in breast cancer and detectable on mRNA and protein level from FFPE tissue. It provides independent predictive information for therapy response and survival after neoadjuvant chemotherapy. In clinical immunotherapy trials, MUC1 expression may serve as a predictive marker.

RNA-based determination of ESR1 and HER2 expression and response to neoadjuvant chemotherapy.

BACKGROUND: Hormone and human epidermal growth factor receptor 2 (HER2) receptors are the most important breast cancer biomarkers, and additional objective and quantitative test methods such as messenger RNA (mRNA)-based quantitative analysis are urgently needed. In this study, we investigated the clinical validity of RT-PCR-based evaluation of estrogen receptor (ESR1) and HER2 mRNA expression. PATIENTS AND METHODS: A total of 1050 core biopsies from two retrospective (GeparTrio, GeparQuattro) and one prospective (PREDICT) neoadjuvant studies were evaluated by quantitative RT-PCR for ESR1 and HER2. RESULTS: ESR1 mRNA was significantly predictive for reduced response to neoadjuvant chemotherapy in univariate and multivariate analysis in all three cohorts. The complete pathologically documented response (pathological complete response, pCR) rate for ESR1+/HER2- tumors was 7.3%, 8.0% and 8.6%; for ESR1-/HER2- tumors it was 34.4%, 33.7% and 37.3% in GeparTrio, GeparQuattro and PREDICT, respectively (P < 0.001 in each cohort). In the Kaplan-Meier analysis in GeparTrio patients with ESR1+/HER2- tumors had the best prognosis, compared with ESR1-/HER2- and ESR1-/HER2+ tumors [disease-free survival (DFS): P < 0.0005, overall survival (OS): P < 0.0005]. CONCLUSIONS: Our results suggest that mRNA levels of ESR1 and HER2 predict response to neoadjuvant chemotherapy and are significantly associated with long-term outcome. As an additional option to standard immunohistochemistry and gene-array-based analysis, quantitative RT-PCR analysis might be useful for determination of the receptor status in breast cancer.

[NF-kappaB subunit p65/RelA expression in ovarian carcinoma: prognostic impact and link to COX-2 overexpression]. / Expression der NF-kappaB-Untereinheit p65/RelA im Ovarialkarzinom: Einflubeta auf Prognose und Uberexpression der COX-2.

AIMS: NF-kappaB has been demonstrated to activate proliferative, inflammatory, and angiogenic processes in ovarian cancer cells in vitro. To add translational information on the situation in vivo, we determined the expression pattern of p65, an important subunit of the classic NF-kappaB pathway, in ovarian carcinoma tissue, and investigated in vivo and in vitro whether this pathway is implicated in the known overexpression of cyclooxygenase-2 (COX-2). METHODS: p65 siRNA, chemiluminescent NF-kappaB transcription factor assay, Taqman PCR, as well as immunoblotting were performed with OVCAR-3 ovarian cancer cells. 83 primary ovarian cancinomas as well as 17 cases of benign ovarian tissue were analyzed by p65 and COX-2 immunohistochemistry using a tissue microarray. RESULTS: DNA-binding avtivity as well as COX-2 mRNA and protein expression were strongly inducible by IL-1beta treatment in OVCAR-3 cells, while p65 siRNA inhibited IL-1beta-dependent p65 activity (p = 0.037) as well as COX-2 expression on the mRNA (p < 0.03) and on the protein level. In human tumor tissue, p65 protein expression was significantly associated with COX-2 expression (p = 0.002) as well as tumor grading (p = 0.005). Furthermore, p65 expression was a significant prognostic indicator of a reduced patient survival both in univariate (p = 0.038) and in multivariate analysis (p = 0.014). CONCLUSION: Our study indicates a deregulation of the classical NF-kappaB pathway in ovarian cancer, which results in the overexpression of the NF-kappaB target gene COX-2. Components of this pathway might constitute novel attractive targets for a specific therapy of advanced ovarian cancer.

Radiation-induced apoptosis in human non-small-cell lung cancer cell lines is secondary to cell-cycle progression beyond the G2-phase checkpoint.

PURPOSE: To characterize the relationship between cell-cycle progression and radiation-induced apoptosis in NSCLC cell lines with different p53 status. MATERIALS AND METHODS: Cell lines with functional (H460, A549) and non-functional p53 (H661 and H520) were irradiated with 20 Gy. Multiparameter flow-cytometry was used to follow the progression of synchronized cells through the cell cycle after irradiation. RESULTS: Delayed apoptosis was observed after cell-cycle progression beyond the G2 block, either in the late G2/M-phase of the same cell cycle being irradiated (H661, H520) or in the G1-phase of the subsequent cell cycle (H460, A549). The apoptotic fraction in H661 and H520 was 60-80% at 144h after irradiation, higher than in A549 and H460 (5 and 35%, respectively). As an alternative to apoptosis in cells cycling beyond the G2 restriction point, hyperploid cells were generated by all cell lines. Inhibition of cell-cycle progression through the G2/M-phase efficiently reduced the induction of late apoptosis. After irradiation in S-phase, 50-60% of cells with functional p53 remained arrested at the G2 restriction point until 144 h post-irradiation, while only 20% of the H661 or H520 did so. CONCLUSIONS: These data characterize radiation-induced apoptosis in NSCLC cell lines as a removal pathway of clonogenically inactivated cells secondary to cell-cycle progression beyond G2/M, and is unlikely to be a critical factor for cellular radiation sensitivity.

The novel synthetic inhibitor of matrix metalloproteinases Ro 28-2653 induces apoptosis in Dunning tumor cells.

The effect of synthetic inhibitors of matrix metalloproteinases (MMP) has been shown against a variety of tumors in preclinical models. Ro 28-2653, a novel synthetic MMP inhibitor, is able to reduce tumor growth in orthotopic prostatic cancer in rats (R3327 Dunning tumor). However, at present this inhibitory mechanism in tumor inhibition in vivo can only be partly explained by the inhibition of the catalytic activity of MMPs overexpressed in cancereous tissue. Using the flow cytometric method, we have investigated the effect of various concentrations of Ro 28-2653 on the Dunning tumor cells with regard to the staining of F-actin and DNA as markers of apoptosis. In combination with fluorescence microscopy we detected the loss of F-actin and the degradation of internucleosomal DNA. This effect of Ro 28-2653 on apoptosis was dose- and time-dependent increasing with concentration between 10 and 100 microg/ml as well as with time of treatment between 24 and 48 h.

Effects of Bacteriophages on the Population Dynamics of Four Strains of Pelagic Marine Bacteria.

Viral lysis of specific bacterial populations has been suggested to be an important factor for structuring marine bacterioplankton communities. In the present study, the influence of bacteriophages on the diversity and population dynamics of four marine bacterial phage-host systems was studied experimentally in continuous cultures and theoretically by a mathematical model. By use of whole genome DNA hybridization toward community DNA, we analyzed the dynamics of individual bacterial host populations in response to the addition of their specific phage in continuous cultures of mixed bacterial assemblages. In these experiments, viral lysis had only temporary effects on the dynamics and diversity of the individual bacterial host species. Following the initial lysis of sensitive host cells, growth of phage-resistant clones of the added bacteria resulted in a distribution of bacterial strains in the phage-enriched culture that was similar to that in the control culture without phages after about 50-60 h incubation. Consequently, after a time frame of 5-10 generations after lysis, it was the interspecies competition rather than viral lysis of specific bacterial strains that was the driving force in the regulation of bacterial species composition in these experiments. The clonal diversity, on the other hand, was strongly influenced by viral activity, since the clonal composition of the four species in the phage-enriched culture changed completely from phage-sensitive to phage-resistant clones. The model simulation predicted that viral lysis had a strong impact on the population dynamics, the species composition, and the clonal composition of the bacterial community over longer time scales (weeks). However, according to the model, the overall density of bacteria in the system was not affected by phages, since resistant clones complemented the fluctuations caused by viral lysis. Based on the model analysis, we therefore suggest that viral lysis can have a strong influence on the dynamics of bacterial populations in planktonic marine systems.

Cryopreserved human amniotic membrane for ocular surface reconstruction.

BACKGROUND: Amniotic membrane transplantation is used for the reconstruction of the ocular surface in the context of, for example, corneal ulcers or conjunctival scarring. The mechanisms by which preserved amniotic membrane grafts promote reepithelialization are unknown. As a first step the viability and proliferative capacity of amnion cells following cryopreservation of membranes in glycerol is investigated. METHODS: Fresh and cryopreserved (in 50% glycerol) amniotic membranes were investigated histologically and by vital stains. Following enzymatic digestion, amniotic cells were stained for viability and cultured in DMEM+10% FBS. In addition, explant cultures were established from fresh and cryopreserved membranes. RESULTS: Histological examination showed no significant morphological alteration following cryopreservation. While fresh membranes contained predominantly vital cells, no such cells were detected following cryopreservation. Also, cells removed enzymatically from cryopreserved membranes were not viable and did not grow in culture. While both epithelial and fibroblastic cells grew from fresh membranes, no growth was seen from cryopreserved membranes. CONCLUSION: The results suggest that the technique for preservation which is most widely used for ophthalmological amniotic membrane transplantation significantly impairs viability and proliferative capacity. This supports the clinical finding that neither immunological reactions nor signs of ingrowth of amniotic cells are observed in patients. Furthermore amniotic membrane grafts seem to function primarily as matrix and not by virtue of transplanted functional cells.

Relation of oestradiol-mediated growth stimulation with the expression of c-erbB-2 protein in xenotransplanted oestradiol-receptor-positive and -negative breast carcinomas.

Attempts were made to correlate growth effects induced by oestradiol and tamoxifen with the hormonal regulation of c-erbB-2 protein in experiments in vivo. We report here the responsiveness of four xenotransplanted oestrogen-receptor(ER)-positive and four ER-negative human mammary carcinomas to oestradiol and tamoxifen. Oestradiol in a dose of 0.5 mg/kg significantly increased the growth of the ER-positive mammary carcinomas 3366, MCF-7, 4134 and 4049, but not the ER-negative tumours 4000, 4296 and MT-3. However, within the group of the ER-negative breast carcinomas the tumour 4151 ES deviates from this growth behaviour, as we could prove an estrogen induced growth. The stimulation of tumour growth by oestradiol was always accompanied by a down-regulation of c-erbB-2 protein both in the ER-positive mammary carcinomas and in the ER-negative mammary carcinoma 4151 ES. Tamoxifen significantly inhibited the growth of the ER/PR-positive mammary carcinomas 3366 and MCF-7 but not the ER-positive/PR-negative mammary carcinomas 4049 and 4134. In the group of ER-negative mammary carcinomas only the growth of the oestrogen-responsive tumour 4151 ES was significantly inhibited by tamoxifen. The inhibition of tumour growth by tamoxifen was correlated with a reversion of the oestradiol-induced down-regulation of c-erbB-2, also in the ER-negative/oestradiol-responsive mammary carcinoma 4151 ES. From our results we hypothesize that the oestrogen-dependent growth of ER-negative breast carcinoma 4151 ES could also be correlated with the oestradiol-regulated expression of c-erbB-2 protein.