Comparing gene expression in deep infiltrating endometriosis with adenomyosis uteri: evidence for dysregulation of oncogene pathways.

BACKGROUND: The pathogenesis of deep infiltrating endometriosis (DIE) is poorly understood. It is considered a benign disease but has histologic features of malignancy, such as local invasion or gene mutations. Moreover, it is not clear whether its invasive potential is comparable to that of adenomyosis uteri (FA), or whether it has a different biological background. Therefore, the aim of this study was to molecularly characterize the gene expression signatures of both diseases in order to gain insight into the common or different underlying pathomechanisms and to provide clues to pathomechanisms of tumor development based on these diseases. METHODS: In this study, we analyzed formalin-fixed and paraffin-embedded tissue samples from two independent cohorts. One cohort involved 7 female patients with histologically confirmed FA, the other cohort 19 female patients with histologically confirmed DIE. The epithelium of both entities was microdissected in a laser-guided fashion and RNA was extracted. We analyzed the expression of 770 genes using the nCounter expression assay human PanCancer (Nanostring Technology). RESULTS: In total, 162 genes were identified to be significantly down-regulated (n = 46) or up-regulated (n = 116) in DIE (for log2-fold changes of < 0.66 or > 1.5 and an adjusted p-value of < 0.05) compared to FA. Gene ontology and KEGG pathway analysis of increased gene expression in DIE compared to FA revealed significant overlap with genes upregulated in the PI3K pathway and focal adhesion signaling pathway as well as other solid cancer pathways. In FA, on the other hand, genes of the RAS pathway showed significant expression compared to DIE. CONCLUSION: DIE and FA differ significantly at the RNA expression level: in DIE the most expressed genes were those belonging to the PI3K pathway, and in FA those belonging to the RAS pathway.

[Relevant mutations in predictive breast cancer pathology]. / Prädiktive Mutationsdiagnostik bei Mammakarzinomen.

Whereas predictive immunohistochemistry has represented a core element of breast cancer classification for decades, predictive molecular pathology, with the exception of in situ hybridization for assessment of HER2 amplification, has only recently gained importance because novel drugs have been approved for treatment of metastatic disease. For the use of PARP inhibitors, proof of BRCA1 or BRCA2 mutation is mandatory. When mutation of the catalytic subunit α of the phosphatidylinositol­4.5­bisphosphate 3­kinase gene (PIK3CA) is present, which can be encountered in up to 40% of luminal breast cancers, the option for treatment with the specific inhibitor alpelisib arises. The HER2 -encoded growth factor receptor contributes to neoplastic transformation not only by amplification and overexpression but also by activating the mutation of the kinase domain, which is responsive to tyrosine kinase inhibitors of the tucatinib/neratinib type. Up to 30% of metastatic and endocrine treated luminal breast cancers acquire an activating mutation of the estrogen receptor gene ESR1, resulting in an autocrine and ligand-independent growth stimulation resistant to aromatase inhibitors. Larotrectinib-sensitive mutation of tropomyosinreceptor kinase is present in up to 50% of secretory breast cancers, whereas the other histologic subtypes display an incidence of below 1%. In conclusion, predictive molecular pathology has gained importance in metastatic breast cancer.

Prognostic gene expression signature for high-grade serous ovarian cancer.

BACKGROUND: Median overall survival (OS) for women with high-grade serous ovarian cancer (HGSOC) is ∼4 years, yet survival varies widely between patients. There are no well-established, gene expression signatures associated with prognosis. The aim of this study was to develop a robust prognostic signature for OS in patients with HGSOC. PATIENTS AND METHODS: Expression of 513 genes, selected from a meta-analysis of 1455 tumours and other candidates, was measured using NanoString technology from formalin-fixed paraffin-embedded tumour tissue collected from 3769 women with HGSOC from multiple studies. Elastic net regularization for survival analysis was applied to develop a prognostic model for 5-year OS, trained on 2702 tumours from 15 studies and evaluated on an independent set of 1067 tumours from six studies. RESULTS: Expression levels of 276 genes were associated with OS (false discovery rate < 0.05) in covariate-adjusted single-gene analyses. The top five genes were TAP1, ZFHX4, CXCL9, FBN1 and PTGER3 (P < 0.001). The best performing prognostic signature included 101 genes enriched in pathways with treatment implications. Each gain of one standard deviation in the gene expression score conferred a greater than twofold increase in risk of death [hazard ratio (HR) 2.35, 95% confidence interval (CI) 2.02-2.71; P < 0.001]. Median survival [HR (95% CI)] by gene expression score quintile was 9.5 (8.3 to -), 5.4 (4.6-7.0), 3.8 (3.3-4.6), 3.2 (2.9-3.7) and 2.3 (2.1-2.6) years. CONCLUSION: The OTTA-SPOT (Ovarian Tumor Tissue Analysis consortium - Stratified Prognosis of Ovarian Tumours) gene expression signature may improve risk stratification in clinical trials by identifying patients who are least likely to achieve 5-year survival. The identified novel genes associated with the outcome may also yield opportunities for the development of targeted therapeutic approaches.

[Update of the German S3 breast cancer guideline : What is new for pathologists?] / Update der S3-Leitlinie Mammakarzinom : Was gibt es Neues für Pathologen?

The relevant content changes for pathologists in the updated interdisciplinary S3 guideline "Early detection, diagnosis, therapy and aftercare of breast cancer" are explained and discussed in the context of the most recent evidence. These include recommendations for risk assessment using Ki-67 and the use of multigene tests in the decision for or against adjuvant chemotherapy in estrogen receptor(ER)­/progesterone receptor(PR)-positive and human epidermal growth factor receptor 2 (HER2)-negative invasive breast cancer. In addition, the assessment of HER2 status is explained. It is described which threshold distance or resection margin status is considered sufficient for resection of ductal carcinoma in situ (DCIS) and invasive breast carcinoma. Finally, recommendations concerning the clinical consequences to be drawn from the detection of a risk lesion in a core needle or vacuum biopsy or at the resection margin of a breast surgical specimen are discussed.

Gonadoblastoma Y locus genes expressed in germ cells of individuals with dysgenetic gonads and a Y chromosome in their karyotypes include DDX3Y and TSPY.

STUDY QUESTION: Which Y genes mapped to the 'Gonadoblastoma Y (GBY)' locus on human Y chromosome are expressed in germ cells of individuals with some Differences of Sexual Development (DSD) and a Y chromosome in their karyotype (DSD-XY groups)? SUMMARY ANSWER: The GBY candidate genes DDX3Y and TSPY are expressed in the germ cells of DSD-XY patients from distinct etiologies: patients with mixed gonadal dysgenesis (MGD) and sex chromosome mosaics (45,X0/46,XY; 46,XX/46,XY); patients with complete androgen insensitivity (CAIS), patients with complete gonadal dysgenesis (CGD; e.g. Swyer syndrome). WHAT IS KNOWN ALREADY: A GBY locus was proposed to be present on the human Y chromosome because only DSD patients with a Y chromosome in their karyotype have a high-although variable-risk (up to 55%) for germ cell tumour development. GBY was mapped to the proximal part of the short and long Y arm. TSPY located in the proximal part of the short Y arm (Yp11.1) was found to be a strong GBY candidate gene. It is expressed in the germ cells of DSD-XY patients with distinct etiologies but also in foetal and pre-meiotic male spermatogonia. However, the GBY region extends to proximal Yq11 and therefore includes probably more than one candidate gene. STUDY DESIGN, SIZE, DURATION: Protein expression of the putative GBY candidate gene in proximal Yq11, DDX3Y, is compared with that of TSPY in serial gonadal tissue sections of 40 DSD-XY individuals from the three DSD patient groups (MGD, Complete Androgen Insensitivity Syndrome [CAIS], CGD) with and without displaying malignancy. Expression of OCT3/4 in the same tissue samples marks the rate of pluripotent germ cells. PARTICIPANTS/MATERIALS, SETTING, METHOD: A total of 145 DSD individuals were analysed for the Y chromosome to select the DSD-XY subgroup. PCR multiplex assays with Y gene specific marker set score for putative microdeletions in GBY Locus. Immunohistochemical experiments with specific antisera mark expression of the GBY candidate proteins, DDX3Y, TSPY, in serial sections of the gonadal tissue samples; OCT3/4 expression analyses in parallel reveal the pluripotent germ cell fraction. MAIN RESULTS AND THE ROLE OF CHANCE: Similar DDX3Y and TSPY protein expression patterns were found in the germ cells of DSD-XY patients from each subgroup, independent of age. In CAIS patients OCT3/4 expression was often found only in a fraction of these germ cells. This suggest that GBY candidate proteins are also expressed in the non-malignant germ cells of DSD-XY individuals like in male spermatogonia. LIMITATIONS, REASONS FOR CAUTION: Variation of the expression profiles of GBY candidate genes in the germ cells of some DSD-XY individuals suggests distinct transcriptional and translational control mechanisms which are functioning during expression of these Y genes in the DSD-XY germ cells. Their proposed GBY tumour susceptibility function to transform these germ cells to pre-malignant GB/Germ Cell Neoplasia in Situ (GB/GCNIS) cells seems therefore to be limited and depending on their state of pluripotency. WIDER IMPLICATIONS OF THE FINDINGS: These experimental findings are of general importance for each individual identified in the clinic with DSD and a Y chromosome in the karyotype. To judge their risk of germ cell tumour development, OCT3/4 expression analyses on their gonadal tissue section is mandatory to reveal the fraction of germ cells still being pluripotent. Comparative expression analysis of the GBY candidate genes can be helpful to reveal the fraction of germ cells with genetically still activated Y chromosomes contributing to further development of malignancy if at high expression level. STUDY FUNDING/COMPETING INTEREST(S): This research project was supported by a grant (01GM0627) from the BMBF (Bundesministerium für Bildung und Forschung), Germany to P.H.V. and B.B. The authors have no competing interests.

[Metastatic breast cancer in the spine : Molecular predictors for choosing adequate treatment strategies]. / Spinale Mammakarzinommetastasen aus Sicht der Wirbelsäulenchirurgie : Prädiktoren auf Molekularebene zur Wahl der adäquaten Therapiestrategie.

Breast cancer is the most common malignancy affecting women and the spinal column is most likely affected by metastases. Modern oncologic treatment options have significantly prolonged survival times in the last decade. Therefore, treatment of vertebral metastases has been of special interest in spine surgery. Different scores are described to evaluate prognosis and to choose correct treatment strategies, which however only differentiate tumor entities and not specific tumor phenotypes. Breast cancer has been classified into five intrinsic subtypes with different survival rates since the turn of the millennium. The aim of this review was to describe molecular predictors of breast cancer malignancy and to better estimate expected survival times and invasiveness of therapies with regard to spinal metastases.

Long-term correction of hemophilia A mice following lentiviral mediated delivery of an optimized canine factor VIII gene.

Current therapies for hemophilia A include frequent prophylactic or on-demand intravenous factor treatments which are costly, inconvenient and may lead to inhibitor formation. Viral vector delivery of factor VIII (FVIII) cDNA has the potential to alleviate the debilitating clotting defects. Lentiviral-based vectors delivered to murine models of hemophilia A mediate phenotypic correction. However, a limitation of lentiviral-mediated FVIII delivery is inefficient transduction of target cells. Here, we engineer a feline immunodeficiency virus (FIV) -based lentiviral vector pseudotyped with the baculovirus GP64 envelope glycoprotein to mediate efficient gene transfer to mouse hepatocytes. In anticipation of future studies in FVIII-deficient dogs, we investigated the efficacy of FIV-delivered canine FVIII (cFVIII). Codon-optimization of the cFVIII sequence increased activity and decreased blood loss as compared to the native sequence. Further, we compared a standard B-domain deleted FVIII cDNA to a cDNA including 256 amino acids of the B-domain with 11 potential asparagine-linked oligosaccharide linkages. Restoring a partial B-domain resulted in modest reduction of endoplasmic reticulum (ER) stress markers. Importantly, our optimized vectors achieved wild-type levels of phenotypic correction with minimal inhibitor formation. These studies provide insights into optimal design of a therapeutically relevant gene therapy vector for a devastating bleeding disorder.

Novel GP64 envelope variants for improved delivery to human airway epithelial cells.

Lentiviral vectors pseudotyped with the baculovirus envelope protein GP64 transduce primary cultures of human airway epithelia (HAE) at their apical surface. Our goal in this study was to harness a directed evolution approach to develop a novel envelope glycoprotein with increased transduction properties for HAE. Using error-prone PCR, a library of GP64 mutants was generated and used to prepare a diverse pool of lentiviral virions pseudotyped with GP64 variants. The library was serially passaged on HAE and three GP64 mutations were recovered. Single-, double- and the triple-combination mutant envelope glycoproteins were compared with wild-type GP64 for their ability to transduce HAE. Our results suggest that lentiviral vectors pseudotyped with evolved GP64 transduced HAE with greater efficiency than wild-type GP64. This effect was not observed in primary cultures of porcine airway epithelial cells, suggesting that the directed evolution protocol was species specific. In summary, our studies indicate that serial passage of a GP64 mutant library yielded specific variants with improved HAE cell tropism, yielding tools with the potential to improve the success of gene therapy for airway diseases.

Lean Umbilical Cord - a Case Report.

The "lean" umbilical cord (also known as thin-cord syndrome) is a comparatively rare anomaly of the umbilical cord, which has seldom been described in the medical literature. We report on a 35-year-old women who presented to us at 29 + 4 weeks gestation with vaginal bleeding and cervical incompetence subsequently complicated not only by premature rupture of membranes but also acute placental insufficiency requiring emergency caesarean section under general anaesthesia at 31 + 2 weeks gestation. At surgery no obvious cause for the acute placental insufficiency - such as placental abruption, cord prolapse or true knot of the umbilical cord - was found. Other possible causes such as vasa praevia or placenta praevia had previously been excluded sonographically on admission for vaginal bleeding. The only notable intraoperative finding was a macroscopically extremely thin umbilical cord.

Changes in chemotherapy usage and outcome of early breast cancer patients in the last decade.

BACKGROUND: During the last decade, neoadjuvant chemotherapy (NACT) of early breast cancer (EBC) evolved from a therapy intended to enable operability to a standard treatment option aiming for increasing cure rates equivalent to adjuvant chemotherapy (ACT). In parallel, improvements in the quality control of breast cancer care have been established in specialized breast care units. PATIENTS AND METHODS: This study analyzed chemotherapy usage in patients with EBC treated at the Heidelberg University Breast Unit between January 2003 and December 2014. RESULTS: Overall, 5703 patients were included in the analysis of whom 2222 (39 %) received chemotherapy, 817 (37 %) as NACT, and 1405 (63 %) as ACT. The chemotherapy usage declined from 48 % in 2003 to 34 % in 2014 of the cohort. Further, the proportion of NACT raised from 42 to 65 % irrespective of tumor subtype. In addition, frequency of pathologic complete response (pCR) defined as no tumor residues in breast and axilla (ypT0 ypN0) at surgery following NACT increased from 12 % in 2003 to 35 % in 2014. The greatest effect was observed in HER2+ breast cancer with an increase in patients achieving pCR from 24 to 68 %. CONCLUSIONS: The results mirror the refined indication for chemotherapy in EBC and its preferred usage as NACT in Germany. The increase in pCR rate over time suggests improvement in outcome accomplished by a multidisciplinary decision-making process and stringent measures for quality control.

Multigene Assays for Classification, Prognosis, and Prediction in Breast Cancer: a Critical Review on the Background and Clinical Utility.

Gene signatures which are based on multigene profiling assays have been developed for the purpose to better define the prognosis and prediction of therapy results in early-stage breast cancer. These assays were designed to be more specific than conventional clinico-pathologic parameters in the selection of patients for (neo-)adjuvant treatment and in effect help to avoid unnecessary cytotoxic treatment. In this review we describe molecular risk scores, for which tests are commercially available (PAM50®, MammaTyper®, MammaPrint®, Oncotype DX®, Endopredict®, Genomic Grade Index®) and IHC risk scores (Mammostrat® and IHC4), and discuss the current evidence of their clinical use.

Outcome analysis of patients with primary breast cancer initially treated at a certified academic breast unit.

INTRODUCTION: Evaluation of oncological outcome and prognostic factors of patients with primary breast cancer treated at a certified academic breast unit. PATIENTS AND METHODS: We prospectively collected data of 3338 patients, diagnosed with primary breast cancer between 01.01.2003 and 31.12.2010 and treated at the Breast Unit Heidelberg, Germany, in order to analyze outcome in clinical practice. We evaluated local control rate (LCR), disease-free survival (DFS), distant disease-free survival (DDFS), observed overall survival (OS) and age-adjusted relative overall survival (ROS). In addition, the impact of known prognostic factors on these outcome variables was examined in univariate and multivariate analyses. RESULTS: Of all patients, 368 (11.0%) had carcinoma in situ (CIS) and 197 (5.9%) had bilateral cancers. For the 2970 patients with invasive cancer, of which 49 patients (1.7%) had metastastic disease at time of diagnosis, DFS, LCR, DDFS, OS and ROS at 5 years were 79.8%, 84.7%, 81.2%, 86.3%, and 89.8%, respectively. In multivariate analysis age, pT category, nodal status, hormone receptor status and grading were identified as independent prognostic factors for OS. CONCLUSION: Compared with recent population-based reports from Germany, more favourable patient characteristics and nominally higher survival was found among this large cohort of patients with primary breast cancer treated at a single certified breast unit.

Deubiquitination of EGFR by Cezanne-1 contributes to cancer progression.

Once stimulated, the epidermal growth factor receptor (EGFR) undergoes self-phosphorylation, which, on the one hand, instigates signaling cascades, and on the other hand, recruits CBL ubiquitin ligases, which mark EGFRs for degradation. Using RNA interference screens, we identified a deubiquitinating enzyme, Cezanne-1, that opposes receptor degradation and enhances EGFR signaling. These functions require the catalytic- and ubiquitin-binding domains of Cezanne-1, and they involve physical interactions and transphosphorylation of Cezanne-1 by EGFR. In line with the ability of Cezanne-1 to augment EGF-induced growth and migration signals, the enzyme is overexpressed in breast cancer. Congruently, the corresponding gene is amplified in approximately one third of mammary tumors, and high transcript levels predict an aggressive disease course. In conclusion, deubiquitination by Cezanne-1 curtails degradation of growth factor receptors, thereby promotes oncogenic growth signals.

Variable expression of the Fragile X Mental Retardation 1 (FMR1) gene in patients with premature ovarian failure syndrome is not dependent on number of (CGG)n triplets in exon 1.

BACKGROUND: Increased expression of the Fragile X Mental Retardation 1 (FMR1) gene in blood cells has been claimed to be associated with variable (CGG)(n) triplet numbers in the 5' untranslated region of this gene. Increased CGG triplet numbers, including that of the so-called premutation range (n= 55-200), were shown to have a risk of <26% to impair ovarian reserve leading to primary ovarian insufficiency and premature ovarian failure (POF). METHODS: DNA and RNA samples were isolated from 74 patients with idiopathic POF to evaluate quantitatively the expression of FMR1 in leukocytes and CGG triplet number on FMR1 gene alleles. mRNA levels were normalized and compared with those of control women. Expression of the encoded protein (FMRP) was analysed by immunohistochemistry on ovarian biopsy tissue sections. RESULTS: A large variance of the FMR1 transcript level was found in the leukocyte RNA samples, but only in patients with POF, and this variability did not correlate to variance of CGG triplet numbers found on both FMR1 alleles (19 < n > 90). During normal folliculogenesis, FMRP is predominantly expressed in granulosa cells. CONCLUSIONS: Our data suggest that FMR1 expression during human folliculogenesis is probably a quantitative trait. Proper function of FMRP in granulosa cells seems to depend on an optimal transcript level. All women with CGG triplet numbers outside the range associated with normal folliculogenesis (26 < n > 34) are therefore expected to have a relaxed FMR1 transcription control. FMR1 transcript levels in leukocytes might therefore be diagnostic for altered FMRP levels in granulosa cells, which will affect the process of folliculogenesis.

FGFR4 Arg388 genotype is associated with pathological complete response to neoadjuvant chemotherapy for primary breast cancer.

BACKGROUND: A single-nucleotide polymorphism (SNP) in the FGFR4 gene is associated with poor prognosis in solid tumors. A recent study presented the first evidence that FGFR4 Arg388 could predict resistance to adjuvant chemotherapy in breast cancer. The present study evaluates the potential of this SNP to predict response to neoadjuvant chemotherapy (NCT) for primary breast cancer (PBC). METHODS: As part of a randomized phase II trial, 257 patients received either doxorubicin-cyclophosphamide (AC) or doxorubicin-pemetrexed (AP) followed by docetaxel (Doc; Taxotere) as NCT for T2-4/N0-2/M0 PBC. FGFR4 genotype analyzed on germline DNA was correlated with clinicopathologic variables, clinical response, and pathological complete response (pCR) using univariate and multivariate analyses. RESULTS: Only axillary lymph node status was associated with FGFR4 Arg388 [odds ratio (OR) 1.82, P = 0.03]. Joint analysis of both treatment arms revealed a correlation of FGFR4 Arg388 with clinical response (OR 2.14, P = 0.03) but not with pCR. In the AC-Doc arm, however, FGFR4 Arg388 was a strong predictor of pCR in the multivariate analysis (OR 3.79, P = 0.03). A significant interaction between FGFR4 genotype and treatment (P = 0.01) was found, indicating a therapy-specific effect. CONCLUSION: We provide the evidence that FGFR4 388Arg is an independent predictor of pCR following AC-Doc as NCT in PBC.

Progress and prospects: prospects of repeated pulmonary administration of viral vectors.

Pulmonary gene therapy may ultimately cure diseases such as cystic fibrosis, alpha1-antitrypsin deficiency, lung cancer and pulmonary hypertension. Efficient expression of delivered genes in target cell types is essential for the achievement of this goal. To this end, re-administration of viral vectors may be required (1) to increase the percentage of transduced airway epithelial cells, (2) to direct gene transfer to individual lobes during successive delivery sessions or (3) to boost attenuated expression over time. Immune responses to viral proteins or viral-encoded proteins are the greatest barrier to repeated vector administration.

Gene therapy progress and prospects: development of improved lentiviral and retroviral vectors--design, biosafety, and production.

Replication defective vectors derived from simple retroviruses or the more complex genomes of lentiviruses continue to offer the advantages of long-term expression, cell and tissue specific tropism, and large packaging capacity for the delivery of therapeutic genes. The occurrence of adverse events caused by insertional mutagenesis in three patients in a gene therapy trial for X-linked SCID emphasizes the potential for problems in translating this approach to the clinic. Several genome-wide studies of retroviral integration are now providing novel insights into the integration site preferences of different vector classes. We review recent developments in vector design, integration, biosafety, and production.

Characteristics associated with long-term progression-free survival following high-dose chemotherapy in metastatic breast cancer and influence of chemotherapy dose.

BACKGROUND: The purpose of this study was to characterize long-term progression-free survivors (LTPFS) of metastatic breast cancer (MBC) following high-dose chemotherapy (HDCT) with autologous stem cell transplantation (ASCT) and to assess the influence of chemotherapy dose in order to identify patients who derive major benefit from this approach. PATIENTS AND METHODS: We compared patient and tumor characteristics of 16 LTPFS with the characteristics of 118 MBC patients who received HDCT with ASCT at our institution between 1992 and 2000. To estimate the cumulative dose of chemotherapy received, the summation dose intensity product (SDIP) of the different chemotherapy regimens was calculated as recently described by Hryniuk et al. The SDIP of the induction regimens was added to that of the HDCT regimens to yield the total SDIP of the chemotherapy received. Multivariate analysis was performed to describe the influence of the total SDIP and other prognostic factors on progression-free survival (PFS). RESULTS: LTPFS were mostly < or = 50 years of age and had limited, chemotherapy-sensitive, hormone-responsive MBC. Due to an apparent dose-survival relationship, an increase by 10 units (U) in the SDIP increased the PFS time by 3 months. Independent predictors of an improved PFS were positive estrogen receptors (P = 0.001), positive combined hormone receptors (P = 0.020), and a complete remission/no evidence of disease status after HDCT (P < 0.001). In patients who had a disease-free interval (DFI) >24 months after primary surgery, an SDIP of >55 U was independently associated with a longer PFS [hazard ratio (HR) = 2.73; 95% confidence interval 1.29-5.81; P = 0.009]. CONCLUSION: HDCT can achieve long-term PFS in young MBC patients with limited, hormone-responsive and chemotherapy-sensitive disease. After a DFI >24 months, a longer PFS is associated with a higher chemotherapy dose as measured by SDIP. These retrospective analyses suggest SDIP might be a tool for studying cumulative dose as a determinant of outcome of MBC chemotherapy. Thus far, however, we cannot clearly identify any subgroup of MBC patients in whom HDCT with ASCT is of particular benefit.

Tandem high-dose chemotherapy in high-risk primary breast cancer: a multivariate analysis and a matched-pair comparison with standard-dose chemotherapy.

Stem cell-supported high-dose chemotherapy (HDCT) is currently being evaluated in patients with high-risk primary breast cancer (HRPBC), as defined by extensive axillary lymph node involvement. Conclusive results from randomized studies with sufficient patient numbers and follow-up are pending. We retrospectively analyzed 144 HRPBC patients enrolled in a single-arm trial of tandem HDCT at the University of Heidelberg to evaluate the prognostic value of nodal ratio, HER2/neu status, and cytokeratin-positive bone marrow cells and to compare the outcomes of these patients with those of a conventionally treated control group of 91 patients matched by nodal ratio, tumor size, combined hormone-receptor status, and HER2/neu status. The tandem HDCT regimen consisted of 2 cycles of induction chemotherapy followed by 2 cycles of blood stem cell-supported high-dose ifosfamide, 12 g/m2; carboplatin, 900 mg/M2; and epirubicin, 180 mg/m2. Conventionally treated patients received a regimen containing anthracycline without taxanes (52 patients) or CMF (cyclophosphamide, methotrexate, and 5-flurouracil; 39 patients). With a median follow-up of 3.8 years, disease-free, distant disease-free, and overall survival rates were 62%, 65%, and 84%, respectively. In univariate analysis, besides the hormone receptor status (P = .007), HER2/neu overexpression was the strongest predictor of earlier death (P = .017). In multivariate analysis, a nodal ratio of > or =0.8 was found to be the only independent predictor of relapse (relative risk [RR] = 2.09; 95% confidence interval [CI], 1.21-3.60; P = .008) and only the absence of hormone receptors was associated with earlier death (RR = 3.59; 95% CI, 1.45-8.86; P = .006). Despite a trend toward later distant relapse after HDCT compared with standard-dose chemotherapy with a median follow-up of 3 years (P = .059), thus far, matched-pair analysis has not demonstrated significantly better survival rates after HDCT in all matched patients (P = .786) or in the subgroups of anthracycline-treated patients and patients with and without overexpression of HER2/neu. So far, the follow-up time has been too short to draw definite conclusions; however, patients with a nodal ratio of > or =0.8, receptor-negative tumors, or HER2/neu overexpression are at high risk for relapse and death, irrespective of the kind of adjuvant chemotherapy.

Identification of three human renin mRNA isoforms from alternative tissue-specific transcriptional initiation.

We have reported that mice transgenic for 140- and 160-kb P1 phage artificial chromosomes (PACs) containing the human renin gene express the gene in a highly tissue-restricted and regulated manner. Herein, we demonstrate that the transgene is also expressed appropriately throughout development. In the course of this investigation, we identified the existence of three transcriptional isoforms of human renin mRNA derived from the utilization of alternative transcription start sites. The first isoform is the kidney-specific isoform, which utilizes the classic renin promoter. The second is a brain-specific isoform, which when previously identified in rats and mice was due to a transcription initiation site within intron A. However, the start site in the human gene resides approximately 1,325 bp upstream of the classic promoter and encodes a new exon 1 (termed exon 1b) that splices directly to exon 2. The third isoform is lung specific and is due to transcriptional initiation 79 bp directly upstream of exon 2, fusing additional DNA within intron A (termed exon 1c) directly to exon 2 without splicing. Importantly, the alternative first exons observed in the PAC transgenic mice were identical to those used to transcribe renin in human fetal kidney, brain, and lung, suggesting these sites are bona fide isoforms of human renin mRNA and not artifacts of transgenesis. Moreover, the subtle differences in tissue-specific transcriptional initiation observed in the renin gene of rats and humans can be faithfully and accurately emulated in a transgenic model.