Ataxia-telangiectasia gene (ATM) mutation heterozygosity in breast cancer: a narrative review.

Background: Despite the fact that heterozygosity for a pathogenic ATM variant is present in 1%-2% of the adult population, clinical guidelines to inform physicians and genetic counsellors about optimal management in that population are lacking. Methods: In this narrative review, we describe the challenges and controversies in the management of women who are heterozygous for a pathogenic ATM variant with respect to screening for breast and other malignancies, to choices for systemic therapy, and to decisions about radiation therapy. Results: Given that the lifetime risk for breast cancer in women who are heterozygous for a pathogenic ATM variant is likely greater than 25%, those women should undergo annual mammographic screening starting at least by 40 years of age. For women in this group who have a strong family history of breast cancer, earlier screening with both magnetic resonance imaging and mammography should be considered. High-quality data to inform the management of established breast cancer in carriers of pathogenic ATM variants are lacking. Although deficiency in the ATM gene product might confer sensitivity to dna-damaging pharmaceuticals such as inhibitors of poly (adp-ribose) polymerase or platinum agents, prospective clinical trials have not been conducted in the relevant patient population. Furthermore, the evidence with respect to radiation therapy is mixed; some data suggest increased toxicity, and other data suggest improved clinical benefit from radiation in women who are carriers of a pathogenic ATM variant. Conclusions: As in the 2017 U.S. National Comprehensive Cancer Network guidelines, we recommend high-risk imaging for women in Ontario who are heterozygous for a pathogenic ATM variant. Currently, ATM carrier status should not influence decisions about systemic or radiation therapy in the setting of an established breast cancer diagnosis.

Activated c-Kit receptor in the heart promotes cardiac repair and regeneration after injury.

The role of endogenous c-Kit receptor activation on cardiac cell homeostasis and repair remains largely unexplored. Transgenic mice carrying an activating point mutation (TgD814Y) in the kinase domain of the c-Kit gene were generated. c-Kit(TgD814Y) receptor was expressed in the heart during embryonic development and postnatal life, in a similar timing and expression pattern to that of the endogenous gene, but not in the hematopoietic compartment allowing the study of a cardiac-specific phenotype. c-Kit(TgD814Y) mutation produced a constitutive active c-Kit receptor in cardiac tissue and cells from transgenic mice as demonstrated by the increased phosphorylation of ERK1/2 and AKT, which are the main downstream molecular effectors of c-Kit receptor signaling. In adult transgenic hearts, cardiac morphology, size and total c-Kit(+) cardiac cell number was not different compared with wt mice. However, when c-Kit(TgD814Y) mice were subjected to transmural necrotic heart damage by cryoinjury (CI), all transgenic survived, compared with half of wt mice. In the sub-acute phase after CI, transgenic and wt mice showed similar heart damage. However, 9 days after CI, transgenic mice exhibited an increased number of c-Kit(+)CD31(+) endothelial progenitor cells surrounding the necrotic area. At later follow-up, a consistent reduction of fibrotic area, increased capillary density and increased cardiomyocyte replenishment rate (as established by BrdU incorporation) were observed in transgenic compared with wt mice. Consistently, CD45(-)c-Kit(+) cardiac stem cells isolated from transgenic c-Kit(TgD814Y) mice showed an enhanced endothelial and cardiomyocyte differentiation potential compared with cells isolated from the wt. Constitutive activation of c-Kit receptor in mice is associated with an increased cardiac myogenic and vasculogenic reparative potential after injury, with a significant improvement of survival.

Positive test dose in a neonate with a caudally placed epidural catheter.

We present a case of a neonate with an epidural catheter placed via the caudal route after induction of general anaesthesia in whom the test doses of epinephrine-containing local anaesthetic was positive on two occasions. Remarkable tachycardia was noted after each of two separate injections through the catheter. Blood was never aspirated from the catheter and placement was without difficulty. After the catheter was removed, blood was noted at the tip.

Analysis of SYT-SSX fusion transcripts and bcl-2 expression and phosphorylation status in synovial sarcoma.

Synovial sarcomas (SS) are characterized by a chromosomal translocation t(X;18)(p11.2;q11.2) which usually fuses the SYT gene from chromosome 18 to SSX1 or SSX2 genes on chromosome X. Also, a variant SYT-SSX4 fusion gene has recently been shown in a single SS case. In addition to these cytogenetic changes, bcl-2 expression, as assessed by immunohistochemistry, has been reported to be an almost general constitutive alteration of SS. In the present work, we analyze a series of 36 SS surgical samples (from 34 patients) by RT-PCR for the presence of the SYT-SSX1 or the SYT-SSX2 fusion transcript. The analysis was extended to SYT-SSX4 on SYT-SSX1-negative and SYT-SSX2-negative cases only. Our results showed a significant correlation between the SYT-SSX2 fusion and the monophasic SS histologic subtype. SYT-SSX1 fusion transcripts were present in both monophasic and biphasic tumors. The SYT-SSX4 fusion type was detected in a single monophasic SS. In the same series of SS cases, we also confirmed and extended the previously reported constitutive expression of bcl-2 protein, by using both immunohistochemical and western blot analysis. Moreover, we demonstrated that the BCL-2 gene is not rearranged or amplified at genomic level, indicating that the high levels of bcl-2 expression observed in SS might result from transcriptional activation of the gene and/or protein stabilization. Finally, we show that bcl-2 is not phosphorylated in tumors from patients who had been preoperatively treated with radio/chemotherapy, in tumors from untreated patients, or in an SS cell line (CME-1) after in vitro treatment with cytotoxic concentrations of DNA-damaging agents or taxanes. These data indicate that SS cells are unable to activate an apoptosis pathway involving bcl-2 phosphorylation/inactivation and may provide a possible explanation for the limited effectiveness of conventional pharmacological treatments of this tumor type.

Detection of ornithine decarboxylase mRNA in human breast cancer MCF-7 cells by in situ RT-PCR.

We have studied the expression of ornithine decarboxylase (ODC) mRNA by in situ hybridization and in situ RT-PCR in human breast cancer MCF-7 cell line. In situ RT-PCR demonstrated the overexpression of ODC mRNA, localized over the cytoplasm, while a very low signal was detected by in situ hybridization. Our findings indicate that in situ RT-PCR could represent a useful tool to study different levels of ODC expression in normal and tumor tissues. Since ODC expression is regulated by c-Myc oncoprotein, this model could be useful to monitor in vivo the effects of new anti-neoplastic molecules, specific inhibitors of c-Myc.

Molecular detection of codon 12 K-RAS mutations in circulating DNA from serum of colorectal cancer patients.

Point mutations of the K-RAS gene at codon 12 are found in about 40% of cases with colorectal cancer. The diagnostic implications of the detection of these mutations and their clinical utility are still unclear. The aim of this study was to test both the feasibility of the detection of the mutated K-RAS gene in serum and its potential role in colorectal cancer detection and monitoring. Codon 12 K-RAS mutations were examined in DNA extracted from the serum of 35 patients with colorectal cancer and were compared with the K-RAS status in the corresponding primary tumor. Molecular detection was performed by the mutant-enriched PCR (ME-PCR) assay, a sensitive method capable of distinguishing a small quantity of mutated DNA in the presence of abundant wild-type DNA. The occurrence of mutations was compared with clinicopathological parameters as well as CEA and CA19.9 serum levels. We found codon 12 K-RAS mutations in the tissue of 13/35 (37%) patients. Serum mutations were detected in 5/13 (38.5%) patients with mutated K-RAS in the tissue. 26/35 (74%) patients showed an identical K-RAS pattern in tissue and serum. No codon 12 K-RAS alterations were found in serum samples of 22 patients with benign gastrointestinal diseases. Elevated serum CEA levels were detected in 16 patients, four of whom also presented serum RAS mutations. Our results confirm that K-RAS mutations can be found in circulating DNA extracted from serum samples of patients with colorectal cancer and show that there is a correspondence between serum and tissue K-RAS patterns.

In situ RT-PCR allows the detection of ornithine decarboxylase mRNA in paraffin embedded archival human hyperplastic breast tissues.

Expression of ornithine decarboxylase (ODC) is induced by c-Myc oncoprotein and is required for cell proliferation and tumour growth. We have studied the expression of ODC mRNA by in situ hybridisation and in situ RT-PCR in archival human hyperplastic breast tissues. A very low signal was detected by in situ hybridisation, while the in situ RT-PCR on human breast archival tissues demonstrated an over-expression of ODC mRNA in epithelial cells characterised by some degree of hyperplasia, maintaining the morphology of the archival tissue intact despite the multiple steps of fixation, permeabilization and thermal cycling.

Inhibition of cancer cell growth and c-Myc transcriptional activity by a c-Myc helix 1-type peptide fused to an internalization sequence.

c-Myc is a nuclear protein with important roles in cell transformation, cell proliferation, and gene transcription. It has been previously shown that a 14-amino acid (aa) modified peptide (H1-S6A,F8A) derived from the helix 1 (H1) carboxylic region of c-Myc can interfere in vitro with specific c-Myc DNA binding. Here, we have linked the above Myc-derived 14-aa peptide to a 16-aa sequence from the third helix of Antennapedia (Int). It has been repeatedly reported that this 16-aa Antennapedia peptide is able to cross mammalian cell membranes and to work as a vector for short peptides. Using fluorescent (dansylated or rhodaminated) peptides, we have shown that the fusion peptide with the Antennapedia fragment (Int-H1-S6A,F8A) but not the c-Myc derived fragment alone (H1-S6A,F8A) was capable of internalization inside MCF-7 human breast cancer cells. Int-H1-S6A,F8A and H1-S6A,F8A were the only two peptides capable of inhibiting coimmunoprecipitation of the c-Myc/Max heterodimer in vitro. We have treated (continuously for 10-11 days) MCF-7 cells with four different peptides: Int, H1-S6A,F8A, Int-H1-S6A,F8A, and Int-H1wt [a peptide differing from Int-H1-S6A,F8A by 2 aa (S6 and F8) in the H1 region]. In intact MCF-7 cells, Int-H1-S6A,F8A was the only active peptide capable of inducing the following biological effects: (a) inhibition of cloning efficiency on plates; (b) inhibition of cell growth and induction of apoptosis in subconfluent/confluent cells; and (c) inhibition of transcription of two c-Myc-regulated genes (ODC and p53). Int-H1-S6A,F8A was active in the 1-10 microM range. Int-H1-S6A,F8A may represent a lead molecule for peptidomimetic compounds that have a similar three-dimensional structure but are more resistant to peptidases and, therefore, suitable for in vivo treatment of experimentally induced tumors.

Mutation frequencies at codon 248 of the p53 tumour suppressor gene are not increased in colon cancer cell lines with the RER+ phenotype.

The replication-error positive (RER+) phenotype characterizes tumour cells with microsatellite instability. This 'mutator phenotype' is thought to induce spread mutations throughout the genome, thus increasing the risk of tumour development. Here we analyse spontaneously arising mutations at the tetranucleotide CCGG ( Msp I recognition site), at positions 14 067-14 070 of the p53 gene sequence, in three colon cancer cell lines, two with microsatellite instability and one without this characteristic. This restriction site covers hot-spot codon 248, which is often mutated in colon carcinomas. Using the Msp I RFLP-PCR assay we found that the mean mutation frequency at this site was not different among the cell lines considered. Taking the substitutions separately, none of the mutations involving codon 248 arose with significantly higher frequency in each of the RER+ cell lines (HCT116 and DLD1) compared with the RER-one (SW480). Only the CG transversion at nt 14 067 (codon 247) occurred with a slightly higher, but biologically insignificant, frequency in one of the RER+ cell lines (HCT116). Our in vitro data support the previously reported lack of correlation between microsatellite instability and p53 mutations in RER+ tumour specimens.

Methods for predicting carcinogenic hazards: new opportunities coming from recent developments in molecular oncology and SAR studies.

Without epidemiological evidence, and prior to either short-term tests of genotoxicity or long-term tests of carcinogenicity in rodents, an initial level of information about the carcinogenic hazard of a chemical that perhaps has been designed on paper, but never synthesized, can be provided by structure-activity relationship (SAR) studies. Herein, we have reviewed the interesting strategies developed by human experts and/or computerized approaches for the identification of structural alerts that can denote the possible presence of a carcinogenic hazard in a novel molecule. At a higher level of information, immediately below epidemiological evidence, we have discussed carcinogenicity experiments performed in new types of genetically engineered small rodents. If a dominant oncogene is already mutated, or if an allele of a recessive oncogene is inactivated, we have a model animal with (n-1) stages in the process of carcinogenesis. Both genotoxic and receptor-mediated carcinogens can induce cancers in 20-40% of the time required for classical murine strains. We have described the first interesting results obtained using these new artificial animal models for carcinogenicity studies. We have also briefly discussed other types of engineered mice (lac operon transgenic mice) that are especially suitable for detecting mutagenic effects in a broad spectrum of organs and tissues and that can help to establish mechanistic correlations between mutations and cancer frequencies in specific target organs. Finally, we have reviewed two complementary methods that, while obviously also feasible in rodents, are especially suitable for biomonitoring studies. We have illustrated some of the advantages and drawbacks related to the detection of DNA adducts in target and surrogate tissues using the 32P-DNA postlabeling technique, and we have discussed the possibility of biomonitoring mutations in different human target organs using a molecular technique that combines the activity of restriction enzymes with polymerase chain reaction (RFLP/PCR). Prediction of carcinogenic hazard and biomonitoring are very wide-ranging areas of investigation. We have therefore selected five different subfields for which we felt that interesting innovations have been introduced in the last few years. We have made no attempt to systematically cover the entire area: such an endeavor would have produced a book instead of a review article.

Functional maturity of the coagulation system in children: an evaluation using thrombelastography.

There are quantitative deficiencies in the coagulation system for at least the first 6 mo of life. Clinical experience, however, does not indicate an increased risk of excessive bleeding during surgical procedures. Thrombelastography, a test providing a functional evaluation of coagulation, was used to assess the hemostatic system of pediatric patients under 2 yr of age. Thrombelastographic data were obtained from 237 healthy pediatric patients less than 2 yr of age undergoing elective noncardiac surgery. Five groups were distinguished: under 30 days, 1-3 mo, 3-6 mo, 6-12 mo, and 12-24 mo. Thrombelastography revealed no defects in coagulation when these groups were compared to each other or to adults, indicating a functionally intact hemostatic process even in neonates. Indeed, children less than 12 mo of age were found to initiate and develop clot faster than adults, with the coagulation process slowing to adult rates after 1 yr of age. In addition to defining functional integrity, our data represents a set of pediatric control thrombelastographic values that have not been previously reported and that may become important in understanding coagulation changes that accompany disease states and surgery in pediatric patients.

Chromium as an industrial carcinogen: Part I.

Successive cohorts by year of hire at the same chromate plant (1931-1932, 1933-1934, 1935-1937) and the combined cohort (1931-1937) of 332 employees were followed through 1993. A total of 283 deaths (85%) of the total cohort were identified. In the combined cohort (1931-1937), 66 lung cancers were found, constituting 23.3% of all deaths and 64.7% of all cancers. The lung cancer mortality rates are shown over a span of decades, from 15 years to over 55 years, with progressive rise. Observations of lung cancer identified, employees not found, and cancer risk by age at hire are cited. Lung cancer death rates increased by gradient level of exposure to insoluble (trivalent) chromium and to soluble (hexavalent) chromium, with a pattern of increase by total chromium. Age-specific death rates for lung cancer according to the same gradient exposure range for total, insoluble, and soluble chromium are presented. The potential cancer risk extends to all forms of chromium and to total chromium.

Chromium as an industrial carcinogen: Part II. Chromium in human tissues.

A continuation of the findings in a study of workers hired in 1931-1937 in a chromate plant, directed at the evaluation of the carcinogenic risk of insoluble, soluble, and total chromium. Chemical analyses of tissues of autopsies, identified by age at hire, cumulative exposure to insoluble, soluble, and total chromium and interval since last exposure are cited for three lung cancer cases. Histological identification of insoluble chromium was demonstrated. Marked deposition and retention of concentrations of chromium was noted 18.0 years since last exposure. In one case with cumulative exposure to insoluble chromium of 10.74 mg versus 0.63 mg for soluble chromium and histological demonstration of insoluble chromium, chrysotile was also identified.

A fast and simple in situ PCR method to detect human papilloma virus infection in archival paraffin-embedded tissues.

We describe a simple and fast method for the detection and localization of low copy numbers of HPV DNA in formalin-fixed and paraffin-embedded archival tissues. We have developed a protocol for direct bl situ-PCR in order to demonstrate its convenience in rapid and reproducible assessment of HPV infection in unknown biopsies. The morphological aspect of the tissues has been maintained, despite the multiple steps of fixation, permeabilization and thermal cycling, and positivity has been detected only in virus target cells.

Endogenous tumor necrosis factor enhances topoisomerase II inhibitors activity in human ovarian cancer cell lines.

In this study, we demonstrated that tumor necrosis factor (TNF), secreted endogenously by four human ovarian cancer cell lines (A2774, IGROV-1, OVCAR-8, SW626), is biologically active against L929 cells and its activity is specifically inhibited by anti-TNF antibodies. Its endogenous production is increased by treatment for 24 h with phorbol myristate acetate (PMA)/ Ionomycin (Iono). All cell lines express TNF high-affinity receptors and release only 60-kdalton soluble TNF receptor, both spontaneously and after stimulation with PMA/Iono. TNF endogenously secreted by human ovarian cancer cell lines is very efficient in potentiating the activity of DNA topoisomerase II inhibitors (doxorubicin, mitoxantrone, VP16). The activity of vinblastine and bleomycin is not potentiated and, more interestingly, cisplatin's activity is inhibited. In 24-h PMA/Iono-stimulated A2774 cells, mitoxantrone specifically generated more cleavable complexes than in unstimulated cells. This result could provide an important tool in the therapy of human ovarian cancer secreting TNF protein, previously considered as a negative prognostic factor.

A general overview of the process of carcinogenesis.

A general synthetic overview of the process of carcinogenesis is presented. The following points are discussed: the uniqueness of tumor disease with respect to other pathologies; tumors viewed as a pathology of the transduction system of signals that regulate the communal life of the cells of multicell organisms; the tumor as a genetic disease of somatic cells; carcinogenesis as a multistage event; the fundamental role of physiologic and pathologic rhythms of cell proliferation in the modulation of tumor incidence; mechanisms entailed in the maintenance of genome integrity; mechanisms involved in the protection of genome integrity from exogenous and endogenous causes of degradation of the genetic message.