The Anti-Proliferative Lichen-Compound Protolichesterinic Acid Inhibits Oxidative Phosphorylation and Is Processed via the Mercapturic Pathway in Cancer Cells.

The lichen compound protolichesterinic acid (PA) has an anti-proliferative effect against several cancer cell lines of different origin. This effect cannot be explained by the known inhibitory activity of PA against 5- and 12-lipoxygenases. The aim was therefore to search for mechanisms for the anti-proliferative activity of PA. Two cancer cell lines of different origin, both sensitive to anti-proliferative effects of PA, were selected for this study, T-47D from breast cancer and AsPC-1 from pancreatic cancer. Morphological changes were assessed by transmission electron microscopy, HPLC coupled with TOF spectrometry was used for metabolomics, mitochondrial function was measured using the Agilent Seahorse XFp Real-time ATP assay and glucose/lactate levels by radiometry. Levels of glutathione, NADP/NADPH and reactive oxygen species [ROS] were measured by luminescence. Following exposure to PA both cell lines showed structural changes in mitochondria that were in line with a measured reduction in oxidative phosphorylation and increased glycolysis. These changes were more marked in T-47D, which had poorer mitochondrial function at baseline. PA was processed and expelled from the cells via the mercapturic pathway, which consumes glutathione. Nevertheless, glutathione levels were increased after 24 hours of exposure to PA, implying enhanced synthesis. Redox balance was not much affected and ROS levels were not increased. We conclude that PA is metabolically processed and expelled from cells, leading indirectly to increased glutathione levels with minimal effects on redox balance. The most marked effect was on mitochondrial structure and metabolic function implying that effects of PA may depend on mitochondrial fitness.

Proton pump inhibitor use and risk of breast cancer, prostate cancer, and malignant melanoma: An Icelandic population-based case-control study.

PURPOSE: Increased expression of Vacuolar-type H+ ATPases (V-ATPases), in the plasma membrane of cancer cells has been suggested to contribute to the development of aggressive cancer phenotypes by promoting acidic tumor microenvironments. Accumulating data suggest that proton pump inhibitors (PPIs) may elicit a chemopreventive effect via V-ATPase inhibition in some cancers, but evidence is still limited. Therefore, we aimed to explore a potential preventive role of PPIs in this study. METHODS: In this population-based case-control study, we identified incident cases of breast cancer (n = 1739), prostate cancer (n = 1897), and malignant melanoma (n = 385) in Iceland between 2005 and 2014 from the Icelandic Cancer Registry. We assessed varying levels of PPI use through record linkages to the Icelandic Medicines Registry. For each case, we selected up to 10 age-matched, sex-matched, and calendar-matched population controls using risk-set sampling. Using conditional logistic regression, we calculated odds ratios (ORs) and 95% confidence intervals (CIs) controlling for NSAID use. RESULTS: Adjusted ORs associated with ever use of PPIs were 1.03 (95% CI: 0.92-1.16) for breast cancer, 1.12 (95% CI: 1.00-1.25) for prostate cancer, and 0.84 (95% CI: 0.69-1.12) for malignant melanoma. Analyses of high use of PPIs (≥1000 DDDs) yielded ORs of 0.97 (95% CI: 0.78-1.19), 1.20 (0.99-1.47), and 0.59 (0.40-1.13) for breast cancer, prostate cancer, and malignant melanoma, respectively. Analyses of cumulative exposure to PPIs did not support a dose-response relationship for any of the three cancer types. CONCLUSIONS: Our findings do not support a chemopreventive effect of PPI use on breast cancer, prostate cancer, or malignant melanoma.

Metabolic Profiling as a Screening Tool for Cytotoxic Compounds: Identification of 3-Alkyl Pyridine Alkaloids from Sponges Collected at a Shallow Water Hydrothermal Vent Site North of Iceland.

Twenty-eight sponge specimens were collected at a shallow water hydrothermal vent site north of Iceland. Extracts were prepared and tested in vitro for cytotoxic activity, and eight of them were shown to be cytotoxic. A mass spectrometry (MS)-based metabolomics approach was used to determine the chemical composition of the extracts. This analysis highlighted clear differences in the metabolomes of three sponge specimens, and all of them were identified as Haliclona (Rhizoniera) rosea (Bowerbank, 1866). Therefore, these specimens were selected for further investigation. Haliclona rosea metabolomes contained a class of potential key compounds, the 3-alkyl pyridine alkaloids (3-APA) responsible for the cytotoxic activity of the fractions. Several 3-APA compounds were tentatively identified including haliclamines, cyclostellettamines, viscosalines and viscosamines. Among these compounds, cyclostellettamine P was tentatively identified for the first time by using ion mobility MS in time-aligned parallel (TAP) fragmentation mode. In this work, we show the potential of applying metabolomics strategies and in particular the utility of coupling ion mobility with MS for the molecular characterization of sponge specimens.

Synergistic cytotoxic effect of the microtubule inhibitor marchantin A from Marchantia polymorpha and the Aurora kinase inhibitor MLN8237 on breast cancer cells in vitro.

Macrocyclic bisbibenzyls are a class of characteristic compounds, exclusively produced by liverworts. They are attracting increasing attention due to their wide range of biological activities, including antibacterial, antifungal, and antioxidative properties as well as cytotoxicity. Marchantin A is a cyclic bisbibenzyl that has previously been isolated from Marchantia polymorpha and other liverwort species and has been shown to exert cytotoxic effects. In the present study we found that the Icelandic M. polymorpha species produces marchantin A and through an in vitro cell growth inhibition assay, marchantin A was shown to induce a reduction in cell viability of breast cancer cell lines A256 (IC50 = 5.5 µM), MCF7 (IC50 = 11.5 µM), and T47D (IC50 = 15.3 µM). The effect was considerably increased in all cell lines in a synergistic manner when the Aurora-A kinase inhibitor MLN8237 was added simultaneously. Fluorescence microscopy confirmed the antimicrotubular effect of marchantin A, and cell cycle analysis indicated enhanced cell division failure when combining this mitotic-spindle inhibitor with the checkpoint modulator.

Cytogenetic polyclonality of breast carcinomas: association with clinico-pathological characteristics and outcome.

Routinely used prognostic factors fail to predict clinical outcome in a significant proportion of breast cancer patients, implying that they can not detect some important biological characteristics. Chromosomal changes have been described in breast carcinomas for many years but their significance is not clear. We compared chromosomal changes with clinico-pathological characteristics and clinical outcome in 203 breast cancer patients with a follow-up of 9-18 years. Combining data from classical cytogenetics and flow cytometry revealed chromosomal abnormalities in 142 cases (70%). Of these, 51 (35.9%) contained two or more cytogenetically abnormal clones. Polyclonality was significantly associated with poor breast-cancer-specific survival (P = 0.03) within 5 years, independent of tumor size, lymph node metastases, and hormone receptors. Specific changes were similar to those previously described, but a new finding was a significant association between del 3p12p21 and poor survival. Polyclonality was significantly associated with TP53-mutations but not with a germline BRCA2 mutation. Less than one third of the polyclonal samples were identified by flow cytometry alone. Cytogenetic changes were detected in 17 out of 114 samples from non-tumorous tissue (15%), two of them identical with a clone in the corresponding tumor. Several samples contained clearly unrelated clones within the tumor and outside, implying either multifocal origin or early divergence. In conclusion, the common deletion on Chromosome 3p12p21 was associated with poor clinical outcome. Chromosomal polyclonality is common in breast carcinomas and predicts poor survival. Polyclonality was poorly detected by one-sample flow cytometry. Multiple sampling might improve the detection rate.

Oral Lichen Planus and Mutated TP53-A Road to Cancer?

The malignant potential of oral lichen planus (OLP) has been discussed and disputed for decades. The lesions are often characterized by strong expression of the TP53 protein in the basal layer of the mucosa. In 2002, we reported the presence of TP53 mutations in nine out of 27 OLP lesions tested. At follow-up in 2009, one case of oral squamous cell cancer (OSCC) had occurred in a different site six years later. In contrast, in another case, TP53 mutation persisted for years without malignant transformation. In a longitudinal study of eight selected patients with OSCC or different pre-malignant lesions, it was concluded that TP53 mutations could occur early or late in the development of OSCC. A follow-up in the present, almost 20 years later, revealed that one further case of OSCC had occurred in a TP53-mutated case of OLP, 21 years after the first sample was taken, again in a different site. With this second case, this small study now points towards a risk of developing OSCC in TP53-mutated OLP lesions. A review of recent literature indicates a growing consensus that OLP should be regarded as a potentially pre-malignant lesion. Several protein markers have been studied, but none proved useful for prediction of malignant progression. The great majority of published studies are retrospective, and it has been suggested that multi-centre prospective studies will be needed to reach a definitive answer on the malignant potential of OLP, and particularly, to identify contributing factors. Screening for TP53 mutations could help to identify the subgroup of OLP patients that is truly at risk of developing oral cancer.

Cytotoxic effects of halogenated tin phosphinoyldithioformate complexes against several cancer cell lines.

Organotin complexes are studied as promising alternatives to the anticancer drug cisplatin. We report two monoorganotin(IV) complexes based on a dibenzyl phosphinoyldithioformate (H-DBPTF) ligand, containing either bromide (Sn-DBPTF-1) or chloride (Sn-DBPTF-2) anions. The complexes were characterized by standard analytical techniques and the structural details of these complexes were elucidated by single crystal X-ray diffraction. Sn-DBPTF-1 was cytotoxic at IC50 <10 µg mL-1 against cancer cell lines A549 (lung cancer), Aspc-1 (pancreatic cancer), OVCAR-3 (ovarian cancer), T-47D (breast cancer) and HCT116 (colon cancer), and breast epithelial stem cell line D492. The non-tumorigenic breast epithelial cell line MCF-10 was less sensitive at IC50 = 22 µg mL-1. Sn-DBPTF-2 had limited cytotoxic effect at IC50 13-37 µg mL-1. Sn-DBPTF-1 induced apoptosis and double-strand DNA breaks. Cell cycle arrest in G2 occurred in HCT116 and accumulation in G1 in Aspc-1. The results indicate that the basic effect of Sn-DBPTF-1 is to induce DNA damage, leading to apoptosis and cell cycle arrest depending on the cell line.

Familial monoclonal gammopathy: hyper-responsive B cells in unaffected family members.

BACKGROUND: In Iceland, eight families have been identified with multiple cases of monoclonal gammopathies (MG) and other lymphoproliferative diseases. In one of these families with several cases of monoclonal gammopathy of undetermined significance (MGUS) and Waldenströms macroglobulinemia, in vitro stimulation with poke-weed mitogen revealed hyper-responsive B cells showing increased immunoglobulin production in one-third of disease-free family members. DESIGN AND METHODS: In this study, the families were further traced and the list of names produced was compared with The Icelandic Cancer Registry (ICR) to find all recent cases of lymphoproliferative diseases. First-degree relatives and descendants older than 20yrs of age (n=350) were selected for screening for paraprotein. Selected family members were tested for B-cell hyper-responsiveness and the lymphocyte phenotype was analysed by flow cytometry. RESULTS: Comparison of the total list of 4370 family members with the ICR revealed 22 new cases and screening for serum paraprotein identified nine new cases of MG, eight being first-degree relatives of known probands. Sixty cases of lymphoproliferative diseases are currently known within the eight families, five of them containing both IgG/A and IgM disorders. Twelve hyper-responders (HR) were identified in four families, eight from one family, of whom four were known already. Stimulated B cells from HR had a significantly higher proportion of CD27(+) memory/plasma cells than controls. CONCLUSION: Identification of new affected family members by screening confirms a hereditary predisposition to B-cell proliferative diseases. Contrary to most studies, IgG/A and IgM disorders occurred together in five families. In four families, enhanced B-cell responsiveness was found in healthy subjects clustered around cases.

Centriole movements in mammalian epithelial cells during cytokinesis.

BACKGROUND: In cytokinesis, when the cleavage furrow has been formed, the two centrioles in each daughter cell separate. It has been suggested that the centrioles facilitate and regulate cytokinesis to some extent. It has been postulated that termination of cytokinesis (abscission) depends on the migration of a centriole to the intercellular bridge and then back to the cell center. To investigate the involvement of centrioles in cytokinesis, we monitored the movements of centrioles in three mammalian epithelial cell lines, HeLa, MCF 10A, and the p53-deficient mouse mammary tumor cell line KP-7.7, by time-lapse imaging. Centrin1-EGFP and alpha-Tubulin-mCherry were co-expressed in the cells to visualize respectively the centrioles and microtubules. RESULTS: Here we report that separated centrioles that migrate from the cell pole are very mobile during cytokinesis and their movements can be characterized as 1) along the nuclear envelope, 2) irregular, and 3) along microtubules forming the spindle axis. Centriole movement towards the intercellular bridge was only seen occasionally and was highly cell-line dependent. CONCLUSIONS: These findings show that centrioles are highly mobile during cytokinesis and suggest that the repositioning of a centriole to the intercellular bridge is not essential for controlling abscission. We suggest that centriole movements are microtubule dependent and that abscission is more dependent on other mechanisms than positioning of centrioles.

Cellular mechanisms of the anticancer effects of the lichen compound usnic acid.

The lichen compound usnic acid is used for its antimicrobial activities in cosmetic products and is also a component of slimming agents. Its effect against cancer cells was first noted over 30 years ago. In this study possible mechanisms of this effect were investigated using two human cell lines, the breast cancer cell line T-47D and the pancreatic cancer cell line Capan-2. Pure (+)-usnic acid from CLADONIA ARBUSCULA and (-)-usnic acid from ALECTORIA OCHROLEUCA were shown to be equally effective inhibitors of DNA synthesis, with IC (50) 4.2 microg/mL and 4.0 microg/mL for (+) and (-)-usnic acid against T-47D, and 5.3 microg/mL and 5.0 microg/mL against Capan-2, respectively. Flow cytometric analysis confirmed the inhibited entry into the S-phase and showed reduction in cell size. Classical apoptosis, as assessed by TUNEL staining, was not observed. Necrosis, measured by LDH release, was seen only in Capan-2 after exposure for 48 hours. Staining with the mitochondrial dye JC-1 demonstrated dose-dependent loss of mitochondrial membrane potential following treatment with usnic acid in both cell lines. In conclusion, usnic acid had a marked inhibitory effect on growth and proliferation of two different human cancer cell lines and led to loss of mitochondrial membrane potential. Cell survival was little affected; late necrosis was seen in one of the cell lines. No difference was noted between the two enantiomers.

Use of proton pump inhibitors and mortality among Icelandic patients with prostate cancer.

Proton pump inhibitors (PPIs) are commonly used drugs among cancer patients. Due to conflicting reports on their safety, we aimed to determine whether PPI use is associated with mortality among prostate cancer patients. In this population-based cohort study, we identified incident diagnoses of prostate cancer between 2007 and 2012 (n = 1058). Follow-up was from 12 months after diagnosis until death, emigration or end the of study. Post-diagnosis use was defined as ≥2 filled prescriptions following diagnosis. We used time-dependent Cox proportional hazard regression models to compute hazard ratios (HRs) and 95% confidence intervals (CIs) for prostate cancer-specific and all-cause mortality associated with post-diagnosis use of PPIs. We identified 347 (32.8%) post-diagnosis PPI users and 711 (67.2%) non-users after diagnosis. Of the 347 patients using PPIs after diagnosis, 59 (17.0%) died due to any cause and 22 (6.3%) due to prostate cancer, compared with 144 (20.3%) and 76 (10.7%) among non-users after diagnosis, respectively. Post-diagnosis PPI use was not associated with prostate cancer-specific mortality (HR 0.88; 95% CI: 0.52-1.48) or all-cause mortality (HR 1.02; 95% CI: 0.73-1.43). Contrary to a previous report, this study did not find evidence of an association between post-diagnosis PPI use and mortality among prostate cancer patients.

Endogenous steroid hormone levels in early pregnancy and risk of testicular cancer in the offspring: a nested case-referent study.

According to the leading hypothesis on testicular cancer (TC) etiology exposure to a specific pattern of steroid hormones in utero, in particular, to high levels of estrogens and low levels of androgens is the major determinant of TC risk in the offspring. We performed a case-referent study nested within Finnish, Swedish and Icelandic maternity cohorts exploiting early pregnancy serum samples to evaluate the role of maternal endogenous steroid hormones with regard to the risk of TC. TC cases and referents were aged between 0 and 25 years. For each case-index mother pair, three or four matched referent-referent mother pairs were identified using national population registries. First trimester or early second trimester sera were retrieved from the index mothers of 73 TC cases and 286 matched referent mothers, and were tested for dehydroepiandrosterone sulfate (DHEAS), androstenedione, testosterone, estradiol, estrone, and sex hormone binding globulin (SHBG). Offspring of mothers with high DHEAS levels had a significantly decreased risk of TC (OR for highest vs. lowest DHEAS quartile, 0.18 (95% CI 0.06-0.58). In contrast, offspring of mothers with high androstenedione levels had an increased risk of TC (OR 4.1; 95% CI 1.2-12.0). High maternal total estradiol level also tended to be associated with an increased risk of TC in the offspring (OR 32; 95% CI 0.98-1,090). We report the first direct evidence that interplay of maternal steroid hormones in the early pregnancy is important in the etiology of TC in the offspring.

Familial predisposition to monoclonal gammopathy of unknown significance, Waldenström's macroglobulinemia, and multiple myeloma.

The medical literature contains reports of around 130 families with two or more cases of MM, MGUS, or WM. An Icelandic family with multiple cases of MGUS, WM, and lymphoma was first described in 1978. In vitro testing of peripheral blood lymphocytes revealed increased production of immunoglobulins in response to poke-weed mitogen in 10 out of 35 family members, referred to as hyperresponders (HR). Enhanced B-cell survival after stimulation was associated with prolonged expression of Bcl-2. A population-based cancer registry study of 218 MM patients identified 7 additional families. Nine new cases of monoclonal gammopathy were detected by the screening of 350 family members. Further testing confirmed previously identified HR in the originally described family as well as detecting new cases. Only two HR were found in the recently identified families. The long-term aim is to identify the genetic background(s) and biology predisposing to the emergence of a persistent clone of immunoglobulin-producing cells.

MITF has a central role in regulating starvation-induced autophagy in melanoma.

The MITF transcription factor is a master regulator of melanocyte development and a critical factor in melanomagenesis. The related transcription factors TFEB and TFE3 regulate lysosomal activity and autophagy processes known to be important in melanoma. Here we show that MITF binds the CLEAR-box element in the promoters of lysosomal and autophagosomal genes in melanocytes and melanoma cells. The crystal structure of MITF bound to the CLEAR-box reveals how the palindromic nature of this motif induces symmetric MITF homodimer binding. In metastatic melanoma tumors and cell lines, MITF positively correlates with the expression of lysosomal and autophagosomal genes, which, interestingly, are different from the lysosomal and autophagosomal genes correlated with TFEB and TFE3. Depletion of MITF in melanoma cells and melanocytes attenuates the response to starvation-induced autophagy, whereas the overexpression of MITF in melanoma cells increases the number of autophagosomes but is not sufficient to induce autophagic flux. Our results suggest that MITF and the related factors TFEB and TFE3 have separate roles in regulating a starvation-induced autophagy response in melanoma. Understanding the normal and pathophysiological roles of MITF and related transcription factors may provide important clinical insights into melanoma therapy.

Maternal Epstein-Barr virus and cytomegalovirus infections and risk of testicular cancer in the offspring: a nested case-control study.

During recent decades the incidence of testicular cancer (TC) has increased rapidly around the world. Associated exogenous etiological factors might therefore be identifiable. We performed a case-control study nested within Finnish, Swedish and Icelandic maternity cohorts exploiting early pregnancy serum samples to evaluate the role of congenital or neonatal infections with Epstein-Barr virus (EBV) and cytomegalovirus (CMV) as risk factors of TC in the offspring. For each case-index mother pair, three or four matched control-control mother pairs were identified using national population registries. First trimester sera were retrieved from the index mothers of 66 TC cases and 258 matched control mothers, and were tested for antibodies to EBV and CMV. High level of maternal EBV IgG antibodies was associated with significantly increased risk of TC in the offspring (odds ratio (OR), 2.50; 95% confidence interval (CI), 1.15, 5.40), especially with risk of non-seminoma TC (OR, 2.73; 950% CI, 1.25, 5.99) and non-seminoma TC diagnosed under 8 years of age (OR, 2.72; 95% CI, 1.05, 7.04). In contrast, offspring of CMV IgG-seropositive mothers had a decreased risk of TC diagnosed under 8 years of age (OR, 0.35; 95% CI, 0.14, 0.89). Our results suggest that EBV and CMV infections may be associated with TC.

Altered plasmalogen content and fatty acid saturation following epithelial to mesenchymal transition in breast epithelial cell lines.

Epithelial to mesenchymal transition (EMT) is a developmental event characterized by phenotypic switching from a polarized epithelial phenotype to an unpolarized mesenchymal phenotype. Changes to plasma membrane function accompany EMT yet the differences in lipid composition of cells that have undergone EMT are relatively unexplored. To address this the lipidome of two cell models of EMT in breast epithelial tissue, D492 and HMLE, were analyzed by untargeted LC-MS. Detected masses were identified and their abundance was compared through multivariate statistical analysis. Considerable concordance was observed in eight lipid components between epithelial and mesenchymal cells in both cell models. Specifically, an increase in phosphatidylcholine and triacylglycerol were found to accompany EMT while phosphatidylcholine- and phosphatidylethanolamine plasmalogens, as well as diacylglycerols decreased. The most abundant fatty acid lengths were C16 and C18 but mesenchymal cells had on average shorter and more unsaturated fatty acids. The results are consistent with enhanced cell mobility post EMT and reflect a consequence of oxidative stress pre- and post EMT in breast epithelial tissue.

Proton-pump inhibitors among adults: a nationwide drug-utilization study.

BACKGROUND: The use of proton-pump inhibitors (PPIs) has grown worldwide, and there are concerns about increased unsubstantiated long-term use. The aim of the study was to describe the real-world use of PPIs over the past decade in an entire national population. METHODS: This was a nationwide population-based drug-utilization study. Patterns of outpatient PPI use among adults in Iceland between 2003 and 2015 were investigated, including annual incidence and prevalence, duration of use, and dose of tablet used (lower versus higher), as well as the proportion of PPI use attributable to gastroprotection. RESULTS: We observed 1,372,790 prescription fills over the entire study period, of which 95% were for higher-dose PPIs. Annual incidence remained stable across time (3.3-4.1 per 100 persons per year), while the annual prevalence increased from 8.5 per 100 persons to 15.5 per 100 persons. Prevalence increased with patient age and was higher among women than men. Duration of treatment increased with patients' age (36% of users over 80 years remained on treatment after 1 year compared with 13% of users aged 19-39 years), and was longer among those initiating on a higher dose compared with a lower dose. The proportion of PPI users concurrently using nonsteroidal anti-inflammatory drugs decreased over the study period, while the proportion concurrently using acetylsalicylic acid, oral anticoagulants, or platelet inhibitors increased. CONCLUSIONS: In this nationwide study, a considerable increase in overall outpatient use of PPIs over a 13-year period was observed, particularly among older adults. Patients were increasingly treated for longer durations than recommended by clinical guidelines and mainly with higher doses.

Monoclonal gammopathy: natural history studied with a retrospective approach.

The aim of this study was to examine the natural history of monoclonal gammopathy using a retrospective approach and a long observation period. Protein electrophoresis (PE) and immunofixation (IF) was performed on frozen prediagnosis serum samples from 65 multiple myeloma (MM) and 10 Waldenström's macroglobulinemia (WM) cases. Paraprotein was found in 28% and 46% of the samples from cases using PE and IF respectively. The type of paraprotein was IgA in 33.4% of cases, IgG in 57%, and IgM in 8.5%. Excluding light chain or non-secretory disease, 72 % of MM cases had a prodromal MGUS phase within 10 years of diagnosis MM and WM were preceded by MGUS in at least half of the cases, confirming the premalignant nature of this condition.

Effect of hypoxia and TP53 mutation status and cytogenetics of normal and malignant mammary epithelium.

It has been proposed that hypoxia favors the growth of tumor cells over normal cells, particularly tumor cells carrying TP53 mutations. Cytogenetic studies of breast cancer have shown that highly complex karyotypes seen in direct harvest preparations are rarely detected after short-term culture. In this study, 34 paired samples of breast carcinomas and grossly nontumorous tissue from the same breast were cultured at 20 and 5% (12 samples) or 20 and 0% oxygen (22 samples). Both carcinoma samples and nontumorous tissue survived at 0% oxygen. Recovery for 24 hours at 20% produced good yields for cytogenetic analysis. Lower oxygen levels did not specifically stimulate growth of tumor cells. Samples with TP53 mutations showed a consistently increased growth under anaerobic hypoxic conditions. Culture at 5% oxygen did not generally reveal more karyotypic abnormalities than found at 20%. In the samples cultured at 0 and 20%, karyotypic abnormalities were detected only in anaerobic hypoxic culture in two cases. Of the only four samples where more complex karyotypes were detected in the low-oxygen culture, two were TP53 mutated. Hypoxic treatment followed by recovery at 20% oxygen may thus increase the yield of complex karyotypes from a subset of breast carcinomas, particularly those with mutated TP53.