CDK12 promotes tumorigenesis but induces vulnerability to therapies inhibiting folate one-carbon metabolism in breast cancer.

Cyclin-dependent kinase 12 (CDK12) overexpression is implicated in breast cancer, but whether it has a primary or only a cooperative tumorigenic role is unclear. Here, we show that transgenic CDK12 overexpression in the mouse mammary gland per se is sufficient to drive the emergence of multiple and multifocal tumors, while, in cooperation with known oncogenes, it promotes earlier tumor onset and metastasis. Integrative transcriptomic, metabolomic and functional data reveal that hyperactivation of the serine-glycine-one-carbon network is a metabolic hallmark inherent to CDK12-induced tumorigenesis. Consistently, in retrospective patient cohort studies and in patient-derived xenografts, CDK12-overexpressing breast tumors show positive response to methotrexate-based chemotherapy targeting CDK12-induced metabolic alterations, while being intrinsically refractory to other types of chemotherapy. In a retrospective analysis of hormone receptor-negative and lymph node-positive breast cancer patients randomized in an adjuvant phase III trial to 1-year low-dose metronomic methotrexate-based chemotherapy or no maintenance chemotherapy, a high CDK12 status predicts a dramatic reduction in distant metastasis rate in the chemotherapy-treated vs. not-treated arm. Thus, by coupling tumor progression with metabolic reprogramming, CDK12 creates an actionable vulnerability for breast cancer therapy and might represent a suitable companion biomarker for targeted antimetabolite therapies in human breast cancers.

Pathogenesis of chronic cluster headache and bouts: role of tryptamine, arginine metabolism and α1-agonists.

The aim of this study was to explore the possible role of tryptamine in the pathogenesis of chronic cluster headache along with that of adrenaline and noradrenaline (α-agonists) together with arginine metabolism in the origin of cluster bouts. Plasma levels of tyramine, tryptamine, serotonin, 5-hydroxyindolacetic acid, noradrenalin, adrenalin and the markers of arginine metabolism such as arginine, homoarginine, citrulline, ADMA and NMMA, were measured in 23 chronic cluster headache patients (10 chronic cluster ab initio and 13 transformed from episodic cluster) and 28 control subjects. The plasma levels of tyramine, tryptamine, noradrenalin and adrenalin were found several times higher in chronic cluster headache patients compared to controls, whereas the plasma levels of arginine, homoarginine and citrulline were significantly lower. No differences were found in the plasma levels of serotonin, 5-hydroxyindolacetic, ADMA and NMMA between chronic cluster headache patients and control subjects. These results provide support for a role of tryptamine in the pathogenesis of chronic cluster headache and, in particular, in the duration of the cluster bouts. In addition, the low levels of the nitric oxide substrates together with the high levels of noradrenalin and adrenalin suggest an activation of endothelial TAAR1 receptors followed by the release of nitric oxide in the circulation that may constitute the final step of the physiopathology of cluster crisis.

Migraine with aura white matter lesions: preliminary data on clinical aspects.

A few clinic-based magnetic resonance imaging studies report an increased risk of signal abnormalities in migraineurs brain's white matter, especially in migraine with aura subjects. A vascular genesis has been hypnotized and migraine with aura was considered an independent risk factor for stroke. Available data of magnetic resonance imaging alterations are often nonspecific and sometimes controversial. The aim of our study is to investigate migraine with aura patients with standardized brain magnetic resonance imaging to detect and to quantify the presence of white matter lesions and to analyze their relation with clinical data. We report preliminary data about first 90 subjects. We did not recognize any clinical aspect in close relationship with these alterations. The only clinical feature that seems to play a role in the presence of alterations is the age, and only in migraineurs women.

Difficulties in work activities and the pervasive effect over disability in patients with episodic and chronic migraine.

Migraine is associated with reduced productivity in work-related activities. The degree to which problems with work are, in turn, associated to the level of migraine-related disability as well as to headache frequency has been poorly explored. The aim of the study was to assess if migraine patients with different degrees of work difficulties showed a different level of migraine-related disability. A consecutive sample of patients with episodic migraine (EM) or with chronic migraine (CM) with medication overuse (MO) attending the Headache Centre of the Neurological Institute C. Besta of Milan was studied. All patients completed the MIDAS and the WHODAS 2.0 questionnaires. The total scores of both questionnaires, frequency of headaches, average pain intensity, and the scores of each subscale of the WHODAS 2.0 were calculated separately for EM and CM patients. The score of WHODAS 2.0 "Work difficulties" subscale was used to divide the studied patients into two groups, i.e. those above and those below the median "Work difficulties" subscale score. Independent sample t test was used to compare these two groups as far as all the other studied variables. A total of 296 patients (102 with EM and 194 with CM-MO) were enrolled. Patients with higher work difficulties score also displayed higher scores in the other WHODAS 2.0 subscales; for those with CM-MO, the differences were significant. The results of this study indicate that having more and more severe workplace problems is associated to a higher disability level in migraineurs. Further studies are needed to better understand workplace disability in different migraine forms, particularly in a qualitative way.

Electromyography data in chronic migraine patients by using neurostimulation with the Cefaly® device.

The objective of this observational study is to report clinical and instrumental results obtained in 23 chronic migraine sufferers treated with transcutaneous neurostimulation with the Cefaly(®) device. The electrom yography (EMG) parameters of the patients monitored before and during neurostimulation with the Cefaly(®) device showed a significant increase in the EMG amplitude and frequency values in the frontalis, anterior temporalis, auricularis posterior and middle trapezius muscles. The Cefaly(®) device could act on the inhibitory circuit in the spinal cord thus causing a neuromuscular facilitation and may help reduce contraction of frontalis muscles.

Study of parafunctions in patients with chronic migraine.

The purpose of this paper is to present the results of a questionnaire investigating parafunctions (particularly clenching and grinding) in patients with chronic migraine presenting sign of temporomandibular disorder. The questionnaire was elaborated by the Dental Clinic of the University of Milano and completed by 125 patients experiencing chronic migraine and attending the Neurological Institute Carlo Besta for an inpatient withdrawal protocol to treat medication overuse. Our results showed high percentages of parafunctions, which were present in 80 % of patients. We note that patient information on possible behaviours and coexisting conditions which may be involved in the mechanisms of chronic headaches, as well as education about these factors, are crucial aspects in the management of chronic headache patients. We suggest that patients suffering from chronic migraine with medication overuse headache should be evaluated in relation to the possible presence of parafunctions, and as far as the need for interocclusal devices, in order to limit the role of temporomandibular dysfunctions as trigger factors or coexisting conditions favouring the development/maintaining of headache chronification.

Occipital neurostimulation in primary headaches: update.

A subset of headache patients are chronic and results refractory to standard medical treatments, they are unsatisfied or unable to tolerate the side effects of medications. In the lack of more effective prophylactic treatment, there is need of alternative approach. Migraine is conceptualized as a chronic and potentially progressive disorder. It is conceivable that more aggressive therapeutic efforts could be warranted in drug-refractory chronic migraine. In this prospective, the new, device-based therapies that allow to affect brain function in less invasive ways may represent a therapeutic opportunity. Peripheral occipital neurostimulation resulted in several trials and case reports to be beneficial in a large variety of headache and craniofacial pain disorders, with chronic primary headache the most studied. We comment on our experience in the application of ONS in drug-refractory chronic cluster headache and chronic migraine patients.

A 14-month study of change in disability and mood state in patients with chronic migraine associated to medication overuse.

This paper aims to evaluate changes in disease severity, disability and mood state in patients with chronic migraine associated to medication overuse (CM-MO). MIDAS was used for assessing disease activity, WHO-DAS-2 for disability, DBI-2 for mood state. ANOVA was used to test change over time; t-test to assess follow-up differences in WHO-DAS-2 and BDI-2 between patients with MIDAS ≤20 and ≥21. Change in MIDAS, WHO-DAS-2 and BDI-2 scores were computed: Pearson's index was used to assess correlation between them; linear regression to assess change in WHO-DAS-2, using MIDAS and BDI-2 change as predictors. Mean MIDAS decreased significantly (from 101.9 to 52.0). In 26.1 % of the sample, MIDAS fell below 21 at follow-up: these patients had lower WHO-DAS-2 score. WHO-DAS-2 change was little correlated to MIDAS change and strongly correlated to changes in BDI-2 scores. 57.1 % of WHO-DAS-2 change variance is explained by change in BDI-2 and MIDAS scores. There was a clear clinical improvement 14 months after detoxification, and a modest reduction in disability which is explained by reduced disease activity and improved mood state. An appropriate treatment of CM-MO, based on detoxification and prophylaxis, is likely to reduce disease burden: recognition and treatment of mood problems may be a key factor to reduce disability.

Positive outcome of occlusal freeway space reestablishment in patients with medication overuse due to chronic migraine.

Considering the great chapter of migraines, it is important to note the signs and symptoms caused by an alteration of the relationship of the facial musculature and the occlusal freeway space (FWS) that is the distance from maximal intercuspation to the habitual rest position (measurable in 1.4-2.5 mm). To any mandible position changing (detected by periodontal, muscle and joint proprioceptors), there is an influence on the neuromuscular system and then an alteration of the FWS. A group of 60 patients with chronic migraine (CM) underwent a withdrawal of overused medication and were subjected to electromyographic and kinesiographic evaluation. All those who presented an alteration of the FWS at rest position have been subjected to treatment with orthosis device for about 12 months. The aim of our work is to obtain and define a correct mandibular position, physiological, in agreement with the neuromuscular structures of the patient. Clinical results obtained on this pool of patients, in terms of reduction of the VAS, let us understand the importance of the inviolability of the FWS and to eliminate any type of mandibular deviation with respect to the closure trajectory induced by TENS in patients suffering from CM.

Circulating microRNAs: next-generation biomarkers for early lung cancer detection.

Early diagnosis of lung cancer by low-dose computed tomography is an effective strategy to reduce cancer mortality in high-risk individuals. However, recruitment of at-risk individuals with asymptomatic lung cancer still remains challenging. We developed a minimal invasive serum test, based on the detection of circulating microRNAs, which can identify at-risk individuals with asymptomatic early stage non-small cell lung carcinomas with 80% accuracy.

Deliberative ethics in a biomedical institution: an example of integration between science and ethics.

The deliberative ethics guidelines elaborated and implemented by members of the IFOM-IEO Campus (Firc Institute of Molecular Oncology (IFOM) and the European Institute of Oncology (IEO)). These should serve the dual purpose of establishing a minimal set of standard rules for bioethical debate and any ensuing decision-making process, especially for the perspective of providing real instruments to foster public engagement and public awareness on the ethical issues involved in biomedical research. It is shown that these guidelines instantiate the scheme of one of the correct ways of debating formalised by the western thought.

Prep1 (pKnox1)-deficiency leads to spontaneous tumor development in mice and accelerates EmuMyc lymphomagenesis: a tumor suppressor role for Prep1.

The Prep1 homeodomain transcription factor is essential for embryonic development. 25% of hypomorphic Prep1(i/i) embryos, expressing the gene at 2% of the normal levels, survive pregnancy and live a normal-length life. Later in life, however, these mice develop spontaneous pre-tumoral lesions or solid tumors (lymphomas and carcinomas). In addition, transplantation of E14.5 fetal liver (FL) Prep1(i/i) cells into lethally irradiated mice induces lymphomas. In agreement with the above data, haploinsufficiency of a different Prep1-deficient (null) allele accelerates EmuMyc lymphoma growth. Therefore Prep1 has a tumor suppressor function in mice. Immunohistochemistry on tissue micrroarrays (TMA) generated from three distinct human cohorts comprising a total of some 1000 human tumors revealed that 70% of the tumors express no or extremely low levels of Prep1, unlike normal tissues. Our data in mice are thus potentially relevant to human cancer.

Understanding biological dynamics: following cells and molecules to track functions and mechanisms.

The dissection of the molecular circuitries at the base of cell life and the identification of their abnormal transformation during carcinogenesis rely on the characterization of biological phenotypes generated by targeted overexpression or deletion of gene products through genetic manipulation. Fluorescence microscopy provides a wide variety of tools to monitor cell life with minimal perturbations. The observation of living cells requires the selection of a correct balance between temporal, spatial and "statistical" resolution according to the process to be analyzed. In the following paper ad hoc developed optical tools for dynamical tracking from cellular to molecular resolution will be presented. Particular emphasis will be devoted to discuss how to exploit light-matter interaction to selectively target specific molecular species, understanding the relationships between their intracellular compartmentalization and function.

Alterations of ubiquitin ligases in human cancer and their association with the natural history of the tumor.

Protein ubiquitination is critical for many cellular processes, through its ability to regulate protein degradation and various signaling mechanisms. In the ubiquitin (Ub) system, substrate specificity is achieved through the E3 family of Ub ligases. Because alterations of the ubiquitination machinery have been reported in human cancers, the selective interference with Ub ligases might represent a powerful therapeutic tool. Here, we report the first wide survey of misregulation of Ub ligases in cancer. We analysed 82 Ub ligases in nine types of cancer by in situ hybridization on tissue microarrays. We found 27 instances in which an Ub ligase was altered in a given type of tumor, when compared with normal tissues: 21 cases of overexpression and 6 cases of underexpression. We further analysed selected Ub ligases in large cohorts of breast and non-small-cell lung carcinomas. In five, of six, of these extended analyses (HUWE1, CCNB1IP1, SIAH1 and SIAH2 in breast cancer and CCNB1IP1 in lung cancer), we found that the levels of Ub ligases correlated significantly with relevant prognostic factors, and with clinical outcome. Our findings show that the alteration of Ub ligases is a frequent event in cancer and identify candidate targets for molecular therapies.

Breast cancer metastases are molecularly distinct from their primary tumors.

Metastases have been widely thought to arise from rare, selected, mutation-bearing cells in the primary tumor. Recently, however, it has been proposed that breast tumors are imprinted ab initio with metastatic ability. Thus, there is a debate over whether 'phenotypic' disease progression is really associated with 'molecular' progression. We profiled 26 matched primary breast tumors and lymph node metastases and identified 270 probesets that could discriminate between the two categories. We then used an independent cohort of breast tumors (81 samples) and unmatched distant metastases (32 samples) to validate and refine this list down to a 126-probeset list. A representative subset of these genes was subjected to analysis by in situ hybridization, on a third independent cohort (57 primary breast tumors and matched lymph node metastases). This not only confirmed the expression profile data, but also allowed us to establish the cellular origin of the signals. One-third of the analysed representative genes (4 of 11) were expressed by the epithelial component. The four epithelial genes alone were able to discriminate primary breast tumors from their metastases. Finally, engineered alterations in the expression of two of the epithelial genes (SERPINB5 and LTF) modified cell motility in vitro, in accordance with a possible causal role in metastasis. Our results show that breast cancer metastases are molecularly distinct from their primary tumors.

Gene expression analysis of early and advanced gastric cancers.

Gastric carcinoma is one of the major causes of cancer mortality worldwide. Early detection results in excellent prognosis for patients with early cancer (EGC), whereas the prognosis of advanced cancer (AGC) patients remains poor. It is not clear whether EGC and AGC are molecularly distinct, and whether they represent progressive stages of the same tumor or different entities ab initio. Gene expression profiles of EGC and AGC were determined by Affymetrix technology and quantitative polymerase chain reaction. Representative regulated genes were further analysed by in situ hybridization (ISH) on tissue microarrays. Expression analysis allowed the identification of a signature that differentiates AGC from EGC. In addition, comparison with normal gastric mucosa indicated that the majority of alterations associated with EGC are retained in AGC, and that further expression changes mark the transition from EGC to AGC. Finally, ISH analysis showed that representative genes, differentially expressed in the invasive areas of EGC and AGC, are not differentially expressed in the non-invasive areas of the same tumors. Our data are more directly compatible with a progression model of gastric carcinogenesis, whereby EGC and AGC may represent different molecular stages of the same tumor. Finally, the identification of an AGC-specific signature might help devising novel therapeutic strategies for advanced gastric cancer.

Prognostic implications of NUMB immunoreactivity in salivary gland carcinomas.

The gene numb encodes for a protein (Numb) involved in cell fate decisions in Drosophila, with proposed endocytic and developmental functions in mammalians. The distribution pattern of Numb in human tissues however, has not been fully characterized. We set out to explore the immunohistochemical expression of Numb in normal and neoplastic (28 adenoid cystic and 34 mucoepidermoid carcinomas) salivary glands, and correlated the results with the clinico-pathologic features of the neoplasms. Intense Numb immunoreactivity was detected in normal ductal cells and in a subset of acinar cells. In salivary carcinomas, we detected diffuse and intense Numb immunostaining in 5 adenoid cystic and 8 mucoepidermoid carcinomas. By contrast, the majority of adenoid cystic and mucoepidermoid cancers showed only moderate (14 and 5 cases) or focal staining (9 and 21 cases), respectively. The corresponding expression of Numb mRNA was documented in normal parotid gland and adenoid cystic carcinoma. Numb immunoreactivity was inversely correlated with the histological grade and Ki-67 immunoreactivity of both adenoid cystic and mucoepidermoid carcinomas. In addition, while tumor grade, stage, Ki-67 and Numb immunoreactivity were associated with disease-free survival in univariate analysis, only Numb and Ki-67 immunoreactivities retained independent prognostic significance in multivariate analysis. These data suggest that loss of Numb is implicated in aberrant differentiation programs of salivary gland carcinomas and may serve as a prognostic indicator in patients treated for these neoplasms.

Nickel-titanium rotary instrument fracture: a clinical practice assessment.

AIM: To prospectively determine the incidence of nickel-titanium rotary instrument fracture in an endodontic clinical practice setting. METHODOLOGY: Eleven second year endodontic residents, using four nickel-titanium rotary instrument systems (ProFile, ProTaper, GTRotary and K3Endo) according to the recommendations of the manufacturers, instrumented 3181 canals in 1403 teeth of 1235 patients, in a dental school post-graduate endodontic clinic, in 1 year. The incidence of instrument fracture was determined based on the number of instruments used. When fracture occurred, data were collected concerning the type, size, taper and prior use of the fractured instruments, the length and location of the fragment within the root canal and the curvature of the canal. RESULTS: The overall incidence of instrument fracture was 0.39%. The incidence of fracture for ProFile, ProTaper, GTRotary and K3Endo files was 0.28%, 0.41%, 0.39% and 0.52%, respectively. There was no statistically significant difference between instrument systems. The percentage of teeth in which instruments fractured was 1.9% (0.28% for anterior teeth, 1.56% for pre-molars and 2.74% for molars). A total of 26 instruments fractured, of which 23 had tapers of 0.06 or greater. Most of the fragments were located in the apical third of the root canal, and both the median and mode amongst the fragment lengths were 2 mm. CONCLUSIONS: The low incidence of nickel-titanium rotary instrument fracture supports the continued use of these instruments in root canal treatment.

Fracture of ProFile nickel-titanium rotary instruments: a laboratory simulation assessment.

AIM: To determine the incidence of ProFile nickel-titanium rotary instrument fracture in an endodontic laboratory simulation. METHODOLOGY: Three hundred and sixty dental students used 2880 ProFile nickel-titanium rotary instruments to prepare 1440 simulated root canals in 720 plastic teeth, and another 2880 ProFile nickel-titanium rotary instruments to prepare 1440 natural root canals in 720 extracted teeth. A standardized crown-down rotary instrumentation technique was used, which included measures to prevent fracture. Rotary instrument fracture was monitored during and after completion of the laboratory simulation exercises to determine the incidence of fracture. When fracture occurred, data were collected concerning the size of the instrument, the length of the instrument fragment, the location of the fragment in the canal and the curvature of the canal in which the instrument fractured. RESULTS: The incidence of instrument fracture was 0.41% in plastic simulated canals and 0.31% in natural root canals. The overall incidence of instrument fracture was 0.36%. Of the instruments that fractured 67% were size 25, 0.04 taper; and 81% of the fragments were located in the apical third of the canal. The mean, median and mode of the fragment lengths were all 3 mm. CONCLUSIONS: The low overall incidence of fracture in this study suggests that ProFile rotary instruments are safe for use by dental students in laboratory simulations and that if preventive measures are taken the incidence of instrument fracture can be minimized.