A need exists for easily administered assessment tools to detect mild cognitive changes that are more comprehensive than screening tests but shorter than a neuropsychological battery and that can be administered by physicians, as well as any health care professional or trained assistant in any medical setting. The Toronto Cognitive Assessment (TorCA) was developed to achieve these goals.n = 303), determined test reliability, developed an iPad version, and validated the TorCA against neuropsychological assessment for detecting amnestic mild cognitive impairment (aMCI) . For the normative study, healthy volunteers were recruited from the Rotman Research Institute registry. For the validation study, the sample was comprised of participants with aMCI or normal cognition based on neuropsychological assessment. Cognitively normal participants were recruited from both healthy volunteers in the normative study sample and the community.We obtained normative data on the TorCA . In an exploratory logistic regression analysis, indices of Immediate Verbal Recall, Delayed Verbal and Visual Recall, Visuospatial Function, and Working Memory/Attention/Executive Control, a subset of the domains assessed by the TorCA, correctly classified 92% of participants . Paper and iPad version scores were equivalent.The TorCA can improve resource utilization by identifying patients with aMCI who may not require more resource-intensive neuropsychological assessment. Future studies will focus on cross-validating the TorCA for aMCI, and validation for disorders other than aMCI. Brief tests such as the Mini-Mental State Examination (MMSE)  and the Our objectives were to obtain normative data on the TorCA and to validate this test for detection of amnestic mild cognitive impairment (aMCI). In addition to the paper version, we developed an electronic application for the iPad and assessed equivalency between the two versions. The advantages of an electronic application include automatic scoring, automatic point-of-care data collection for potential data entry into a clinical or research registry, a printable summary of results, and graphical representation of percentile performance on each cognitive domain.OrientationThe TorCA consists of 27 subtests within seven cognitive domains—Orientation, Immediate Recall, Delayed Recall, Delayed Recognition, Visuospatial Function, Working Memory/Attention/Executive Control, and Language Table —and can 2.Immediate Verbal RecallThere are 12 items included: year, month, day, date, season, place/building, floor, city, province, country, Prime Minister, and Premier of the province.3.Delayed Verbal and Visual RecallThe CERAD 10-Word list  is prese4.Delayed Verbal and Visual RecognitionDelayed recall of the CERAD Word List and the Benson Figure Copy  are asse5.Visuospatial FunctionRecognition of whether words appeared in the CERAD list and which one of four complex figures was copied are assessed.6.Working Memory/Attention/Executive ControlThis scale consists of Clock Drawing  and the 7.LanguageWorking memory and attention are assessed by Digit Span and Serial Subtractions. Executive control  is asses8.TorCA Sum IndexThere are eight subtests included: Verbal Fluency , confrontation naming of 15 items from the Multilingual Naming Test (MINT) , SentencConsistent with standard practice in neuropsychology, there is no upper limit on Verbal Fluency for “F” words and animals. Therefore, there is no maximum on the Sum Index.n = 303) were recruited from the Rotman Research Institute (RRI) registry. There were four age groups: 50–59, 60–69, 70–79, and 80–89 years. Exclusion criteria were history of neurological disease, drug abuse, head injury with loss of consciousness, attention deficit hyperactivity disorder, active psychiatric illness, or use of medication containing any opioid. Non-native English speakers were included if they could understand all instructions. For test items, and administration and scoring instructions, see the Toronto Dementia Research Alliance website (www.tdra.ca). Figure The study was approved by the Research Ethics Board at Baycrest Health Sciences. Healthy volunteers (r) calculated between the first and second tests. Internal consistency was determined by calculating Cronbach’s α for domain and Sum Index scores from the normative data study.To assess test stability, the TorCA was readministered to 29 participants after a median interval of 73 days (range 28–120) with mean difference, percentage score change, and stability coefficients  based on neuropsychological assessment. From 220 consecutive referrals from all sites, 25 refused clinical services, 7 were inappropriate, and 188 were assessed by a neuropsychologist. Of those assessed, 108 did not have MCI or NC and four met exclusion criteria, yielding 50 participants with aMCI (single domain/multiple domain = 13/37) and 26 with NC. Figure As it proved difficult to find individuals with normal cognition in memory clinics, the remaining 31 normal participants were recruited from the current normative study sample and the community. The paper version of the TorCA was administered prior to neuropsychological assessment in all but three instances. The interval between neuropsychological assessment and TorCA was within six months.As assessments were conducted in a clinical context, the neuropsychologists were aware of the TorCA scores and differential diagnoses. The majority of neuropsychological assessments were conducted by trained assistants not directly involved in the diagnostic process, although one of the neuropsychologists tested 42 participants. The TorCA was conducted by trained nurses, medical trainees, or research assistants who were blinded to the neuropsychological assessment results.Exclusion criteria for the validation study were medical or neurological disorders that could cause cognitive deficits including untreated sleep apnea, traumatic brain injury with loss of consciousness greater than 30 min, history of stroke, attention deficit hyperactivity disorder requiring medication, substance abuse, or other significant psychiatric disorders.Kaplan–Baycrest Neurocognitive Assessment (KBNA) .Trail Making Test Forms A and B .Wechsler Adult Intelligence Scale—III (WAIS-III) Digit Symbol .WAIS—III Digit Span .Wechsler Memory Scale—Revised (WMS-R) Logical Memory I and II subtests (Story A or B) .Wechsler Abbreviated Scale of Intelligence (WASI) Vocabulary , Similarities, and Matrix Reasoning subtests .Boston Naming Test  .Delis–Kaplan Executive Function System (D-KEFS) Color-Word Interference Test .Multifactorial Metamemory Questionnaire—Memory Mistakes scale .Lawton and Brody ADL questionnaire .Hospital Anxiety and Depression Scale .The following were administered as part of the neuropsychological battery:All participants with aMCI met published criteria . ObjectiConcurrent validity was determined by the ability of the TorCA to discriminate between aMCI and NC participants. Construct validity was determined by correlations between TorCA subtests and neuropsychological tests in the aMCI and NC groups and by testing for expected group differences on TorCA indices and subtests.n = 22, female/male = 17/5; mean (SD) age = 73.6 (7.7) years) was recruited from the normative sample and was administered the paper version first . The second group  age = 70.6 (10.1) years) was recruited from the RRI registry and was administered the iPad version first .Forty-five normal participants were tested using paper and iPad versions and were divided into two groups. One group (2(df = 1) = 4.26, p = 0.04), 60–69 year group (χ2(df = 1) = 14.14, p = 0.001), and 70–79 year group (χ2(df = 1) = 16.33, p = 0.001) but not for the 80–89 year group (χ2(df = 1) = 1.08, p = 0.30).Table Normative TorCA test scores are categorized into ≤ 5th percentile (impaired), 6th–24th percentile (borderline), or ≥ 25th percentile . Median time to complete the TorCA was 34 min (range 25–63). Tables F = 6.45, p = 0.001) (Table d = 0.31) [F = 7.36, p = 0.007). Age and education were weakly, but significantly, correlated with Sum Index , each accounting for approximately 5% of the variance.The Sum Index was significantly affected by age   for gendThe results of the test–retest study using the paper version in normal participants are presented in Table The intratest reliabilities of the TorCA indices are presented in Table χ2 = 6.33, p < 0.02), whereas the aMCI group had an approximately equal gender balance .Table Effect sizes based on difference between group means and standard deviations for neuropsychological tests used to determine group membership are provided in Fig. F = 36.86, p < 0.001). A MANOVA on the remaining seven domain indices revealed a significant effect for group  = 23.78, p < 0.001). Pairwise comparisons, with Bonferroni correction for seven multiple comparisons at p ≤ 0.05/7 (0.007), revealed significant differences for orientation, immediate memory recall, delayed memory recall, and delayed memory recognition indices.Table Prior to analyzing TorCA subtest scores for group differences, boxplots for each subtest were inspected. Distribution of scores on Trail Making , Alternating Sequences, Similarities, Sentence Repetition and Comprehension, Single Word Reading and Comprehension, and Semantic Knowledge showed a marked negative skew with a ceiling effect for both groups. Kolmogorov–Smirnov tests on these subtests revealed no differences in distribution of scores between the two groups. Therefore, these subtests were dropped from further between-group analyses.F, p < 0.001). Table Scores on Verbal Learning, Verbal Recall, Verbal Recognition, Visual Recall, Serial Subtractions, Digit Span, Trail Making A and B completed times measure, Benson Figure Copy, Clock Drawing, Verbal Fluency—F Words, Verbal Fluency—Animals, and MINT Naming were analyzed with a MANOVA for between-group differences Table . There wχ2 = 31.5, p < 0.0001, AUC = 0.84 (95% CI 0.75–0.92)). The sensitivity, specificity, likelihood ratio of a positive response (LRPR), likelihood ratio of a negative response (LRNR), positive predictive value (PPV), negative predictive value (NPV), Youden index, and correct classification of each Sum Index from 209 to 319 was calculated. The optimum cutoff value was determined by considering the maximum correct classification, LRPR, and Youden index combined with a view to minimizing false positives and maximizing classification accuracy. A Sum Index cutoff value of 275 was optimal and yielded an overall classification accuracy of 79% (95% CI 70–86%), sensitivity of 80% (95% CI 66–89%), specificity of 79% (95% CI 66–88%), LRPR of 3.80 (95% CI 2.26–6.40), and LRNR of 0.25 (95% CI 0.14–0.45). Given the aMCI prevalence of 47% in our sample, the 275 cutoff value yielded a PPV of 0.77 (95% CI 0.63–0.87) and NPV of 0.82 (95% CI 0.69–0.90). Agreement between the TorCA, using this cutoff value, and classification achieved by standard clinical and neuropsychological criteria was weak to moderate [The TorCA Sum Index discriminated between the aMCI and NC groups .To explore which TorCA indices best discriminated between aMCI and NC, indices for Orientation, Immediate Memory Recall, Delayed Memory Recall, Delayed Memory Recognition, Visuospatial, Working Memory/Attention/Executive Control, and Language were entered into a backward, stepwise logistic regression that generates a posttest probability of aMCI Table . Four inThe neuropsychological tests were grouped into nine domains: Immediate Recall, Delayed Recall, Delayed Recognition, Visuospatial, Cognitive Flexibility, Attention/Concentration, Executive Control, Verbal Fluency, and Language. Correlations between TorCA and neuropsychological domains are presented in Table r(43) = 0.86, p < 0.001) and no difference between TorCA Sum Index on paper  and iPad  versions (t(44) = − 0.56, p = 0.58). There was a trend (t(44) = 2.00, p = 0.052) for the mean Sum Index to be slightly lower on the first administration  compared to the second . Test–retest reliability between first and second administration was good (r(43) = 0.87, p < 0.001). There was no association between test–retest interval and change in Sum Index on first and second testing (r(43) = 0.04, p = 0.77). In addition to lack of a linear relationship between the change in Sum Index and test–retest interval, neither a quadratic (p = 0.90) nor a logarithmic model (p = 0.66) fit the data. In addition, the mean TorCA Sum Index did not differ for the group that took the paper version first  compared to the group that took the iPad version first  (t(43) = 1.5, p = 0.14).There was a strong correlation between paper and iPad versions  with mean increase of only 3.3 points. Age and education accounted for only 5% of the variance in total score. Although age-adjusted norms are available for each decade from 50 to 89 years, the TorCA can be administered across this range with minimal need for age correction. Paper and iPad version scores were not significantly different. The iPad version provides easier administration with near automation of scoring and graphical representation of percentile scores  reflected increases in the immediate and delayed memory indices (14.3% and 10.7% respectively) with no other index exceeding an increase of 1.5% (Language).Internal consistency of the Sum Index was adequate and reflected the heterogeneous nature of individual tests. Low internal consistency reflected the diverse nature of cognitive abilities on Delayed Recall and Working Memory/Attention/Executive Control. The former combines verbal and visual memory, whereas the latter combines heterogeneous measures related to frontal system function. Low internal consistency also reflected restricted range in scores on Orientation, Delayed Recognition, and Visuospatial Function.www.tdra.ca). This is automatically calculated with the iPad version of the TorCA. It should be emphasized that the correct classification of 92% arises from four domains of the TorCA rather than the total score on the entire test. In contrast, correct classification was 79% based on the Sum Index .We validated the TorCA for detection of aMCI based on a need for cognitive assessment tools that can identify early decline, that are much shorter than typical neuropsychological batteries, and that can be administered by any health professional or trained assistant. A combination of TorCA subscores yielded correct classification, sensitivity, and specificity of over 90%. Logistic regression revealed that scores in four domains—Immediate Recall, Delayed Verbal and Visual Recall, Visuospatial Function, and Working Memory/Attention/Executive Control—correctly classified 92% of participants, and yielded an easily applied formula to calculate the probability of aMCI  decreases and increases as pretest probability increases. Conversely, NPV increases with decrease in pretest probability and decreases as pretest probability increases. PPVs and NPVs listed earlier for the optimal value relate only to the pretest probability of aMCI in our sample (50/107 = 0.47). Table Based on the validation data for TorCA Sum Index reported in this article, the TorCA is comparable to published data on the MoCA for detection of MCI. A meta-analysis of 20 studies conducted by Ciesielska et al.  reportedThe TorCA has potential resource allocation implications in centers with neuropsychology resources by identifying patients who do not require neuropsychological assessment due to a high probability of aMCI or because this disorder is effectively ruled out. Although the logistic regression was exploratory, a reasonable strategy might be to rule out aMCI if probability, based on the logistic regression formula, is below 0.55. Due to the likelihood that the logistic regression formula overestimates classification , we recoStudy limitations should be acknowledged. First is the need for cross-validation. Whereas the validation study revealed that the use of the logistic regression formula would refine the identification of aMCI, this represents an initial, exploratory result and further cross-validation of the formula is needed to confirm critical values and stability of constituent indices. A second limitation is that the logistic regression formula for probability of aMCI applies only to differential diagnosis of aMCI vs normal aging. Future studies are needed to validate the TorCA for differentiating aMCI from other cognitive disorders, and to determine whether it performs equally well for identifying single vs multiple domain aMCI. A third limitation is that participants in the validation study had relatively high IQs. Studies are needed to determine validity of the TorCA for diagnosing aMCI in participants with lower IQs. In addition, a caution is that interpretation of positive or negative cases must take into account differences between patients’ estimated pretest probabilities of a condition and prevalence of the condition in validation studies. A fourth limitation is that the orientation items consisting of Prime Minister, Premier, and season are country specific. This will be addressed in future by translating the TorCA into languages other than English and carrying out normative and validation studies using the translated tests. Ideally, normative and validation studies should also be carried out in English-speaking countries other than Canada. Finally, this study focused only on aMCI from a diagnostic perspective. Future studies will be needed to validate the TorCA for diagnosis of other forms of mild cognitive decline. It is likely that the discriminating indices on the TorCA will differ from those that predict aMCI.The TorCA is a relatively short cognitive assessment tool for identification of early cognitive decline and can be administered by any health care professional or assistant with appropriate training. It also has the potential to save both time and physical resources by identifying patients who may not require neuropsychological assessments for diagnosing aMCI. Future studies will focus on cross-validating the TorCA for aMCI and validating this test for disorders other than aMCI.


Hypophosphatemia has many causes, and is often encountered during DKA (Diabetic Ketoacidosis) treatment. However, it rarely requires clinical intervention.Ventricular arrhythmia was observed in a 10-year-old girl with newly diagnosed type 1 diabetes mellitus and hypophosphatemia while undergoing treatment for ketoacidosis. Oral phosphate supplementation ceased ventricular arrhythmia almost completely.The clinical signs of hypophosphatemia are potentially life-threatening. Therefore, physicians should be vigilant when treating patients who are at risk of hypophosphatemia. Severe hypophosphatemia accompanied by clinical symptoms requires oral or intravenous supplementation of phosphate. Diabetic ketoacidosis (DKA) is a life-threatening condition that requires immediate treatment. Type 1 diabetes mellitus is an autoimmune disease, in which beta-cell destruction process starts as cellular reaction that leads to beta-cell damage that stimulates autoimmune humoral reaction. As the level of anti-pancreatic autoantibodies rises, insulin secretion is impaired leading to complete lack of insulin. As a result, hyperglycemia occurs and leads to ketoacidosis. The initial symptoms such as polydipsia, polyuria and nycturia lead to dehydration. As acidosis progresses, the patient presents elevated heart rate, elevated breathing rate and Kussmaul breathing pattern occurs. These symptoms are often accompanied by stomach ache, nausea and vomiting. As condition worsens, neurological symptoms, ranging from confusion and sleepiness to coma, may occur. The laboratory criteria for DKA diagnosis are: Hyperglycemia (plasma glucose > 200 mg/dL), serum pH < 7,3 or serum bicarbonate level < 15 mmol/L and also the presence of serum ketones and ketonuria. DKA severity is determined by the level of acidosis: in mild DKA pH is 7,2–7,3 or bicarbonate level is 10–15 mmol/L; in moderate DKA pH is 7,1–7,2 or bicarbonate level is 5–10 mmol/L; in severe DKA pH < 7,1 or bicarbonate level < 5 mmol/L. Essential treatment requires fluid resuscitation and human recombinant insulin administartion in continuous IV infusion. Patient overall condition should be monitored continuously, blood glucose and neurological status should be checked every hour, serum electrolytes should be checked every 2 h after starting IV fluids. Additionally ECG should be monitored for abnormal T waves and T waves changes. Cerebral edema and hypokalemia are the most common complications during DK treatment. Other possible complications include severe hypophosphatemia, hypocalcaemia, hypomagnesaemia, hypoglycemia, heart arrhythmias, deep vein thrombosis, pulmonary embolism, sepsis, pulmonary edema, acute respiratory distress syndrome, rhabdomyolisis, ischemic renal necrosis, acute renal failure, acute pancreatitis [Phosphorus is an element which is essential for life. The human body contains 11 to 14 g/kg of lean body mass . The majFood is a rich source of phosphorus, especially dairy products, meat and beans. It is estimated that 1 ml of milk contains 1 mg of phosphorus. A typical diet provides 800–1400 ng of phosphorus daily, and approximately 65% of ingested phosphorus is absorbed in the intestines [The normal serum concentration of phosphate fluctuates with age, with the normal concentration for adults ranging between 0.8 and 1.3 mmol/L. Mild hypophosphatemia is defined as a phosphate level between 0.32 and 0.65 mmol/L, while severe hypophosphatemia is diagnosed when the phosphate level drops below 0.32 mmol/L [A shift in phosphate level is expected to occur in DKA, resulting both from the pathogenesis of the disease and insulin action. Although changes in serum phosphate level are widely described , 8, therThere are multiple issues related to the interpretation of serum inorganic phosphorus (Pi) levels. Firstly, there is no simple translation of serum phosphate levels to the severity of clinical signs . SecondlThe life-threatening clinical manifestations of hypophosphatemia are associated with the energetic role of phosphate. As a part of ATP, phosphate is a key element in cell energy turnover. Furthermore, 2,3-DPG deficiency in red blood cells promotes oxygen binding to hemoglobin, therefore oxygen is less likely to be released to the tissues that need it most. Ischemia occurs as a result, which affects both tissues and organs.The main clinical manifestations of hypophosphatemia include metabolic encephalopathy , impaired cardiac contractility (including cardiomyopathy), heart arrhythmia , respiratory failure, impaired skeletal muscle contractility , rhabdomyolysis, dysphagia and hemolysis , 10–14.A 10-year-old girl was admitted to our clinic due to ketoacidosis associated with newly diagnosed type 1 diabetes. Prior to admission, she had suffered from polyuria, polydipsia, and nycturia for about 2 weeks, and presented with fatigue, drowsiness and a lack of appetite for the last two days. Her history did not include any chronic diseases, and she had normal growth and development.On admission, she was in a serious state, suffering from vomiting and dehydration, and displaying Kussmaul breathing, a respiratory rate of 45 breaths/min and tachycardia. The blood test revealed a blood glucose level of 26.8 mmol/L (482 mg/dl), pH 6.902, base excess (BE) of − 29.3 mmol/L, HbA1c of 12.9%, sodium level of 142 mEq/L, potassium level of 4.11 mEq/L and ketone level of 6.1 mmol/L. The patient was treated according to the recommendations of the International Society of Pediatric and Adolescent Diabetes and the Polish Diabetes Association .Initially the patient received 500 ml of 0.9% NaCl IV infusion. Next, a continuous IV infusion of short acting insulin was commenced with 2,5 U/ h dose, taking into consideration that the patient weight was 40 kg. IV Insulin infusion was sustained for 51 h with dosages ranging from 1,5 U/h to 4 U/h. Overall the patient received 81,75 units of IV insulin during the first 24 h of treatment. Moreover, potassium was supplemented intravenously accordingly to changes in electrolytes results. Although she remained in a serious condition during the first 24 h of treatment, she stopped vomiting and showed gradual improvement in blood test results, with pH normalization and a decrease in the level of ketones. The glucose level was maintained between 8.3 and 13.9 mmol/L (150–250 mg/dl) with intravenous insulin and glucose infusions. The neurological status was checked hourly, and was reported as stable. An ECG was performed within the first few hours of admission, which did not reveal any crucial abnormalities aside from tachycardia. The patient received 65 units of IV insulin during the second 24 h f treatment. During the whole continuous insulin infusion the patient fluid input was 7850 ml  and diuresis was 3400 ml.The patient’s overall condition improved during the second day of treatment, despite the fact that she was still receiving intravenous insulin. That day irregular heartbeats were noticed on auscultation during the physical examination and on the ECG monitor. A second ECG showed premature ventricular complexes  with LBBB (left bundle branch block) morphology and a normal axis Fig. . ContinuThe ECG performed after the first recognition of the patient’s the irregular heart rhythm Fig.  showed: 2HPO4 and 4.88 g NaH2PO4 in 100 ml of distilled water, containing 1.6 mmol of inorganic phosphate per 1 ml). As 1 mmol of inorganic phosphate weighs 31 mg, our patient was receiving 62 mg Pi/kg body weight every 24 h from the phosphate mixture. She continued to present an irregular heart rate, thus a 24-h ECG Holter monitor examination was performed. The results showed 38,000 monomorphic PVCs, 1615 occurrences of ventricular bigeminy, 1230 of ventricular trigeminy and 453 of ventricular quadrigeminy, which comprised 38% of all the heart electrical activity recorded.Potassium was supplemented both orally and intravenously. Overall the patient received 123 mEq of potassium during the continuous insulin therapy. As far as phosphorus is concerned, the patient was fed with liquid dairy products that were rich in phosphate, as she was only able to swallow small amounts of non-solid foods due to odynophagia. She was also started on oral phosphate supplementation , ventricular tachycardia and atrial fibrillation, which authors describe as multi etiological –29.Arrhythmia associated with hypophosphatemia are mainly supraventricular arrhythmia, and may also include single preliminary ventricular complexes, ventricular bigeminy and trigeminy . VentricMany authors , 5, 15 hPhosphate may be administered orally or intravenously, depending on the patient’s condition Table . Oral adThe recommended oral dosage of phosphate is 30–40 mg Pi/kg of body weight every 24 h, administered in four to five doses , 32. It Intravenous administration of phosphate solution should be used for critically ill patients and those who are nil by mouth. Both potassium and sodium phosphates are available. These should be administered together with sodium chloride or glucose solutions, as low-soluble salt would precipitate in solutions containing calcium. High dose or rapid phosphate infusion may lead to hypophosphatemia, hypotension, hypocalcaemia with tetanus, renal failure and ECG abnormalities. Most of the data about dosage has come from research on adults, thus each case must be approached with caution. A dosage of 0.08 mmol/kg body weight every 6 h is recommended for short-term uncomplicated hypophosphatemia, whereas a dosage of 0.16 mmol/kg body weight is advised for long-term hypophosphatemia of complex origin. Some manufacturers recommend a maximum single phosphate dosage of 0.24 mmol/kg body weight, although some authors have reported the use of doses of 0.4–0.5 mmol/kg body weight every 6 h in specific conditions, up to a maximum dose of 50 mmol. Doses should be individually adjusted to the patient’s clinical condition, hypophosphatemia severity, and their reaction to phosphate infusion , 33.It is recommended that blood calcium and phosphate levels be carefully monitored in DKA patients in a serious condition. The blood phosphate level decreases after the initiation of DKA treatment, which is potentiated by insulin. Clinical symptoms usually occur at a blood phosphate level lower than 0.32 mmol/L, although the intracellular phosphate level also plays a crucial role. Hypophosphatemia commonly appears on the second day of treatment, thus it is vital to monitor ECG. Severe hypophosphatemia with clinical symptoms requires oral or intravenous supplementation of phosphate. So far, there is no evidence of any benefits of routine administration of phosphate in patients with DKA. Patients with DKA who are at risk for hypophosphatemia include those with low blood pH, extremely high blood glucose level and very low BE, nil by mouth patients who have received intravenous insulin for longer than 24 h, cachectic or malnourished patients, and those with a vitamin D deficiency.


Pediatric retroperitoneal tumors in the renal bed are often large and heterogeneous, and their diagnosis based on conventional imaging alone is not possible. More advanced imaging methods, such as diffusion‐weighted (DW) MRI and the use of intravoxel incoherent motion (IVIM), have the potential to provide additional biomarkers that could facilitate their noninvasive diagnosis.To assess the use of an IVIM model for diagnosis of childhood malignant abdominal tumors and discrimination of benign from malignant lesions.Retrospective.n = 32 malignant, n = 10 benign), verified by histopathology.Forty‐two pediatric patients with abdominal lesions .1.5T MRI system and a DW‐MRI sequence with six D), fast diffusion coefficient (D*), and perfusion fraction (f) were computed using a segmented fitting model. Histograms were constructed for whole‐tumor regions of each parameter.Parameter maps of apparent diffusion coefficient (ADC), and IVIM maps of slow diffusion coefficient  analysis.Comparison of histogram parameters of and their diagnostic performance was determined using Kruskal–Wallis, Mann–Whitney D* and f were significantly higher in neuroblastoma compared to Wilms' tumors (P < 0.05). The ROC analysis showed that the best diagnostic performance was achieved with D* 90th percentile  and f mean values  in discriminating between neuroblastoma (n = 11) and Wilms' tumors (n = 8). Discrimination between tumor types was not possible with IVIM D or ADC parameters. Malignant tumors revealed significantly lower ADC, D, and higher D* values than in benign lesions .IVIM parameters IVIM perfusion parameters could distinguish between malignant childhood tumor types, providing potential imaging biomarkers for their diagnosis.Level of Evidence: 4Technical Efficacy: Stage 2J. Magn. Reson. Imaging 2018;47:1475–1486. Malignant abdominal tumors in children are often diagnosed using a combination of conventional imaging and histology.Magnetic resonance imaging (MRI) is increasingly used in the diagnosis, staging, and management of pediatric solid tumors due to its relatively high resolution and lack of nonionizing radiation.b‐value DW‐MRI can account for the pure diffusion characteristic (D), and separate the pseudodiffusion (D*) effect caused by microcirculation or blood perfusion, and determine the perfusion fraction (f) corresponding to the fraction of signal arising from the vascular component.D in comparison to ADC.The ADC approach for heterogeneous tissues such as those found in abdominal tumors can be relatively simplistic and does not maximize the information that can be potentially extracted from the DWI.The heterogeneous nature of abdominal tumors can be difficult to characterize based on imaging measures such as the mean ADC value alone.Therefore, the aim of this study was to retrospectively evaluate the feasibility and diagnostic potential of using IVIM and ADC histogram analysis for discriminating between individual malignant and between benign and malignant pediatric tumor types.n = 1), incomplete or improper MRI scans (n = 10), or small lesions where single‐slice largest area <3 cm2 or total volume <6 cm3 (n = 2). The final population included 42 patients, comprised of 10 benign and 32 malignant cases , operating under the rules of Declaration of Helsinki 1975 (and as revised in 1983). Informed parental consent was obtained from all subjects after the image acquisition. A computerized search of medical records identified 55 patients who underwent abdominal MRI including DWI between June 2012 and September 2016. All cases were reviewed by the tumor study board for suspected solid malignancy. The following exclusion criteria were applied to the cohort: having received treatment prior to imaging (chemotherapy or surgery), nonsolid pathology (n = 1), Ewing's sarcoma (n = 1), germ cell tumor (n = 1), hepatoblastoma (n = 4), nephroblastomatosis (n = 1), neuroblastoma (n = 11), ovarian immature teratoma (n = 1), rhabdoid (n = 2), rhabdomyosarcoma (n = 2), and Wilms' tumor (n = 8). The benign lesions consisted of indolent abdominal mass (n = 1), ganglioneuroma (n = 3), hematocolpos (n = 1), lipoma (n = 1), hemangioma (n = 1), mesoblastic nephroma (n = 1), osteomyelitis (n = 1), and vascular malformation (n = 1). The mean size of the malignant tumors was 32.1 cm (range 8.5 ± 53.1 cm) and benign lesions 16.8 cm (range 7.2 ± 47.7 cm). The cohort demographics are summarized in Table The cohort comprised 16 female patients  and 26 male patients . The age range for the whole group was 0–14 years, with a mean age ± SD 3.7 ± 2.8 years. The malignant cases included those with clear cell sarcoma (1‐weighted turbo spin‐echo (repetition time / echo time [TR/TE] 760 to 817/7.7 msec), axial T2‐weighted turbo spin‐echo (TR/TE 3000 to 5640/67 to 87 msec) and DWI acquisition. The DWI protocol used a spin‐echo planar imaging (EPI) sequence with six b‐values , EPI factor = 174, three averages (NSA = 3), TR/TE 3200 to 9900/92 msec, parallel imaging (GRAPPA) with an acceleration factor of two, and 75% partial Fourier encoding. The diffusion‐weighting was applied in three orthogonal directions, of which an average image was derived in the axial acquisition plane. Depending on patient size, the field‐of‐view (FOV) was 221 to 350 × 172 to 317 mm, matrix size 122 to 192 × 128 to 192, slice thickness 5 mm with no gap between slices and voxel size of 1 to 2.34 mm2 × 5 mm. The DWI protocol acquisition time ranged from 4 min 52 sec to 7 min 35 sec.MRI was performed with a Siemens Avanto 1.5T  scanner and a 4‐channel body receive coil at Birmingham Children's Hospital. The imaging protocol included fat‐suppressed axial and coronal pre‐ and postgadolinium Tb‐values can be described by a biexponential relationship introduced by Le BihanS0 and Sb are the signal intensities at b = 0 and b = 50, 100, 150, 600, or 1000 s/mm2, respectively. Using this relationship, a nonlinear least‐squares fit was applied to the data on a voxel‐by‐voxel basis. A stepwise fitting was used to increase the stability of the biexponential fitting and the reliability of the IVIM parameters as reported by previous studies.b‐values >100 s/mm2 the perfusion effects are assumed negligible as D* ≫D, and linear regression can be performed to compute the D parameter from the gradient of the fit.f parameter from extrapolating the fit to the y‐axis and taking the difference to the b0 signal. The biexponential fitting was then performed using the Levenberg–Marquardt algorithm with the predefined values of D and f to find the D* parameter.Postprocessing of the DW MR data was performed using an in‐house imaging analysis tool developed in MeVisLab platform . The analysis algorithm for IVIM was developed in Python (v. 2.7). The relationship between the diffusion signal intensities and the b‐values 0 and 1000 s/mm2:The computation of ADC values was performed using a monoexponential linear fit of the th, 25th, 75th, and 90th percentiles, skewness, kurtosis, and entropy were calculated from the normalized histograms based on values extracted from the ROI. The histograms were normalized to the maximum value due to differences in ROI sizes, and to compute average histograms for individual tumor types.The ADC and IVIM parameter maps were analyzed using in‐built histogram and ROI drawing modules. For each case, mean, median, 5b = 0 images by an author with clinical experience (K.M.), after which they were refined by a consultant radiologist . Any changes to the ROIs were made in consensus of the two authors (K.M. and K.F.). Conventional MR images (T1 and T2) were used to aid delineation of tumors at each consecutive slice, excluding peritumoral edema. On each slice, sections of tumor that were >50% of the more central adjacent slice and ≥3 cm were included to minimize any partial volume effects.Whole tumor areas were included in the ROIs, including cystic and necrotic areas. The ROIs were drawn manually on DW U‐test was used to establish differences between the malignant and benign lesions. Bonferroni correction was used.All statistical analyses were performed using SPSS Statistics  software. A nonparametric Kruskal–Wallis test was used for the comparison of IVIM and ADC histogram parameters from the malignant tumors and Dunn's test was performed to determine which tumors gave rise to the difference. Mann–Whitney Receiver operating characteristic (ROC) curves were used to determine how well the diffusion parameters discriminated individual tumor types, and benign from malignant lesions. The cutoff value that demonstrated the greatest Youden index on the estimated curves was used to determine the sensitivity and specificity. The area under the ROC curve (AUC) was determined to assess the diagnostic performance of the IVIM and ADC histogram parameters.f was between 25–40%, with Pearson correlation coefficient, r. This was performed for cases of neuroblastoma (n = 5) and Wilms' tumors (n = 4).Voxelwise correlations between perfusion fraction and diffusion parameters were assessed for whole tumor ROIs and tumor regions where P < 0.05 was considered to indicate a significant difference.All tests performed were two‐sided. Numerical values are reported as median ± SD. Distributions of median ADC and IVIM parameter values of malignant and benign lesions are presented in Fig. D, D*, and f histogram parameters are presented in Table D* (P 0.007–0.021) and f (P < 0.001–0.008) between the malignant tumors. For both D* and f these included histogram parameters mean, median, and 75th/90th percentiles. Additionally, D* skewness and f 25th percentile, kurtosis, and entropy showed significant differences. No differences were observed for ADC and D histogram parameters. The Dunn's test revealed that the D* values of neuroblastoma were significantly higher in comparison to Wilms' tumor (P 0.005–0.012) and skewness was significantly higher for Wilms' (P = 0.017) compared to neuroblastoma. Similarly, the f values in neuroblastoma were significantly higher in comparison to Wilms' tumors (P < 0.001–0.002) and higher kurtosis (P = 0.006) and entropy (P < 0.001) were seen for neuroblastoma.Comparison between the individual malignant tumor types and their ADC, f histograms averaged for the malignant tumor types are shown in Fig. Normalized D* and f histogram parameters to study the diagnostic performance of discriminating neuroblastoma from Wilms' tumor, with the results summarized in Table D* histogram parameters mean, median, 75th/90th percentiles, and skewness could discriminate the two tumor types with AUC values >0.900 . The f histogram parameters mean, median, 5th/25th/75th/90th, kurtosis, and entropy could discriminate Wilms' from neuroblastoma, with mean, median, 25th/75th/90th, and entropy demonstrating AUC values >0.900 .ROC analysis was performed for D maps were similar in appearance, although D was generally lower in comparison to ADC and some regions suggested drops in DW signal when b‐value >0, seen as a lower intensity on D map in comparison to ADC. For both Wilms' and neuroblastoma, the D* maps appeared the most heterogeneous, indicating greater variability of values, which accounted for the high histogram entropy. In reference to the healthy kidneys seen on the f maps, a lower vascular character was suggested for the Wilms' case , suggesting that the higher f and D* values resulted from pathological origins rather than image artifacts. However, the overall voxelwise correlations in neuroblastoma and Wilms' cases demonstrated a negative correlation for D and f, while ADC and f showed positive correlation.No correlation between IVIM and ADC parameters was found in the image regions where D, and D* parameters (Supplementary Table S1). The ADC, D, D*, and f for benign lesions were as follows: ADC = 1597 ± 484 (×10−6 mm2/s), D = 1552 ± 622 (×10−6 mm2/s), D* = 11,610 ± 3385 (×10−6 mm2/s), and f = 16 ± 6.7%. Both ADC and D were found to be significantly lower and D* significantly higher in malignant tumors. Most ADC histogram parameters demonstrated a significant difference with lower mean (P = 0.007), median (P = 0.001), 5th percentile (P = 0.005), 25th percentile (P = 0.007), 75th percentile (P = 0.007) and higher kurtosis (P < 0.001), skewness (P < 0.001), and entropy (P = 0.036) for malignant tumors. The D parameter was only found to discriminate malignant tumors with lower median (P = 0.049), 25th percentile (P = 0.045), and higher skewness (P = 0.018). While the f parameter was not found to not show difference between the benign and malignant lesions, the D* parameter demonstrated higher median (P = 0.039) and entropy (P = 0.002) in malignant tumors.The discrimination between benign and malignant lesions was possible with ADC, D, and D* are presented in Fig. −6 mm2/s, D 1242 × 10−6 mm2/s, and D* 11,104 × 10−6 mm2/s were the most accurate cutoff levels, with sensitivity and specificity of 80.0% and 81.3% for ADC, 66.7% and 87.9% for D, and 55.6% and 93.8% for D*. The ADC was found to have better diagnostic performance than D or D* for discriminating benign from malignant tumors, with AUC values 0.825  for ADC, 0.717  for D and 0.736  for D*.ROC analysis was performed for the diffusion parameters that demonstrated a significant difference between benign and malignant lesions (summarized results can be found in Supplementary Table S2). The ROC curves corresponding to median histogram values together with the highest resulting AUC values for ADC, D and entropy for D* with AUC values 0.919 , 0.758 , and 0.840 , respectively. The optimal cutoff values were ADC skewness: 3.30 × 10−2 , D skewness: 3.56 × 10−2 , and D* entropy 9.32 .The best diagnostic performance was achieved with skewness for ADC and D* and f histogram parameters were revealed for Wilms' tumors and neuroblastomas. No significant differences were observed with D or ADC histogram parameters for the individual tumor types. Discrimination of benign from malignant lesions was possible with the diffusion coefficients ADC, D, and D*, but not with f histogram parameters. These results suggest that the complex, heterogeneous structures of abdominal tumors can be characterized using histogram analysis, to potentially facilitate their noninvasive diagnosis.The present study investigated the diffusion and perfusion characteristics of pediatric abdominal tumors based on ADC and IVIM models. We have demonstrated ADC and IVIM‐derived histogram parameters to be helpful in discriminating between malignant neuroblastoma and Wilms' tumors as well as differentiating benign from malignant lesions. Distinctive IVIM‐derived The use of an IVIM model or IVIM histogram analysis for pediatric abdominal tumors has not been reported previously. The histogram approach has been increasingly used for heterogeneous tissues, allowing interpretation of the complex nature and features seen in tumors.D* and f were found to be significant predictors of tumor type, while both ADC and D parameters were unable to discriminate between them. Although these tumors generally display different characteristic features, both in terms of clinical presentationD* and f parameters. The lack of correlation between IVIM and ADC parameters in regions where f was 20–40% suggests that higher f and D* values resulted from pathology rather than image artifact. This finding is of clinical importance, as it could potentially be applied to other tumors, which are more difficult to discriminate clinically, such as distinguishing Wilms' from renal rhabdoid or clear‐cell sarcoma of the kidney, or mesoblastic nephroma. This is particularly relevant given the recommendation to avoid pretreatment biopsy in these tumors.Our results indicate that IVIM‐derived histogram parameters may facilitate discrimination between malignant neuroblastoma and Wilms' tumors. Comparison of Wilms' tumors and neuroblastoma indicated significant differences in the tumor perfusion characteristics. The perfusion influenced parameters D* and higher entropy for f were observed in neuroblastoma. This is suggestive of a more irregular and heterogeneous vasculature of neuroblastoma compared to Wilms' tumors. Diagnostic performance with sensitivity and specificity above 85% was achieved with D* mean, median, 75th/90th percentiles, and skewness, and with f using mean, median, 75th/90th percentiles, kurtosis, and entropy.In addition to the higher values of the IVIM perfusion parameters, higher skewness for f. Interestingly, voxelwise correlations in neuroblastoma and Wilms' cases demonstrated this to be true for D and f, with negative correlation shown, whereas ADC and f showed positive correlation. This agrees with the IVIM model and the proposed “true” tissue coefficient D, and the contribution of both diffusion and vascular characteristics to the measure of ADC.Cellularity and vascularity have been hypothesized to increase in a similar manner, corresponding to a decrease in ADC and increase in f values differing from others in the same tumor group. Tumors behaving particularly aggressively tended to have higher f values, whereas those following a more indolent course had lower f values. The mean f value for Wilms' tumors in our cohort was 11.0 ± 0.8%; the patient with the highest mean f value (14.4%) died following a very aggressive disease process with multiple relapses. Rhabdoid tumors are typically aggressive malignancies with poor survival. The two patients in our cohort with rhabdoid tumors had mean f values of 23.2% and 8.4%, the former died 3 months after presentation, whereas the latter remains in remission 2 years following completion of treatment. This is suggestive of f being a potential prognostic biomarker, with high‐risk tumors having high f values. This is biologically plausible, with increased tumor vascularity reflected through an increase in f. Although our numbers are too small to draw firm conclusions, this interesting observation deserves further exploration in a larger cohort.A review of our results in a clinical context revealed the interesting observations that tumors following an unusual clinical course often had D, and D*. Of these parameters, the best performance was observed with the ADC, which performed well across the percentiles (≤75th percentile). It is possible that ADC, influenced by both D and D*, has a higher diagnostic performance due to the combinations of diffusion and perfusion characteristics into a single parameter. Interestingly, ADC and D were lower in malignant tumors, while D* was higher. Quantitative assessment of ADC is simpler than performing IVIM, and would be feasible to incorporate into clinical practice, with considerable potential to improve patient care. The robust performance of ADC across the percentiles also suggests that the use of histogram analysis might be less relevant, and the use of mean or median might be sufficient for discrimination of benign from malignant lesions. This finding should be tested with a cohort including a larger number of benign cases.Many of the benign lesion types included in this study can be commonly diagnosed on conventional MRI. However, the benign cases in our cohort were a subset of these that were suspected for malignancy and their diagnosis based on clinical information and conventional MRI was not possible. Therefore, all cases required histological verification. Additional imaging methods such as DW‐MRI can provide particularly useful information for diagnostically difficult cases. In our cohort, the discrimination of benign from malignant lesions was feasible with the histogram parameters of ADC, f parameter failed to reach significance in discrimination of benign from malignant tumors. A wide range of f values was observed for the benign cohort. This may reflect differences in vasculature rather than cellularity, particularly as benign lesions evaluated included a hemangioma and vascular malformation. Although the f parameter may not determine benign from malignant cases, it may be useful in evaluating the vascular environment within lesions.The 2 correction2 relaxation times of blood and tissue. Third, the group sizes were unbalanced for discrimination of benign from malignant tumor types and further larger and multicenter studies are required to confirm these results. Finally, while the IVIM model is becoming more popular in abdominal applications, the biophysical origins of the IVIM parameters require further exploration and justification. Promising results were shown in a previous study of the pancreas, which was able to verify the vascular contribution to the diffusion signal by varying the echo time of the MR acquisition.Our study has several limitations. First, the sample size was relatively small for the comparison of individual tumor types. However, the preliminary results are promising and suggested discrimination may be possible, although further validation is required in a larger cohort. Second, no Tb‐value DW‐MRI allowed the computation of IVIM parameters, and the whole‐tumor ROI approach ensured that the heterogeneity of the tumors was taken into consideration. Our initial results in the childhood abdominal tumors suggest that the use of IVIM perfusion parameters, and in particular the perfusion fraction, could facilitate the diagnosis of individual tumor types, and therefore provide a set of noninvasive imaging biomarkers for their characterization.In conclusion, our results suggest that IVIM parameters and their histogram analysis can provide useful insight into the complex structures of pediatric abdominal tumors. The use of multi Additional supporting information may be found in the online version of this articleSupporting InformationClick here for additional data file.


However, little is known about the effect of Cd administration routes on the testicular structure. Thus, this study investigated the testicular impact of Cd exposure comparing both i.p. and oral routes, both single dose (SD), in addition to the oral route in fractional doses (FD). Swiss adult male mice received CdCl2 1.5 mg/kg i.p., 30 mg/kg oral SD, and 4.28 mg/kg oral FD for 7 consecutive days. The Cd bioaccumulation was observed in all routes, mainly in the oral FD route. The concentrations of testicular Ca and Cu decreased in all animals exposed to Cd, while Zn and Mn decreased only in the i.p. route. Testicular SOD activity was reduced in both routes of oral administration, while CAT increased in the i.p. route, and GST increased in all animals exposed to Cd. Changes in the tubular parameters and cell viability were observed in both routes of Cd administration but were more intense in the oral route, mainly in the FD. Serum testosterone concentration was reduced in both routes of oral administration. Tubular damage, such as the vacuolization of the seminiferous epithelium, germ cell detachment, and seminiferous tubule degeneration, occurred in all groups exposed to Cd. Therefore, the oral Cd administration presented greater potential to promote testicular damage, mainly when the metal was given in a fractionated way.The toxic effects of cadmium (Cd) on reproductive parameters are widely described in the literature. Experimental models often make use of the intraperitoneal route ( Cadmium (Cd) is a nonessential metal that can contaminate by occupational and nonoccupational exposure. Occupational exposure is associated with paint, plastic, glass, metal alloy production, and mining activities . NonoccuIt is known that Cd exposure can affect organs, such as the liver, lung, and kidneys, and the testicles are particularly sensitive to toxicity mediated by this pollutant , 5. ChanWhen ingested, Cd is absorbed in the duodenum by the divalent metal receptor (DMT-1), which absorbs microminerals , 11. Thii.p.) (1.5 mg/kg) and at the corresponding oral dose (30 mg/kg), both in single doses, a severe acute hepatic intoxication is observed in the i.p. route. However, Mouro et al. [i.p. (1.2 mg/kg) route in the subchronic evaluation. The authors did not observe histomorphometric changes in the testicular tissue, at the used doses, in the subacute evaluation. However, it is still unknown whether the dose, route, and frequency of administration  cause similar testicular damage. Therefore, the present study is aimed at verifying if Cd exposure, by oral and i.p. single dose and fractionated oral route, may lead to the same changes in testicular reproductive parameters in adult mice in the subacute evaluation after Cd exposure.Matovic et al.  have shoo et al.  found thad libitum. The experimental procedures were approved by the Ethics Committee on Animal Use of the Federal University of Viçosa (protocol 058/2016) and were performed in accordance with the guidelines issued by the National Council for the Control of Animal Experimentation (CONCEA).Twenty Swiss adult male mice (8 weeks old) were maintained under controlled illumination (12-12 h light/dark) and ambient temperature (21 ± 1°C). Water and feed, standard rodent diet, were offered n = 5 animals/group): one control group and three groups exposed to Cd. The control group received distilled water by gavage, while the other groups received a solution of cadmium chloride  at the following concentrations: 1.5 mg/kg (0.92 mg Cd/kg) i.p. single dose (i.p. SD), 30 mg/kg (18.33 mg Cd/kg) orally by gavage single dose , and 4.29 mg/kg/day (2.62 mg Cd/kg/day) orally by gavage for seven consecutive days . After one week, both groups exposed to oral Cd received a total of 30 mg/kg CdCl2. The dose of 1.5 mg/kg of CdCl2 was chosen based on a previous study carried out by our laboratory (unpublished data). The oral dose of 30 mg/kg was chosen because Cd absorption into duodenal cells is approximately 5% of that which was orally ingested [i.p. Furthermore, this amount is considered a safe standard rate of absorption after Cd exposure [After 1 month of adaptation, the mice were randomized in four experimental groups (ingested –17. Thusi.p.) and euthanized by deepening the anesthesia  followed by cardiac puncture and exsanguination. The testes were collected, dissected, and weighed.The 7-day period was adopted to observe subacute effects induced by Cd exposition before a possible rehabilitation process could change the degree of damage . On the The left testis of each animal was immersed in Karnovsky fixative solution, in which approximately half of each testis was destined for histological evaluation and cell viability whereas the other half was used to determine the dosage of Cd and microminerals. The right testis of each animal was frozen (-80°C) for assessment of oxidative and nitrosative stress markers.3) and 0.5 mL of perchloric acid . Afterward, they were transferred to the hot plate. The temperature was gradually raised to 90°C so that the samples had a complete digestion. Then, the samples were diluted in deionized water in a 10 mL volumetric flask and filtered using filter papers. The concentrations of cadmium (Cd), zinc (Zn), calcium (Ca), magnesium (Mn), manganese (Mg), copper (Cu), and iron (Fe) were determined using an atomic absorption spectrophotometer (SpectrAA 220FS Varian) [Samples of the testis designated to this were weighed in an analytical balance  and dried (70°C), until the dry weight was constant. The dried samples were placed in Erlenmeyer flasks with 1.5 mL of concentrated nitric acid (HNO Varian) .The frozen testes were homogenized with potassium phosphate buffer  containing 1 M EDTA, in the proportion of 100 mg of tissue for 1 mL of buffer, followed by centrifugation at 3000 g (6200 rpm) for 10 min. The analyses were performed in the supernatant and in duplicate.−2) reaction in hydrogen peroxide (H2O2), monitored at 570 nm in a microplate spectrophotometer (PowerWave X) [2O2 for 60 seconds, in a spectrophotometer, at 240 nm. The glutathione S-transferase (GST) activity was estimated spectrophotometrically, at 340 nm [The superoxide dismutase (SOD) activity was determined by the pyrogallol method, which is based on the ability of SOD to catalyze the superoxide radical (OrWave X) . The catrWave X) , by meast 340 nm , and cal3PO4), at room temperature, for 10 min. The absorbance was measured spectrophotometrically at 570 nm. The nitrite concentration was calculated with reference to the standard curve (0.0610 to 125 μM) of sodium nitrite (NaNO2).For determining MDA levels, the samples were placed to react with thiobarbituric acid reactive substance (TBARS) solution  for 40 min in a water bath (90°C). TBARS formation was monitored at 535 nm in a microplate spectrophotometer (PowerWave X) . Tissue −1, pH 7.4) [μm) were obtained using a rotatory microtome , with at list 40 μm between sections. Then, the histological slides were stained with toluidine blue/sodium borate (1%). The digital images were obtained using a light-field photomicroscope  connected to a digital camera . Finally, the histomorphometric assessment was performed using ImageJ®  software.The left testis was immersed in Karnovsky fixative solution   for 24 hFor the histopathological evaluation of the testis, the proportion of normal and pathological seminiferous tubules was estimated by counting 200 random tubules per animal. The pathological tubules were separated as follows: mild pathologies  and severe pathologies (absence of germ cells and/or Sertoli cells). This procedure was adapted from the Johnsen index .μm were stained with acridine orange  and propidium iodide  for evaluating cellular morphological changes [Histological slides containing testicular sections of 1  changes . AO is a changes .4 μm2 per testis was used to calculate the percentage of nonviable germ cells within the seminiferous tubules [Viable cells stained by AO exhibit intact green nuclei, while nonviable cells costained by AO and PI show a dense yellow/orange/red nucleus due to chromatin condensation and the degree of loss membrane integrity . Digital tubules .The gonadosomatic index (GSI) was determined according to Amann  using thThe proportion among the components that constitute the testicular parenchyma was obtained using square grids placed over digital images (100x magnification). A total of 2,660 intersection points were counted per animal classifying those that were on seminiferous tubules and their components  and interstitial tissue. The percentage of each component was determined by the following equation: volume density (%) =  × 100. The volume (mL) of each component was obtained by the following equation: (%tubule or intertubule × testicular parenchyma weight)/100 . ConsideThe tubulesomatic (TSI) and epithelium somatic (ESI) indexes were also estimated using the following equations, respectively: TSI = STV/BW × 100, in which STV is the seminiferous tubule volume and BW is the body weight , and ESIThe seminiferous tubule diameter (STD) was considered the mean of 20 random circular seminiferous tubule cross sections from each animal regardless the tubule stage because the tubular diameter does not change in adult male mice throughout the seminiferous epithelium cycle . These tπR2, in which R is the tubule radius. The lumen area (LAr) was calculated by the following equation: LAr = LR2, where LR is the luminal radius. The epithelium area (EAr) was obtained by subtracting STAr from LAr. The area results were expressed as square millimeter (mm2). The tubule epithelium ratio (TER) was obtained by dividing the STAr by the EAr [The seminiferous tubule area (STAr) was determined using the following equation: STAr =  the EAr .πR2  [The seminiferous tubule length (STL) was estimated using the following equation: STL = STV/meter/2) . AfterwaThe proportion among the intertubular components  was calculated after counting 1,000 intersection points, per animal, over the intertubular compartment. The following equation was used to calculate the percentage of such components: the percentage of each intertubular component = number of points on the component × 100/1000 . The following equation was applied to calculate the volume (mL) of each component: (percentage of the element in the testes × testicular parenchyma weight)/100 .πR3), R = nuclear diameter/2, and CV = %cytoplasm × NV/%nucleus, respectively) were calculated, followed by the calculation of the single Leydig cell volume  [The diameter of the Leydig cell nucleus was considered the mean of 30 nuclear diameters per animal (400x magnification), which had characteristic perinuclear chromatin and evident nucleoli. Nuclear (NV) and cytoplasmic volumes (CV) (NV = 4/3( in μm3) .The volume occupied by the Leydig cells in the testis was determined as follows: percentage of Leydig cells in the testicular parenchyma × parenchyma weight of the testes/100. Afterward, the volume occupied by Leydig cells in the testis was divided by the testicular weight to obtain the volume that the Leydig cells occupy per gram of testis .μm3)/Leydig cell individual volume (μm3). The total number of Leydig cells per gram of testis was calculated using the following equation: volume that the Leydig cell occupies per gram of testis (μm3)/volume of one Leydig cell (μm3). The Leydigsomatic index (LSI) was calculated using the following equation: LSI = total volume of Leydig cell in the testicular parenchyma/BW × 100, in which BW is the body weight [The total number of Leydig cells in the testes was determined using the following equation: volume occupied by Leydig cells in the testicular parenchyma .p ≤ 0.05. Data were expressed as mean ± standard deviation of the mean (SEM).The percentage values were converted into sine arc to increase the statistical analysis accuracy . Normal p ≤ 0.05). The concentration of manganese (Mn) and zinc (Zn) decreased in animals that received Cd i.p. SD in comparison to the control and oral FD groups, similar to the oral SD (p ≤ 0.05).The concentration of testicular Cd increased in all routes of administration of the metal compared to the control, mainly in the oral FD, while Ca and Cu decreased in all groups exposed to Cd . However, the activity of this enzyme did not differ between the groups that received Cd. MDA and NO levels were not altered by Cd exposure (p > 0.05).SOD activity decreased in the animals that received oral Cd in both routes of administration. CAT activity increased in relation to the control group only in the animals that received Cd p ≤ 0.05). There were no changes either in body weight or in gonadosomatic and parenchymosomatic indexes in all experimental groups (p > 0.05).Testicular and parenchyma weights decreased in animals that received oral Cd in both routes of administration (p ≤ 0.05).The changes in tissue architecture observed in animals exposed to Cd include degeneration of the seminiferous tubules with absence of germ cells and generalized vacuolization of the seminiferous epithelium . Therefop ≤ 0.05).Animals exposed to Cd showed increased proportions of cells with initial damage, evidenced by the overlap of acridine orange and propidium iodide (merge), and positive propidium iodide cells . Howeveri.p. SD (Tables p ≤ 0.05). However, the lumen volume decreased in animals treated with Cd oral SD compared to the control group but was similar to Cd oral FD (p ≤ 0.05). The percentage of the epithelium decreased in animals exposed to Cd i.p. SD (p ≤ 0.05). However, the epithelium volume was reduced only in animals exposed to Cd oral FD compared to the control group, similar to the groups exposed to Cd. The epithelium height decreased in the Cd oral FD group in relation to the control and Cd oral SD groups (p ≤ 0.05).The percentage and volume of the lumen increased in the animals that received Cd p ≤ 0.05). The tubule epithelium ratio increased in animals exposed to Cd oral FD in relation to the other experimental groups (p ≤ 0.05). The other tubular morphometric parameters did not change after Cd exposure in both routes (p > 0.05).The volume of seminiferous tubules was reduced in animals in which Cd was administered orally in both single and fractionated doses (i.p. SD (p ≤ 0.05). The percentage of connective tissue was higher in animals exposed to Cd oral SD, while their volume increased in these same animals, compared to the control and Cd oral FD groups (p ≤ 0.05). The percentage of macrophages increased in all routes of Cd exposure, while the volume increased in both forms of oral administration (p ≤ 0.05). The blood vessel volume decreased when Cd was offered orally SD compared to the control animals and those that received Cd i.p. SD (p ≤ 0.05), but it was similar to the oral FD group (p > 0.05). The percentage of the Leydig cytoplasm increased in animals that received Cd i.p. in comparison to the other experimental groups (p ≤ 0.05). The percentage of the Leydig nucleus was higher in the animals that received Cd orally SD when compared to the control (p ≤ 0.05). The Leydig cytoplasm volume decreased when Cd was administered orally FD compared to the control and Cd i.p. groups (p ≤ 0.05). However, the administration of Cd via i.p. increased the cytoplasmic volume of Leydig cells (p ≤ 0.05) in relation to the other experimental groups.The intertubular volume decreased in the animals that received Cd oral FD compared to Cd p > 0.05).There were no changes in the stereological parameters of Leydig cells, such as nuclear diameter, cytoplasmic and nuclear volumes, and cell numbers in animals exposed to Cd (Supplementary p ≤ 0.05). Animals receiving Cd i.p. did not show any change in serum testosterone concentration (Supplementary p > 0.05).The concentration of serum testosterone decreased in animals exposed to oral Cd  in animals receiving Cd oral route fractions, may indicate loss of the germinative epithelium , 7. SuchConsidering the oral FD route, the greater intensity of changes in tubular morphometry may be related to the higher Cd accumulation and not to oxidative stress. Dodson et al.  challeng2i.p. daily, for 15 and 30 days, showed greater alteration in testicular histology compared to those exposed to 2 mg/kg i.p. in a single dose and evaluated after 15 and 30 days of exposure. Cupertino et al. [2, in a single dose i.p., in addition to tubular obstruction in those receiving at least 1.4 mg/kg. These same authors observed increased testicular Cd concentration in all animals exposed to the metal. However, unlike the present study, the authors reported higher germinative epithelium loss, especially in animals that received 1.8 and 2.2 mg/kg of CdCl2, to increased testicular concentration of Ca and consequent dystrophic calcification. Predes et al. [2i.p. and evaluated after 7 days. The authors observed reduced tubular diameter after 56 days of metal exposure, which gives evidence of the progression of tubular alterations caused by Cd, and spermatogenesis impairment.The results found by Sharma and Kaur  corroboro et al.  reporteds et al.  also obsi.p. did not lead to changes in the other tubular parameters. Since proportion is a spot analysis and the values obtained are used to determine the global parameters of the organ, it is concluded that the i.p. route did not cause the same intensity of toxicity shown by the oral route in the tubular parameters.The increased percentage and volume of the lumen associated with reduced percentage of the epithelium observed in animals that received Cd The increased percentage of connective tissue was significant only in animals that received Cd oral SD, although its proportion was also higher in other Cd-administrated groups. It is known that the presence of macrophages induces the synthesis of collagen via fibroblasts . Thus, ti.p. SD administration, may be due to the onset of the necrotic cell death process. According to Majno and Joris [i.p. route. Such loss may have increased the intensity of the tubular changes found in the oral route.The increased percentage of the Leydig cell nucleus in the oral SD route, along with the increased cytoplasmic percentage and cytoplasmic and cell volumes in nd Joris , cells iβ-thalassemia in order to maintain homeostasis due to Fe overload [β-cells after As exposure [The oral FD route presented greater intensity of testicular morphometric changes and lower oxidative stress. The lower oxidative stress may have occurred due to the adaptation to ROS generation caused by continuous exposure to Cd. This adaptive mechanism is reported in patients with overload , in pancexposure , and in exposure . Thus, ii.p. route reduces Mn and Zn concentration. SOD activity decreases only when the metal is administered orally. Changes in the tubular parameters and cell viability are observed in both routes, but it is more intense in the oral route, mainly when fractionated. Serum testosterone concentration decreases when Cd is given orally. The lower serum testosterone concentration may be related to the decreased testicular and parenchyma weights and increased intensity of morphological damage in the testis of animals receiving oral Cd, mainly in the fractioned dose. Thus, the oral administration of Cd showed greater testicular damage potential, mainly when the metal is administered in a fractionated dose.The subacute exposure leads to the bioaccumulation of this metal, regardless of the route of administration, although it is more significant in the oral fractionated route. The route of administration does not affect the reduction of testicular Ca and Cu, whereas the


Phaseolus vulgaris L.) are, however poor, limited by low soil phosphorus (P), nitrogen (N) and poor biological N2-fixation. On-farm experiments were carried out to study the effect of N, P and rhizobia inoculation on common bean yield and yield components during the 2014/2015 and 2015/2016 cropping seasons in Eastern Zimbabwe. Experiments were conducted on five farmers’ fields located in two agroecologies; three fields were considered to be degraded with soil organic carbon (SOC) < 4 g kg−1 and available P < 6 mg kg−1, while the two non–degraded sites had SOC > 7 g kg−1 and available P > 15 mg kg−1. Two common bean varieties (Gloria and NUA45) were tested in a split-plot arranged in randomized complete block design. The main plot factor was the combination of N (0 and 40 kg ha−1) and P (0 and 20 kg ha−1), and the sub-plot factors were variety (Gloria and NUA 45) and inoculation with Rhizobium tropici strain CIAT899 (+/− inoculum). At planting, both N and P were applied at 20 kg ha−1, with an additional 20 kg ha−1 N top dressing applied at flowering. Analysis of variance indicated common bean did not respond to rhizobia inoculation (P > 0.05) whilst P significantly increased the number of nodules and active nodules per plant (P < 0.001), and grain yield. Application of 40 kg ha−1 N significantly increased the number of pods per plant, number of seeds per pod, and grain yields. A significant NP interaction was only observed on grain yield for non-degraded soils. Co-application of N and P in non-degraded sites increased grain yields from 0.27 to 1.48 Mg ha−1during the first season and from 0.37 to 2.09 Mg ha−1during the second season. On degraded sites, NP application resulted in uninspiring grain yield gains of 0.09 to 0.19 Mg ha−1 during the first season, and from 0.16 to 0.28 Mg ha−1 in the second season. In general, effects of N or P were not significantly different, suggesting that farmers could invest in either of these nutrients for increased common bean grain yields. Strategically, P investments would be more logical as residual P effects to rotational cereals improve overall cropping system performance. The response of common bean to inoculation in Zimbabwe still needs to be widely investigated for these and other varieties.Common bean is an important crop with potential to curb malnutrition in poor Sub-Saharan African populations. Yields of common bean ( N2-fixabacteria . Althoug legumes . Inocula legumes . Plant a of pods . Nodule fixation . Nitrogefixation , with GiWhile most grain legumes only require ‘starter’ N to initiate early growth before the N2-fixing symbiosis is established, in Zimbabwe, market-oriented farmers on sandy soils often apply additional top-dressing N fertilizer for high yields. In Brazil, In this study, our intention was to build on the existing knowledge from other studies on common bean nutrition. Our objectives were to: (1) investigate the effect of N, P and rhizobia inoculants on productivity of common beans across different soils and in two agro-ecological zones where common bean is typically grown on smallholder farms, and (2) provide an alternative fertilization strategy for common bean in the context of common crop sequences.22.1−1, and those that had SOC > 7 mg kg−1. We subsequently used this information to narrow our field selection to the five field sites we used  and Murehwa  in Eastern Zimbabwe, during the 2014/15 and the 2015/2016 cropping seasons. We intended to set up experiments on at least two farms, with soils representative of degraded and non-degraded soil fertility conditions in each of the two farming areas. We therefore initially took soil samples and analysed for SOC content for 20 field sites in each of the farming communities. Soil organic carbon is known to be a robust index for soil health e.g. . Prospec we used .Table 1P−1, while Murehwa is located in a relatively drier NR IIb, receiving about 750 mm year−1 annually. Rainfall is unimodal, and is received from November to April. Zimbabwe is divided into five natural agro-ecological regions (NR I-V) with NR I having the most favourable conditions, receiving >1000 mm rainfall while semi-arid NR V receives annual average rainfall of <500 mm  that receives reliable rainfall averaging 900 mm year <500 mm . Rain-feBefore the cropping season, composite soil samples consisting of five sub-samples were collected from the plough layer (0–20 cm depth) along the fields’ diagonal and bulked for each of the five sites. The soil samples were air-dried, and those for total N, available P, extractable bases analysis were passed through 2 mm sieve, while those for SOC analysis were passed through a 0.5 mm sieve. Total SOC was determined by the modified Walkley-Black) , while t2.2i)Control (no fertilizer or rhizobia added),ii)NP + I [ammonium nitrate (34.5% N) + single super phosphate + inoculation],iii)NP (ammonium nitrate + single super phosphate),iv)N + I (ammonium nitrate + inoculation),v)P + I (single super phosphate + inoculation),vi)N (ammonium nitrate only),vii)P (single super phosphate only), andviii)+I (inoculation only).Experimental treatments were designed to explore the interaction of nitrogen (N), phosphorus (P) and rhizobia inoculation (+I) on two common bean varieties (Gloria and NUA45) using site as a random factor. In each field, the experiments were laid out in a split plot arranged in randomized complete block design replicated in three blocks. Main plots were nested within the blocks and sub-plots were nested within the main plots. The main plot treatments were fertilizer management , and subplots were randomly assigned to +/- inoculation and variety, resulting in the following treatments:Rhizobium tropici strain CIAT899) obtained from a local commercial manufacturer  was used to inoculate the bean seed prior to sowing, using a rate of 100 g inoculum per 25 kg of seed as recommended by the manufacturer. Phosphorus was applied at 20 kg ha−1 P at planting, while N was applied in two splits, 20 kg ha−1 at planting plus an additional 20 kg ha−1 N applied at flowering stage, for a total N rate of 40 kg ha−1. All treatments that had P applied also received 12 kg ha−1 sulphur since single super phosphate also contains 5%. Sulphur is generally deficient on these sandy soils.Two improved varieties, Gloria and NUA 45 that are both maintained by Zimbabwe Crop Breeding Institute and locally available on the market in Zimbabwe, were used. The varieties are both determinate and take approximately 90–100 days to reach physiological maturity. A rhizobia inoculum  were cut at soil level and heaped at the centre of the plot. Random sampling of 10 plants was done and all the pods on each plant were counted and recorded to determine the number of pod per plant. A sub-sample of the pods was used to determine the number of seeds per pod. Later, all pods from the net plot were harvested into perforated harvest bags, sun-dried for 14 days and threshed. The grain was then weighed and grain moisture content determined using a John Deere SW moisture meter. Yields reported here are adjusted to 10% moisture content.2.5The number of nodules and active nodules per plant, number of pods per plant and number of seeds per pod were transformed using quantile normalization and subjected to analysis of variance (ANOVA) using R Version i3863.32. 33.1All sites had low clay content, ranging from 8–12%,while two sites that had poor SOC content of less than 0.4% were concomitantly acidic (pH < 5.5) and acutely deficient in available P . These i3.2−1 P was added (p = 0.006). The number of nodules per plant increased from three in the control to eight in the P treatment  and the second season (p = 0.003). Increases in pod number from four in the control to eight in the N only treatment were observed in the first season, and eight to 11 during the second season , while the potential benefits of fertilizing non-degraded soils were 1.5 and 1.93 Mg ha−1 (B1 and B2)  . Inocula44.1While common bean originated from regions with moderately fertile soils, globally, the cultivation of common bean by smallholder farmers on degraded soils has contributed to poor productivity . In manyR. Tropici gave no positive response. Inoculating common bean with rhizobia gave no significant increase in nodulation, biomass or grain production . RebeschPhosphorus fertilization significantly increased nodulation and number of active nodules, but only for non-degraded soils . Strong 4.2Application of N or P had comparable effects on common bean productivity, with no clear benefits of co-application of these nutrients in most of the cases . Market–5Application of N or P had equal magnitude of increasing common bean grain yields with no significant benefits of adding both elements. This result is important as farmers in the study area regularly invest more in N fertilizers for common bean than in P. With well documented P residual benefits to crops in rotations, direct P fertilization to common bean is expected to improve cropping system performance. This study also established that the improved common bean varieties that are currently on the market did not respond to rhizobia inoculum currently marketed in Zimbabwe. We also confirmed the existence of degraded non-responsive soils. While some ‘wonder’ legumes such as cowpea can be successfully grown on infertile soils, attempts to grow common bean on such soils results in very low yields.


P< 0.0001 and P = 0.001, respectively). Results were confirmed when adjusting for propensity score. Among the 216 hormone‐receptor positive patients eligible for BM, a more favorable PFS and OS were observed when maintenance ET was administered (P < 0.0001). Multivariate analysis showed that PS, BM, number of disease sites and maintenance ET were related to PFS, while response and maintenance ET were related to OS. In hormone‐receptor positive patients, BM produced a significant PFS and a trend towards OS benefit only in absence of maintenance ET . In the triple‐negative subgroup, we observed a trend towards a better OS for patients who received BM (P = 0.06), without differences in PFS (P = 0.21). Our results confirmed the efficacy of first‐line paclitaxel‐bevacizumab in real‐world practice; both BM and maintenance ET significantly improved PFS and OS compared to no maintenance therapies. J. Cell. Physiol. 232: 1571–1578, 2017. © 2016 Wiley Periodicals, Inc.Bevacizumab in combination with taxanes in HER2‐negative metastatic breast cancer (MBC) patients has shown improved progression‐free survival (PFS), despite the lack of clear overall survival (OS) benefit. We performed a retrospective analysis to evaluate the impact of paclitaxel‐bevacizumab and of maintenance therapy with bevacizumab (BM) and endocrine therapy (ET) in the real‐world practice. We identified 314 HER2‐negative MBC patients treated in 12 cancer centers. Overall, the median PFS and OS were 14 and 40 months, respectively. Among the 254 patients potentially eligible for BM, 183 received BM after paclitaxel discontinuation until progression/toxicity. PFS and OS were improved in patients who had received BM in comparison with those potentially eligible but who did not receive BM ( Metastatic breast cancer (MBC) is still considered incurable, with a median survival of 2–4 years  regulates tumor angiogenesis by stimulating endothelial cell proliferation and migration, inhibiting apoptosis, remodelling extracellular matrix, and increasing vascular permeability. Bevacizumab is a humanized monoclonal antibody directed against VEGF‐A. It prevents binding of VEGF to receptors on endothelial cells, leading to inhibition of angiogenesis and tumor growth, promoting blood vessels degradation, potentiating the effect of chemotherapy , alone or in combination with maintenance ET in the subset of patients with hormone‐receptor positive tumors.2, plus bevacizumab, 10 mg/kg every 2 weeks or 15 mg/kg every 3 weeks (depending on the physician preference), with or without BM after paclitaxel discontinuation. Treatment was continued until disease progression, unacceptable toxicity, or refusal. Endocrine sequential maintenance treatment was given based on physician choice and according to standard guidelines after paclitaxel discontinuation in patients with hormone‐receptor positive tumors.HER2‐negative MBC patients not enrolled in clinical trials were retrospectively and sequentially identified and recruited from 12 Italian cancer centers. Treatment schedules were paclitaxel administered weekly, at the dose of 80 or 90 mg/mTreatment efficacy was evaluated by conventional Response Evaluation Criteria in Solid Tumors (RECIST) criteria. All the patients signed a written informed consent, and the institutional ethic committees approved the retrospective analysis.Descriptive statistics were used to summarize patient characteristics.The associations between variables were tested by chi–square or Fisher's exact test. Survival estimates were computed and compared by Kaplan–Meier product‐limit and log–rank test. The median follow‐up was estimated with the Kaplan–Meier reverse method.To minimize the differences in patients’ covariates, which could become confounding factors in the examination of treatment effects in a non‐randomized cohort, we used the propensity score match to create groups of patients who were similarly likely to receive a given treatment on the basis of their baseline characteristics . Variables testing significant at the univariate analysis were entered into the model, which also included interaction terms. Enter limit and remove limit were When considering BM, we included in the analysis only patients who had received adequate paclitaxel treatment and who had not experienced disease progression. To determine the role of maintenance ET, we comprised patients with hormone‐receptor positive tumors only. SPSS software  was used for all statistical evaluations.From January 2008 to February 2015, 314 MBC patients were retrospectively identified and enrolled at 12 Italian cancer centres. Included patients had received at least one paclitaxel‐bevacizumab cycle. Main patient and tumor characteristics are listed in Table Overall, 24.8% of patients had received neoadjuvant chemotherapy and 57.6% adjuvant chemotherapy. Two hundred and eleven patients had received adjuvant ET . The remaining 8% of hormone‐receptor positive patients had not received adjuvant ET due to physician decision.Among the 254 patients with hormone‐receptor positive tumors, 39% had received one or more ET for advanced disease , prior to first‐line paclitaxel‐bevacizumab.Overall, the median duration of treatment with paclitaxel and BM was 8 months . After paclitaxel discontinuation, BM was not administered to 60 patients because of disease progression during (27 patients) or at the end of chemotherapy (25 patients), or due to toxicity (8 patients). These patients were not included in the prognostic factors analysis. Further 71 patients did not receive BM due to physician or patient decision, even in absence of progression or toxicity. Overall, 183 patients (58.3%) received BM after paclitaxel discontinuation. Main patient and tumor characteristics according to BM administration are listed in Supplementary Table S1. Among the 254 patients with hormone‐receptor positive tumors, 70.5% received ET as maintenance treatment after paclitaxel discontinuation, which was combined with BM in 51.2% of patients, and administered without BM in 19.3% of patients (Supplementary Table S2).P = 0.13). Disease control rate, defined as CR, PR, and SD lasting ≥6 months, was recorded in 87.3% of patients, with significant differences between hormone‐receptor positive and triple‐negative tumors   (9.9%) and 177 partial responses (PR) (56.4%), for an overall RR of 66.2% . Stable disease (SD) was observed in 80 patients (25.5%). No significant differences in RR were observed by molecular subtype . The median OS was 25 months  in the triple‐negative subgroup, while it was 41 months  in patients with hormone‐receptor positive tumors (P = 0.009). Among the 179 (70.5%) hormone‐receptor positive patients who received maintenance ET, median PFS and median OS were 18.5  and 59  months, respectively. Conversely, among the 75 (29.5%) hormone‐receptor positive patients who did not received maintenance ET, median PFS and median OS were 8.1  and 25  months, respectively. Therefore, maintenance ET after paclitaxel‐bevacizumab offered a significant benefit both in PFS and OS (P < 0.0001).In the overall patient population, at a median follow up of 27 months , median PFS was 14 months , and median OS was 40 months   , respectively  compared with 13  months for patients who did not receive BM . Maintenance ET offered a significant benefit in the median PFS and OS both in ET naive patients and in those who had previously received ET.Figure P = 0.0007). These results were confirmed also in patients with an adequate chemotherapy length (at least 18 paclitaxel administration). Conversely, in patients having received maintenance ET, the administration of BM was associated with a trend in median PFS benefit . However, in patients treated with BM for more than 2 months, the advantage of adding BM became significant, even in presence of maintenance ET (P = 0.04). In patients with an adequate length of chemotherapy and maintenance ET, the advantage in the median PFS of adding BM was lost (P = 0.20), even if the duration of BM was adequate.When exploring BM in hormone‐receptor positive patients, at the multivariate analysis, PS, BM, number of disease sites and maintenance ET were related to PFS, while treatment response and maintenance ET were associated with OS Table . ResultsP = 0.06). Conversely, no advantage from BM was observed in patients having received maintenance ET (P = 0.89).In the subset of hormone‐receptor positive patients not receiving maintenance ET, the administration of BM prolonged median OS from 22 to 28 months (P = 0.21)   , RR was significantly higher with bevacizumab‐containing therapy than with chemotherapy alone , median OS was 18.9 versus 17.5 months, and 1‐year OS rates were 71% versus 65%. . Median PFS was 8.1 months and 6.4 months in the combination group and chemotherapy alone arm, respectively (HR 0.74). Results were confirmed in subgroup analysis by molecular subtype  and OS  in patients having received BM , and no OS benefit, with an increase in serious adverse event in the experimental arm , and median OS was not reached in the arm with maintenance ET  to BM/taxane regimen was evaluated in a recent meta‐analysis including seven randomized trials and 1,124 HER2‐negative advanced breast cancer patients treated in first‐line setting . However, maintenance ET in the subset of hormone‐receptor positive patients certainly affected OS. Indeed, am ong the hormone‐receptor positive patients who received maintenance ET, the median PFS and OS were significantly more favorable than in patients not treated with maintenance ET (P < 0.0001). Moreover, in our patient population, previous ET in adjuvant/advanced setting did not decrease the efficacy of subsequent maintenance ET.In the present study, our findings on the combination of paclitaxel‐bevacizumab administered as first‐line treatment in real‐world practice fairly compares with the results of the ATHENA trial, showing a RR of 66.2%, a median PFS of 14 months, and a median OS of 40 months, whereas in the ATHENA trial these estimates were 52%, 9.5 and 25 months, respectively  and in median OS , compared with patients with no maintenance ET, independently on the administration of BM.In the subset of 216 hormone‐receptor positive patients eligible for BM, maintenance ET offered a clear advantage both in median PFS , and median OS from 38 to 55 months (P = 0.001), confirming the ATHENA results, where median TTP was prolonged from 9.5 to 11.6 months and median OS rose from 25 to 30 months . These latter data seem to support the need of prolonging BM administration. In patients receiving maintenance ET and having received an adequate chemotherapy duration, no advantage in PFS from BM was observed (P = 0.20), but numbers are small.In the present study, overall, the administration of BM prolonged median PFS from 13 to 18 months (P = 0.06), and this trend disappeared when concomitant maintenance ET was delivered (P = 0.89).In the hormone‐receptor positive subgroup the administration of BM, in absence of maintenance ET, prolonged OS , while a trend toward OS benefit was observed (P = 0.06). A possible explanation deriving from our data may be that the prolonged administration of BM allowed an increased number of post‐progression treatment lines, and possibly favored their efficacy due to the sustained anti‐angiogenetic effect.In the small subset of triple‐negative MBC patients, median PFS and OS were 9 and 25 months, respectively, comparing favorably with the ATHENA results where these estimates were 7.2 and 18 months (Thomssen et al., Our results are consistent with previous observational studies, and confirmed the advantage of adding BM to a first‐line chemotherapy with paclitaxel‐bevacizumab in HER2‐negative MBC patients (Smith et al., Results from our subset of triple‐negative patients are intriguing, given the suggestion for longer OS in patients under BM, even if there was no impact on PFS. Although our numbers invite caution, the prolonged administration of BM may have favored post‐progression treatments and impacted OS.Our results confirmed the relevance of adding maintenance ET in hormone‐receptor positive patients. Such relevance seems to exceed the favorable effect of BM in this subgroup of patients.The present work has important limitations, which mainly stem from its retrospective observational nature which makes it prone to confounding and bias. Moreover, our patient population is heterogeneous, several doses and schedules of treatment were used, and the sample size is relatively small when considering subgroups. The existence of possible major differences between patients receiving maintenance therapies versus remaining patients must be taken into account. In our study, the two groups analyzed showed significant differences in terms of PS. To minimize the PS‐related selection bias, survival analysis were adjusted using propensity score. However, case matching by PS could not remove other potentially important causes of bias from unknown confounders possibly including prior/subsequent therapies, comorbidities, and differences in disease biology. In these regards, it is worth mentioning that data concerning co‐morbidities and safety were not available for analysis purposes. However, our study also has important strengths. Among them, the suggestion of the efficacy of both BM and maintenance ET, and the statistical methodology applied, with the use of propensity score to reduce the selection bias. Moreover, data deriving from real‐world studies provide a realistic picture of what really takes place outside clinical trials.In conclusion, our results confirm that paclitaxel‐BM represent one of among the effective therapeutic strategies in real‐world patients with HER2‐negative MBC. In the absence of disease progression or significant toxicity, the continuation of BM, and the addition of ET in the hormone‐receptor positive subgroup, appear to be a reasonable approach for more favorable outcomes.TG and PV contributed to study conception, critical revision of the manuscript for important contents. PV and LP contributed to manuscript drafting, study conduct, data collection, and analysis planning. PM, MB, MMS, SB, and AG critically revised the manuscript for important intellectual content. IS contributed to the statistical analysis. LM, CN, AC, AM, LDL, DS, AF, MGS, EM, ER, VS, AG, DP, AA, CN, FA, AV, AP, CDA, MP, LM, and IB contributed to the data collection, database set up and implementation, contribution to data analysis. All authors read and approved the final version of this manuscript and are responsible for all the aspects of the work.Additional supporting information may be found in the online version of this article at the publisher's web‐site.Supplementary Table S1.Click here for additional data file.Supplementary Table S2.Click here for additional data file.