First trimester pregnancy outcomes in a large IVF center from the Lombardy County (Italy) during the peak COVID-19 pandemic.

At the beginning of 2020, the Italian Lombardy region was hit by an "epidemic tsunami" which was, at that point in time, one of the worst pandemics ever. At that moment the effects of SARS-COV 2 were still unknown. To evaluate whether the pandemic has influenced ART (Assisted Reproduction Techniques) outcomes in an asymptomatic infertile population treated at one of the major COVID-19 epicentres during the weeks immediately preceding lockdown. All ART procedures performed during two time periods were compared: November 1st, 2018 to February 28th, 2019 (non-COVID-19 risk) and November 1st, 2019 to February 29th, 2020 (COVID-19 risk). In total 1749 fresh cycles (883 non-COVID-19 risk and 866 COVID-19 risk) and1166 embryos and 63 oocytes warming cycles (538 and 37 during non-COVID and 628 and 26 during COVID-19 risk, respectively) were analysed. Clinical pregnancies per cycle were not different: 370 (25.38%) in non-COVID versus 415 (27.30%) (p = 0.237) during COVID-19 risk. There were no differences in biochemical pregnancy rates 52 (3.57%) versus 38 (2.50%) (p = 0.089) nor in ectopic pregnancies 4 (1.08%) versus 3 (0.72%) (p = 0.594), spontaneous miscarriages 84 (22.70%) versus 103 (24.82%) p = 0.487, nor in intrauterine ongoing pregnancies 282 (76.22%) versus 309 (74.46%) p = 0.569. A multivariate analysis investigating differences in spontaneous miscarriage rate showed no differences between the two timeframes. Our results support no differences in asymptomatic infertile couples' ART outcomes between the pre COVID and COVID-19 periods in one of the earliest and most severe pandemic areas.

One step further: randomised single-centre trial comparing the direct and afterload techniques of embryo transfer.

STUDY QUESTION: What are the differences in ease of use between two different embryo transfer (ET) techniques: the preload direct approach and the afterload approach. SUMMARY ANSWER: The afterload technique seems to reduce the rate of difficult ETs. WHAT IS KNOWN ALREADY: Numerous published trials now document that the ET procedure has an impact on pregnancy and delivery rates after IVF. Difficult transfers should be avoided, as they reduce implantation and pregnancy rates. Preload direct ETs with soft catheters under ultrasound guidance is currently considered the best procedure. However, when using soft catheters, it is not known which technique is preferable or which one should be implemented to reduce the operator factor. STUDY DESIGN, SIZE, DURATION: This prospective randomised unblinded controlled clinical trial, included 352 ultrasound-guided ETs assigned to either direct ET or afterload ET, between September 2017 and March 2019. The sample size was calculated based on the historical rate of difficult ETs encountered between 2014 and 2015 with a direct ET procedure. PARTICIPANTS/MATERIALS, SETTING, METHODS: The inclusion criteria were women 18-38 years old, with BMI between 18 and 28, receiving a single-thawed blastocyst transfer. The exclusion criteria were use of testicular sperm and preimplantation genetic testing (PGT) cycles. The primary outcome was the rate of difficult or suboptimal transfers defined as: advancement of the outer sheath (specific for the direct transfer), multiple attempts, use of force, required manipulation, use of a stylet or tenaculum, dilatation, or use of a different catheter. The secondary outcome was clinical pregnancy rate. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 352 frozen ETs were randomised, with 176 patients in each group. The two arms were homogeneous for female and male age, female BMI, duration of infertility, secondary infertility, previous deliveries or miscarriages, myomas, previous surgery to the uterine cavity, cycle day at ovulation trigger, freeze all cycles, first transfers, indication for treatment, endometrial preparation protocol and duration, endometrial thickness, and blastocyst grade at vitrification. Across the entire population, 85 (24.1%) ETs were defined as difficult. The rate of difficult transfers was significantly higher in the direct ET group than in the afterload group: 68 (38.6%) versus 17 (9.7%), respectively (OR 0.17, 95% CI 0.09-0.30, P < 0.001). The mean percentage in the rate of difficult transfers per operator was 22.5% (SD ± 14.5%), of which 36.1% (SD ± 23.4%) were in the direct group compared with 8.6% (± 8.2%) in the afterload group (P < 0.001). The difficult transfer rate among operators varied from 0 to 43.8% (0-77.8% in the direct group and 0 to 25.0% in the afterload group). The clinical pregnancy rates (42.0% vs 48.3%, P = 0.239 in the direct and afterload groups, respectively) were not significantly different between the groups. LIMITATIONS, REASONS FOR CAUTION: There were 18 experienced operators who participated in the trial. Conclusions about the pregnancy rate should not be generalised, since the sample analysis was not performed on this outcome and, although clinically relevant, the difference was not significantly different. WIDER IMPLICATIONS OF THE FINDINGS: The rate of difficult transfers was significantly higher in the direct ET group compared with the afterload ET group, although a wide variation was observed among operators. Further studies regarding the association between transfer technique and ART outcomes are required. STUDY FUNDING/COMPETING INTEREST(S): No specific funding was sought and there are no competing interests. TRIAL REGISTRATION NUMBER: NCT03161119. TRIAL REGISTRATION DATE: 5 April 2017. DATE OF FIRST PATIENT'S ENROLMENT: 26 September 2017.

The human factor: does the operator performing the embryo transfer significantly impact the cycle outcome?

STUDY QUESTION: Is Ongoing Pregnancy Rate (OPR) operator-dependent, and can experience improve embryo transfer efficiency? SUMMARY ANSWER: OPR is influenced by the operators who perform the embryo transfer (ET), and experience does not assure proficiency for everyone. WHAT IS KNOWN ALREADY: ET remains the critical step in assisted reproduction. Although many other factors such as embryo quality and uterine receptivity impact embryo implantation, the proper ET technique is clearly an operator-dependent variable and as such it should be objectively standardized. STUDY DESIGN, SIZE, DURATION: Retrospective comparative analysis including all fresh ETs performed between January 1996 and December 2016 at the Humanitas Fertility Center after IVF-ICSI cycles. PARTICIPANTS/MATERIALS, SETTING, METHODS: IVF/ICSI fresh ETs performed by 32 operators, 19 824 cycles in all, were analyzed. All transfers consisting of freehand insertion of a preloaded soft catheter into the uterine cavity under transabdominal ultrasound guidance were considered. Two different statistical analyses were performed. First, a logistic regression model with a random intercept for the operator was used to estimate the heterogeneity of the rate of success among operators, accounting for woman age, FSH, number of oocytes retrieved, fertilization rate, year of the procedure, number and stage of transferred embryos and operator's experience. Second, the relationship between experience and pregnancy rate was estimated separately for each operator by logistic regression, and operator-specific results were combined and compared in a random-effects meta-analysis. In both analyses, the operator's experience at time t was measured in terms of number of embryo transfers performed before t. MAIN RESULTS AND THE ROLE OF CHANCE: The heterogeneity among operators was highly significant (P value <0.001) and explained 44.5% of the total variability. The odds ratio of success of the worst operator in respect to the mean was equal to 0.84. For the best operator, the odds ratio of success was equal to 1.13 in respect to the mean. Based on the meta-analysis of the relationship between operator's experience and success rate, it resulted that, on average, the operators' performance did not improve with additional transfers. LIMITATIONS, REASONS FOR CAUTION: At our center, operators become independent for ET's after performing between 30 and 50 transfers under supervision. It is also possible that other relevant factors, such as embryologists on duty for the ET, have not been included in the present analysis and this may represent a potential bias. Among these, it should be mentioned that the embryologists on duty for the ET were not taken into consideration. WIDER IMPLICATIONS OF THE FINDINGS: Continued performance analysis and the use of a digital simulator could help operators to test their expertise over time and either correct poor performance or avoid doing transfers. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: NCT03561129.

Necessity of therapy for post-thyroidectomy hypocalcaemia: a multi-centre experience.

PURPOSE: Hypoparathyroidism is one of the most common and most feared complications of total thyroidectomy (TT). The aim of this study is to detect possible markers that may facilitate early tracing of hypocalcaemia-prone patients in order to reduce clinical cost by optimizing patient discharge and to avoid unnecessary treatment. METHODS: Over an 18-month period, 995 patients, 23 % male and 77 % female, aged 52.9 ± 13.4 years, underwent TT in ten Lombardy hospitals. The following parameters were analyzed: calcaemia before and 12-24 and 48 h after surgery, pre- and post-operative parathyroid hormone (PTH) at 24 h and pre-operative 25OH vitamin D. RESULTS: Mortality was nil and morbidity was 22.4 %. Mean 24-h calcaemia and PTH were 2.17 ± 0.15 mmol/l and 31.81 ± 20.35 pg/ml, respectively; mean 24-h PTH decay was 36.7 ± 34.12 %. Four hundred seventy-three (47.5 %) patients were hypocalcaemic at discharge; 142 of whom had transient hypoparathyroidism that became permanent in 27. Patients developing hypocalcaemia had significantly higher values of PTH and calcium decay. At multiple logistic regression, only 24-h calcium decay, PTH drop and the presence of symptoms and parathyroid auto-grafting were significantly related to hypoparathyroidism. The association of these factors had a 99.2 % negative predictive value (NPV) for the development of hypoparathyroidism. A 70 % PTH drop had a 93.75 NPV for transient hypoparathyroidism. A 12 % calcaemia decay had a 95.7 NPV for hypoparathyroidism. CONCLUSIONS: Hypocalcaemic asymptomatic patients with less than 70 % PTH and 12 % calcaemia decay may be safely discharged without treatment. Symptomatic patients and those with parathyroid grafting should receive calcium and vitamin D.

Potential impact of the 70-gene signature in the choice of adjuvant systemic treatment for ER positive, HER2 negative tumors: a single institution experience.

PURPOSE: We investigated in a single institution series of 124 women with operable breast cancer whether tumor clinicopathological features could predict the 70-gene signature (Mammaprint, MP) results, and whether MP results could help to make decisions for the use of chemotherapy (CT) in patients (pts) with ER positive breast cancer beyond recommendations of international guidelines. RESULTS: Among the 68 ER/PgR positive, HER2 negative tumors, Ki-67 ≥ 20% was the only significant predictor of a high risk-MP among standard clinicopathological features. In candidates for endocrine therapy with undetermined benefit from CT according to international guidelines, MP results would have led to different treatment decisions in 13/46 (28%) and in 20/68 (29%) pts according to NCCN and St. Gallen recommendations, respectively. CONCLUSIONS: Ki-67 independently predicted high risk-MP in ER/PgR positive, HER2 negative tumors. MP results would have led to discordant treatment recommendations in about 30% of cases, generally increasing indication rate for CT. The results of large randomized trials are warranted in order to understand whether we should rely on multigene assays rather than on standard clinicopathological features for treatment decisions.

Preliminary evidence for high anti-PLAC1 antibody levels in infertile patients with repeated unexplained implantation failure.

OBJECTIVE: Placenta-specific1 (PLAC1) is a trophoblast-specific gene encoding for a protein that is highly expressed in human placenta, on the surface of the syncytiotrophoblast. PLAC1 was found to elicit spontaneous antibody responses in cancer patients. We aimed to determine the levels of anti-PLAC1 antibodies in infertile women with a history of unexplained repeated implantation failure after IVF cycles as compared to fertile women. STUDY DESIGN: An observational case-control clinical study. MAIN OUTCOME MEASURE(S): Two groups of patients were analysed in two different experimental settings: 21 infertile women and 81 control patients were enrolled in the first group, 16 infertile women and 67 fertile controls in the second group. Anti-PLAC1 antibody levels and ranking were analysed by ELISA test. RESULTS: In both groups of infertile patients enrolled, optical densities (OD) from ELISA test ranked significantly higher than those of controls (0.27 ± 0.2 vs. 0.13 ± 0.1 respectively; p = 0.0009 in the first group), (0.62 ± 0.38 vs. 0.39 ± 0.35 respectively; p = 0.0044 in the second experiment). In the first group about one case in four (29%) had OD levels above the 95th percentile (0.337) for healthy controls (p = 0.005). In the second experiment 4 out of 16 cases (25%) had OD levels above the 95th percentile (0.878) for healthy controls (p = 0.023). CONCLUSIONS: Anti-PLAC1 antibodies could represent a biomarker associated with infertility and with high probability of repeated implantation failure after ovarian stimulation and IVF-ET, greatly improving the diagnostic work up of infertile couples.

Five years (2004-2009) of a restrictive law-regulating ART in Italy significantly reduced delivery rate: analysis of 10,706 cycles.

STUDY QUESTION: Was the delivery rate of ART cycles negatively affected by the enactment of the Law 40/2004 by the Italian Parliament which imposed a long list of restrictions for ART procedures? SUMMARY ANSWER: This large and extensive comparative analysis of ART outcomes prior to and after the introduction of the Law 40 revealed a significant reduction in pregnancy and delivery rates per cycle, independent of age or other clinical variables, once the law went into effect. WHAT IS KNOWN ALREADY: Several studies have been published on the effect of Law 40/2004 on ART outcomes, some authors demonstrating a negative impact of the Law in relation to specific etiologies of infertility, other authors showing opposite conclusions. STUDY DESIGN, SIZE, DURATION: Retrospective clinical study of 3808 patients treated prior to the enactment of the Law, September 1996-March 2004 (Group I) and 6898 treated during the Law, March 2004-May 2009 (Group II). PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 10 706 ART cycles were analysed, 3808 performed before and 6898 after the application of the Law. An intention-to-treat statistical analysis was performed to detect pregnancy and delivery rates (pregnancies ≥ 24 weeks) per started cycle. A P value of <0.05 was considered statistically significant. We analysed different outcomes: differences in fertilization, pregnancy and delivery rate, multiple pregnancies and miscarriage rates between the two time periods. MAIN RESULTS AND THE ROLE OF CHANCE: The delivery rate for started cycle was 20% before and 16.0% after the introduction of the Law representing a 25% reduction (P < 0.001). The multivariate analysis, corrected by female age of >38 years, duration of infertility, basal FSH level and number of retrieved oocytes, showed a 16% lower delivery rate (odds ratio: 0.84; confidence interval: 0.75-0.94). This statistical approach removed the risk that the observed effects were due to chance and confirmed unequivocally that the Law was an independent factor responsible for the reduced likelihood of a successful outcome. LIMITATIONS, REASONS FOR CAUTION: This is a retrospective study. A prospective randomized study, with patients treated in the same time period and randomized to restrictions or not, would have minimized potential limitations due to differences in years of treatments. WIDER IMPLICATIONS OF THE FINDINGS: Our findings based on the analysis of such a large number of cycles proved clearly and unequivocally that imposing restrictions on the practice of ART penalized patients. These data represent a relevant clinical contribution for countries still debating the enactment of restrictive limitations of ART.

Traumatic brain injury in the elderly: considerations in a series of 103 patients older than 70.

AIM: Mortality and morbidity due to brain injury in the elderly population is a growing clinical problem: among older patients, those >70 years have a considerably higher risk both in terms of mortality and morbidity. Thereafter, the reasons influencing outcome have not been clearly examined: in the present study we addressed these questions considering the main clinical characteristics exerting a significant impact on the outcome of patients aged > 70, with emphasis for the severity of brain injury and anticoagulant (CAW) treatments. METHODS: We performed a retrospective analysis of 103 consecutive isolated head injury patients older than 70, admitted at our Department in the period November 2004-November 2009. The clinical variables considered were as follow: age, sex, type of TBI, GCS, pre-TBI use of anti-coagulants (aspirin, warfarin, clopidogrel), INR at admission (INR values were subdivided in values >1.25 as at risk for hemorrhagic events and <1.25 as normal), initial CT scan classification looking at the presence of subarachnoid hemorrhage (t-SAH) or mass lesions; the main outcome measure was the Glasgow Outcome Scale. RESULTS: The most frequent cause of TBI was accidental fall (65%): 39 were in CAW therapies and in 36 cases the cause of falling down injury was recorded due to a sincopal event (arterial hypotension, atrial fibrillation); in the older patients an accidental fall is significantly related to the TBI, while in the patients aged 70-75 years, TBI is related to a traffic accident (P=0.002). Moreover the cause of TBI correlates with the CAW treatment, the accidental fall being significantly more frequent in patients in CAW treatment (P=0.003). Overall mortality rate is significantly related to an elevated INR class, to presence of t-SAH (16/50 patients) and subdural hematoma (26/46). CONCLUSION: The results of the present study show that in a population of patients aged > 70, TBI is a high risk event if patient has concurrent treatment with CAW therapies and if an accidental fall is the cause of TBI. In these cases the finding of t-SAH represents a high-risk parameter for mortality but not for morbidity.

Italian Constitutional Court modifications of a restrictive assisted reproduction technology law significantly improve pregnancy rate.

BACKGROUND: In May 2009, the Italian Constitutional Court banned most of the limitations of a restrictive law regulating assisted reproduction technology on the grounds that it limited a couple's right to have access to the best possible medical treatment and reduce any possible higher risk of complications. The aim of the study was to compare our results in fresh cycles before and after this change. MATERIALS AND METHODS: We analysed retrospectively 3274 IVF cycles: 2248 before and 1026 after the law was modified. RESULTS: There was no significant difference between the two groups in terms of age, basal FSH levels, years of infertility, the number of previous cycles or the number of oocytes retrieved but the number of oocytes used (2.7 ± 0.6 versus 4.6 ± 1.8; P = <0.001), the number of embryos obtained (2.0 ± 0.9 versus 3.3 ± 1.8; P = <0.001) and transferred (2.2 ± 0.7 versus 2.3 ± 0.7; P = <0.001) were all higher after the removal of the previous restrictions, as was the pregnancy rate per started cycle (23.49% versus 20.42%; P = 0.047). Before modification of the law, the pregnancies were single in 74.11% of the cases (versus 71.43% afterwards), twins in 23.44% (versus 26.89%; P = 0.318) and triplets in 2.46% (versus 1.68%; P = 0.594). CONCLUSIONS: Our preliminary results after the removal of the previous legal restrictions show a higher pregnancy rate per started cycle (3.7% represents a 15% difference) and a positive (albeit non-significant) trend towards a reduction in the number of multiple pregnancies.

Differential diagnosis and management of focal ground-glass opacities.

Focal pulmonary ground-glass opacities (GGOs) can be associated with bronchioloalveolar carcinoma. The present retrospective study aimed to test the validity of a multistep approach to discriminate malignant from benign localised (focal) GGOs, identifies useful diagnostic features on computed tomography (CT), and suggests appropriate management guidelines. A stepwise approach, including oral antibiotics, follow-up high-resolution CT (HRCT) 40-60 days later and CT-guided core biopsy, was used. All cases with localised GGOs detected since 2001 were reviewed. CT features were described according to a structured scheme. In total, 40 patients were evaluated. Of these, 11 patients were diagnosed with benign GGOs, 19 patients had lung cancer and 10 were undetermined. Nonpolygonal shape, apparent radial growth and clear-cut margins were associated with a malignant histology. The specificity of CT findings was low. Diagnostic accuracy increased after oral antibiotics, follow-up HRCT and percutaneous core biopsy. Overall, 18 patients underwent surgery for lung cancer. In conclusion, malignant ground-glass opacities have a fairly typical appearance, but some benign lesions closely mimic their malignant counterparts. The stepwise approach adopted in the present study increased the diagnostic specificity and reduced time to definitive diagnosis. Segmentectomy might be the ideal resection volume for such tumours.

IGEV regimen and a fixed dose of lenograstim: an effective mobilization regimen in pretreated Hodgkin's lymphoma patients.

We explored the efficacy of the IGEV regimen (ifosfamide, gemcitabine, vinorelbine and prednisone) combined with a fixed dose of lenograstim (263 mug/day) to mobilize peripheral blood stem cells (PBSCs) in 90 Hodgkin's lymphoma patients. The median total CD34+ cells/mul peak, colony-forming units granulocyte-macrophage and white blood cells for all individual collection sets were 85/mul, 12 x 10(4)/kg and 20 700/mul, respectively. An adequate number of CD34+ cells (more than 3 x 10(6) or 6 x 10(6) CD34+ cells/kg depending on whether single or tandem high-dose chemotherapy was used) were collected in 89 out of 90 (98.7%) mobilized patients, whereas the only failure reached 2.3 x 10(6) CD34+ cells/kg. The median CD34+ cell collections were 11 x 10(6)/kg (range 2.3-39 x 10(6)/kg) and 10 x 10(6)/kg (range 6-22.0 x 10(6)/kg) with a median of 1 and 2 leukaphereses for patients eligible for single high-dose treatment and for candidates for tandem transplant, respectively. Target yields were reached in 71.43 and 49.09% and additionally in 17.14 and 43.64% of cases after the first and second apheresis procedures, respectively. Hematological and non-hematological side effects were acceptable, and no toxic deaths occurred. Thirty-four patients received a single and 47 received tandem transplantation with rapid engraftment. These results confirm that the IGEV regimen with lenograstim support can be used successfully and safely to mobilize PBSCs.

Genetic and epigenetic changes in primary metastatic and nonmetastatic colorectal cancer.

Colorectal cancer (CRC) develops as multistep process, which involves genetic and epigenetic alterations. K-Ras, p53 and B-Raf mutations and RASSF1A, E-Cadherin and p16INK4A promoter methylation were investigated in 202 CRCs with and without lymph node and/or liver metastasis, to assess whether gene abnormalities are related to a metastogenic phenotype. K-Ras, B-Raf and p53 mutations were detected in 27, 3 and 32% of the cases, with K-Ras mutations significantly associated with metastatic tumour (P=0.019). RASSF1A, E-Cadherin and p16INK4A methylation was documented in 20, 44 and 33% of the cases with p16INK4A significantly associated with metastatic tumours (P=0.001). Overall, out of 202 tumours, 34 (17%) did not show any molecular change, 125 (62%) had one or two and 43 (21%) three or more. Primary but yet metastatic CRCs were prevalent in the latter group (P=0.023) where the most frequent combination was one genetic (K-Ras in particular) and two epigenetic alterations. In conclusion, this analysis provided to detect some molecular differences between primary metastatic and nonmetastatic CRCs, with K-Ras and p16INK4A statistically altered in metastatic tumours; particular gene combinations, such as coincidental K-Ras mutation with two methylated genes are associated to a metastogenic phenotype.

Clinical phenotype and beta-cell autoimmunity in Italian patients with adult-onset diabetes.

OBJECTIVE: To characterize the phenotype of a large population of Italian patients with adult onset (> or =40 years) diabetes who were attending outpatient clinics and who were screened for glutamic acid decarboxylase 65 autoantibodies (GADA), protein tyrosine phosphatase IA-2 (IA-2A) and IA-2beta/phogrin (IA-2betaA). DESIGN AND METHODS: This was a cross-sectional study comprising a total of 881 patients, aged < or = 70 years, diagnosed with type 2 diabetes after the age of 40 years, and consecutively recruited in five clinics located in different geographic areas of Italy (Milan, Florence, Rome, Naples and Catania). Their mean disease duration was 8.1 (6.9; s.d.) years. GADA, IA-2A and IA-2betaA were measured with radiobinding assays with in vitro translated S-methionine-labelled glutamic acid decarboxylase 65 (GAD65) or IA-2 or IA-2beta. Anthropometric and clinical data were collected and compared amongst patients with or without autoantibodies. RESULTS: Sixty-three (7.1%) patients had one or more autoantibodies, 58 (6.6%) had GADA, 22 (2.5%) had IA-2A, six (0.7%) had IA-2betaA and 19 (2.15%) had two or more autoantibodies. IA-2A or IA-2betaA, in the absence of GADA, were found in only five patients. Autoantibody-positive patients were more often female (63.5 vs 36.5%; P < 0.009), had higher glycated haemoglobin (Hb A1c) (P < 0.001), lower body mass index (BMI; P < 0.0005) and waist/hip ratio (WHR; P < 0.01); female gender being the main contributor to BMI and WHR. We did not observe any differences in age at diagnosis or duration of disease with respect to the presence or absence of islet autoantibodies. The proportion of patients on insulin therapy was higher in patients with two or more antibodies, compared with those with one antibody only, and no antibodies (P for trend < 0.001), and among patients with GADA, in those with higher antibody titre (73.9% in those with > 10 units vs 42.0% in those with < or = 10 units; P < 0.007). CONCLUSIONS: Patients with adult onset diabetes characterized by autoimmunity to beta-cells showed a clinical phenotype with anthropometric features that differed from those classically observed in patients with type 2 diabetes. The number and titre of autoantibodies, which reflect the severity of autoimmunity and beta-cell impairment, amplified this difference. The usefulness of autoantibody screening in adult-onset diabetes is further emphasized by these findings.

EGFR overexpression in malignant pleural mesothelioma. An immunohistochemical and molecular study with clinico-pathological correlations.

The epidermal growth factor receptor (EGFR) is overexpressed in many epithelial malignancies, against which some antitumoral drugs have been developed. There is a lack of information as to EGFR expression in malignant pleural mesothelioma (MPM), an aggressive and fatal cancer poorly responsive to current oncological treatments. Our aim was to: (a) compare EGFR immunohistochemical expression with mRNA levels measured by real time PCR; (b) assess the relationships between EGFR expression and clinico-pathological data including survival; (c) analyze the EGFR mutations. We developed an immunohistochemical method of EGFR evaluation based on the number of immunoreactive cells and staining intensity in 61 MPMs. EGFR immunoreactivity was documented in 34/61 (55.7%) cases. A significant correlation between EGFR protein and mRNA levels (p = 0.0077) was found, demonstrating the reliability of our quantification method of EGFR membrane expression. Radically resected patients (p = 0.005) and those with epithelial histotype (p = 0.048) showed an increased survival. No statistical correlation between EGFR immunoreactivity and patients survival was observed. No EGFR mutation was documented. This study documents EGFR overexpression in MPM at the protein and the transcriptional levels; it proposes a reliable method for EGFR expression evaluation in MPM. EGFR levels are not associated with clinico-pathological features of patients, including survival.

Analysis of epidermal growth factor receptor expression as a predictive factor for response to gefitinib ('Iressa', ZD1839) in non-small-cell lung cancer.

Gefitinib ('Iressa', ZD1839) is an orally active epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that has demonstrated antitumour activity and favourable tolerability in Phase II studies. We investigated whether EGFR expression levels could predict for response to gefitinib in patients with advanced non-small-cell lung cancer (NSCLC), who received gefitinib (250 mg day(-1)) as part of a worldwide compassionate-use programme. Tissue samples were analysed by immunohistochemistry to assess membrane EGFR immunoreactivity. Of 147 patients enrolled in our institution, 50 patients were evaluable for assessment of both clinical response and EGFR expression. The objective tumour response rate was 10% and disease control was achieved in 50% of patients. Although high EGFR expression was more common in squamous-cell carcinomas than adenocarcinomas, all objective responses were observed in patients with adenocarcinoma. Response and disease control with gefitinib were not associated with high EGFR expression. Overall, median survival was 4 months, and the 1-year survival rate was 18%. Strong EGFR staining correlated with shorter survival time for all patients. Gefitinib demonstrated promising clinical activity in this group of patients with NSCLC. These results have also shown that EGFR expression is not a significant predictive factor for response to gefitinib.

Activity of a specific inhibitor, gefitinib (Iressa, ZD1839), of epidermal growth factor receptor in refractory non-small-cell lung cancer.

BACKGROUND: Gefitinib (Iressa(TM), ZD1839) is an orally active, selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor. Phase I studies showed that it is well tolerated, with evidence of tumor regression in patients with advanced non-small-cell lung cancer (NSCLC). Therefore, we aimed to assess the antitumor activity and tolerability of gefitinib in a series of patients with previously treated, advanced NSCLC, as a part of a compassionate use program. PATIENTS AND METHODS: To be eligible, all patients were required to have histologically or cytologically proven advanced or metastatic NSCLC, prior chemotherapy with at least one cisplatin-containing chemotherapy regimen or contraindication to cytotoxic drugs, Eastern Cooperative Oncology Group performance status < or =2, and adequate hematological, renal and hepatic parameters. All patients provided signed informed consent. Patient re-evaluation was performed every 4-6 weeks. RESULTS: Seventy-three consecutive patients were enrolled. Response rate, including complete and partial response, was 9.6%; an additional 43.8% of patients achieved stable disease, for an overall disease control of 53.4%. EGFR1 status was evaluated by immunocytochemistry in 25 patients. According to EGFR1 immunoreactivity all responses were observed with medium/strong imunoreactivity while three out of four responses were observed in high expressive patients. Median survival for all patients was 4 months while it reached 6 months for patients with disease control. The 1-year survival rate was 13.1% for the entire series and 23.2% for patients with disease control. Non-hematological toxicity was generally mild. CONCLUSION: Gefitinib has promising activity with a good toxicity profile in patients with progressive NSCLC who have received one or two prior chemotherapy regimens. A possible relationship within response and EGFR1 expression is suggested.

Intensified CHOP regimen in aggressive lymphomas: maximal dose intensity and dose density of doxorubicin and cyclophosphamide.

BACKGROUND: Following our previous study of CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) intensification in non-Hodgkin's lymphoma (NHL), in the present report we attempted to further increase dose intensity by shortening the between-course intervals with the support of growth factors. PATIENTS AND METHODS: A total of 67 patients were enrolled. With a fixed dose of doxorubicin 75 mg/m(2), cyclophosphamide (CTX) was started at a dose of 1750 mg/m(2) and increased by 250 mg/m(2) in consecutive cohorts of patients provided that no dose-limiting toxicity occurred. After the maximal tolerated dose (MTD) had been identified, this was used to treat more patients in order to confirm the feasibility of the regimen on a large scale, with the number of cycles being varied on the basis of disease extension. RESULTS: Twenty-three cases were enrolled in the CTX dose finding phase. Dose-limiting non-hematological toxicity occurred at 2250 mg/m(2). As the intermediate level of 2000 mg/m(2) had a borderline toxicity profile, a CTX dose of 1750 mg/m(2) was defined as the MTD. A total of 53 patients then received the MTD during the course of the study as a whole. At the MTD, toxicity was acceptable. Only 10 of 189 cycles (4%) required hospitalization due to infection or febrile neutropenia. Seventy-four percent of the patients achieved complete remission. Freedom from progression and overall survival at 12 months were 71% and 86% in the whole series, and 58% and 71% for high-risk cases, respectively. CONCLUSIONS: This intensified CHOP regimen is feasible on an outpatient basis. It can be safely considered a definitive treatment in patients at low and intermediate risk, and as induction before high-dose consolidation in high-risk cases.

Irinotecan and raltitrexed: an active combination in advanced colorectal cancer.

BACKGROUND: Irinotecan and raltitrexed are active agents in metastatic colorectal cancer. Preclinical findings suggest a remarkable synergistic activity between the two drugs and the feasibility of this association has been shown in a recent phase I study. The aim of our phase II trial was to assess the efficacy and tolerability of the combination of irinotecan and raltitrexed in patients with metastatic colorectal cancer untreated with chemotherapy. PATIENTS AND METHODS: From June 1998 to February 2000, 46 patients were enrolled. Patients received irinotecan 350 mg/m(2) on day 1 and raltitrexed 2.6 mg/m(2) on day 2, every 3 weeks, for up to nine courses. Tumour assessment was performed every three cycles. RESULTS: A total of 223 cycles of chemotherapy, with a median number of six (range 1-9) courses per patient, was administered. According to intention-to-treat analysis, the overall response rate was 46% (95% confidence interval 31% to 61%). The median duration of response was 21 weeks (range 11-> or =101), the median time to progression 27 weeks (range 1-> or =116), and the median overall survival 57 weeks (range 1-> or =130). The main toxicities were diarrhoea, with National Cancer Institute common toxicity criteria grade 3/4 in 26% of patients, grade 3/4 neutropenia in 20%, grade 3 nausea-vomiting in 13%, grade 3 asthenia in 11% and grade 3/4 transaminase elevation in 4%. CONCLUSIONS: Results achieved with irinotecan and raltitrexed show that this regimen is active, despite 'not-negligible' toxicity, and may represent a useful regimen for specific subgroups of colorectal cancer patients.