Claudio Zamagni

Phase II multicenter, uncontrolled trial of sorafenib in patients with metastatic breast cancer.

This trial was conducted to assess the efficacy and safety of sorafenib in patients with metastatic breast cancer. In this multinational, open-label phase II study, patients with metastatic breast cancer that had progressed after at least one prior chemotherapy regimen were continuously treated with oral sorafenib, 400 mg twice daily. The primary endpoint was overall best response; a secondary endpoint was percentage of patients with stable disease for ≥16 weeks. Biomarker analyses were also performed. Of the 56 patients enrolled into the study, 54 were treated with at least one dose of sorafenib. Partial response was observed in one patient (2%) and stable disease in 20 patients (37%); no complete responses were observed. Disease stabilization for ≥16 weeks was seen in 12 patients (22%); stabilization for ≥6 months in seven patients (13%). The most common drug-related grade 3 adverse events were rash/desquamation (6%), hand–foot skin reaction (4%), and fatigue (4%). Baseline vascular endothelial growth factor levels, levels of soluble epidermal growth factor receptor during treatment and both baseline and changes in soluble human epidermal growth factor receptor 2 levels correlated significantly with clinical outcomes. Although the primary endpoint of overall response rate showed minimal improvement on sorafenib 400 mg twice-daily treatment, the rate of disease stabilization was encouraging in patients treated with one or more lines of chemotherapy. The treatment had a clinically manageable toxicity profile. Further investigation of single-agent sorafenib in this patient population is not recommended; however, studies investigating combinations of sorafenib with chemotherapeutic agents are warranted and ongoing.

FDG-PET/CT in advanced ovarian cancer staging: Value and pitfalls in detecting lesions in different abdominal and pelvic quadrants compared with laparoscopy

INTRODUCTION AND AIM Ovarian carcinoma (OC) is a common cancer in the Western Countries, and an important cause of death in patients suffering with gynaecologic malignancies. The majority of patients present with advanced disease at the time of diagnosis. Treatment with debulking surgery followed by chemotherapy is the standard approach while chemotherapy is contemplated when surgery is not possible. A correct pre-operative staging is important to ensure a most appropriate management. Laparoscopy (LPS) is the standard diagnostic tool for the assessment of intraperitoneal infiltration but is invasive and requires general anaesthesia. FDG-PET/CT is increasingly used for staging different types of cancer, and the aim of this study is to assess the value of FDG-PET/CT in staging advanced OC and its sensitivity to detect lesions in different quadrants of the abdominal-pelvic area compared to laparoscopy. MATERIALS AND METHODS From September 2004 till April 2008, 40 patients with high suspicion of OC were referred to our hospital for diagnostic LPS to explore the possibility of optimal debulking surgery. Those who were not suitable for surgery were referred for chemotherapy. Before chemotherapy, the patients underwent an FDG-PET/CT scan. The findings in 9 quadrants of abdominal-pelvic area (total 360 quadrants) for PET/CT and LPS were recorded and compared. RESULTS In 14/360 areas (3.8%), surgical evaluation was not possible because of presence of adhesions, thus the number of areas explored by laparoscopy was 346. Tumour was found in 308 quadrants (38 quadrants free of disease). PET/CT was positive in all 40 patients with true negative results in 26/346 quadrants (7.5%), and true positives results in 243/346 quadrants (70.2%). False positive and negative PET/CT results were found in 12/346 and 65/346 quadrants, respectively. False positive PET/CT findings were evenly present in all quadrants. False negative PET/CT findings were present in 31/109 (28.4%) upper abdominal quadrants (epigastrium and diaphragmatic areas). Final analysis showed a sensitivity and specificity for PET/TC of 78.9 and 68.4% respectively with a positive predictive value of 95.3%. A significant difference was noted between mean SUVmax associated with lesions smaller or larger than 0.5 cm (p=0.006). CONCLUSION Our results suggest that PET/CT may prove a useful tool for pre-surgical staging of ovarian cancer with a sensitivity and specificity of 78 and 68%, respectively. However, it may be used in combination with laparoscopy for better results. PET/CT showed an adequate correlation between SUVmax values and laparoscopy findings of lesions>5mm, but a high rate of false negative results in lesions<5mm such as in carcinomatosis. PET/CT should be used carefully in early stage disease, with low risk of peritoneal infiltration, because of high rate of false positive results, to avoid unnecessary therapy procedures.

Early 18F-2-fluoro-2-deoxy-d-glucose positron emission tomography may identify a subset of patients with estrogen receptor-positive breast cancer who will not respond optimally to preoperative chemotherapy

A pathologic complete response (pCR) and minimal residual disease (pMRD) after preoperative chemotherapy (PCT) for early stage or locally advanced breast cancer (BC) correlates with a good prognosis.

Pharmacokinetics of third-generation aromatase inhibitors

The latest generation of nonsteroidal aromatase inhibitors (anastrozole and letrozole) has been approved by the US Food and Drug Administration for use in the first- and second-line treatment of postmenopausal women with hormone receptor-positive (or unknown) breast cancer. The steroidal agent exemestane is approved for second-line treatment and is currently being evaluated for first line. In addition, these agents are being evaluated in the adjuvant and neoadjuvant settings. Because preclinical studies have suggested some differences in the efficacy of these agents to inhibit aromatase, it has been important to evaluate whether these properties actually translate into meaningful differences in the clinical setting. The pharmacokinetic properties of each aromatase inhibitor influences their ability to perform effectively. Examination of the data suggests differences in uptake rates, half-life of elimination, metabolism, and clearance rates that could influence their efficacy. However, the significance of these differences for clinical effectiveness over long-term use remains to be determined.

BRCA-Associated Ovarian Cancer: From Molecular Genetics to Risk Management

Ovarian cancer (OC) mostly arises sporadically, but a fraction of cases are associated with mutations in BRCA1 and BRCA2 genes. The presence of a BRCA mutation in OC patients has been suggested as a prognostic and predictive factor. In addition, the identification of asymptomatic carriers of such mutations offers an unprecedented opportunity for OC prevention. This review is aimed at exploring the current knowledge on epidemiological and molecular aspects of BRCA-associated OC predisposition, on pathology and clinical behavior of OC occurring in BRCA mutation carriers, and on the available options for managing asymptomatic carriers.

Mitochondrial DNA Mutation in Serous Ovarian Cancer: Implications for Mitochondria-Coded Genes in Chemoresistance

Case Report A69-year-oldwomansufferingdyspneaandwidespreadabdominal pain was admitted. Preliminary tests detected mild anemia, leukocytosis, and high levels of CA125 (2,200 U/mL). Abdominal-pelvic ultrasound scan, toraco-abdominal computer tomography (CT) and positron emission tomography (PET) indicated a gastric expansive mass together with two adherent pelvic solid masses, vascularized, strictly contiguous to bowel, omental cake, peritoneal carcinomatosis marks and ascites (Figs 1A and 1C; Figs 1E and 1G, arrows). Esophageal-gastric duodenoscopy was normal. CT-guided biopsy of the pelvic masses led to diagnose a poorly differentiated ovarian carcinoma in July 2010. A six-cycle carboplatin-paclitaxel chemotherapy was administered from August 2010 to December 2010. Radiological CT and PET at the end of the chemotherapyshowednearlycomplete response(Figs1Band1D;1Fand 1H). Diagnostic laparoscopy confirmed the absence of peritoneal carcinomatosis and suggested that surgery had become feasible. In March 2011, the patient was submitted to hysterectomy with bilateral annessiectomy, pelvic and lomboartic lymphadenectomy, omentectomy and peritoneal biopsies with complete macroscopical removal of disease. The histologicalreportshowedpersistenceofmicroscopicfociofdisease inthe ovaries (G3serouspapillaryadenocarcinoma),with lymphnodes,omentum and peritoneum free from disease. No adjuvant chemotherapy was administered because the patient had received six full courses of preoperativechemotherapyandnomacroscopicdiseasewaspresentaftersurgery. The patient was enrolled in the Mitochondria in Progression of Endometrial and Ovarian Cancer (MiPEO) study approved by the local ethical committee at S. Orsola Hospital, Bologna. Signed informed consent was obtained. She was subjected to regular follow-ups every 4 months with CT, pelvic ultrasound, and test of CA125. With the aim to characterize the residual chemoresistant ovarian cancer, hematoxylin/eosin staining showed a clear-cut oncocytic component, undetected in the prechemotherapy biopsy. Therefore, the acquisition of oncocytic change was observed exclusively in the ovarian cancer tissueresidualafterchemotherapy(Fig2).Wehavepreviouslyshownthat mitochondrial DNA (mtDNA) mutations in respiratory complex I (CI) genes are markers of oncocytic transformation, which cause CI derangement and, consequently, impairment of mitochondrial respiration. In order to understand whether mtDNA mutations and CI disassembly underlay the oncocytic phenotype, whole mtDNA sequencing was performed. Sequence analysis revealed the presence of m.10875T C missense mutation of a conserved residue (p.39Leu Pro) in the MTND4 gene encoding a CI subunit (Figs 3 and 4 [boxed in red in Fig 4]). The mutation was nearly homoplasmic, although a minor contamination withadjacentnontumortissuecouldnotbecompletelyruledout.Predictor of amino acid change pathogenic potential PolyPhen2 revealed the mutation to be probably damaging. Proof of pathogenicity was obtained through negative immunohistochemistry (IHC) staining of nuclearencoded CI subunit NDUFB8, which does not integrate within the complex when ND4 is lacking (Fig 2). In order to ascertain that the mtDNA disruptive mutation had accumulated postchemotherapy, normal and prechemotherapy tissues were screened for the same mutation, which was shown to be absent (Fig 3). In order to exclude the presence of a possible low-level heteroplasmy, a sensitive locked nucleic acid–based technique with a detection limit of 0.1% was implemented. Green squares represent standard curve samples (slope: 3.364; r 0.995; PCR efficiency: 97%). Wild-type control is circled in black and prechemotherapy sample in red. Absence of the mutation in prechemotherapy and in nontumor tissue was confirmed (Fig 5), indicating that the homoplasmic m.10875T Cwasapostchemotherapy-specificevent,consistentwiththe NDUFB8-positive staining of prechemotherapy tissue (Fig 2). Concordantly with the previously determined correlation between the occurrence of mtDNA mutations and low proliferation of oncocytic tumors, Ki67 of the postchemotherapy mass was nearly zero (Fig 6). Next, we sought to determine whether the residual mass had retained the original pro-oncogenic lesions, in order to ascertain a modifier role for the MTND4 mutation in keeping the chemoresistant clone under alow-proliferativeconstraint.Screeningofoncogenesfrequentlyinvolved in ovarian carcinogenesis, namely KRAS, BRAF, CTNNB1, PIK3CA, ERBB2 and AKT1 did not reveal mutations. Screening of tumor suppressor genes TP53 and PTEN identified a TP53 pathogenic heterozygous p.220Y C mutation both in preand postchemotherapic tissue, but not in the nontumor lymph node (Fig 7). Moreover, the homozygous TP53 p.72P R variant detected in all three samples (Fig 7) was previously reported to render TP53 a sensitive target for oncogenic protein E6 of human papilloma virus strain 16 (HPV16), occasionally associated with ovarian carcinoma. HPV16 was indeed detected both in tumor and in thenontumorlymphnodeofthepatientas it isevidentfromalignmentof detected amino acidic viral sequence (Fig 8). These data suggested cell transformation was due to the viral oncogenic properties in combination with the TP53 mutations.

Estrogen receptor 1 mRNA is a prognostic factor in ovarian carcinoma: determination by kinetic PCR in formalin-fixed paraffin-embedded tissue

Epidemiological and cell culture studies indicate that ovarian carcinoma growth is dependent on estrogen stimulation. However, possibly due to the lack of a reliable biomarker that helps to select patients according to prognostically relevant estrogen receptor (ER) levels, clinical trials using anti-estrogenic therapeutics in ovarian carcinoma have had inconsistent results. Therefore, we tested if ER expression analysis by a quantitative method might be useful in this regard in formalin-fixed paraffin-embedded (FFPE) tissue. In a study group of 114 primary ovarian carcinomas expression of estrogen receptor 1 (ESR1) mRNA was analyzed using a new method for RNA extraction from FFPE tissue that is based on magnetic beads, followed by kinetic PCR. The prognostic impact of ESR1 mRNA expression was investigated and compared to ERalpha protein expression as determined by immunohistochemistry. In univariate survival analysis the expression level of ESR1 mRNA was a significant positive prognostic factor for patient survival (hazard ratio (HR) 0.230 (confidence interval (CI) 0.102-0.516), P=0.002). ERalpha protein expression was correlated to ESR1 mRNA expression (P=0.0001); however, ERalpha protein expression did not provide statistically significant prognostic information. In multivariate analysis, ESR1 mRNA expression emerged as a prognostic factor, independent of stage, grade, residual tumor mass, age, and ERalpha protein expression (HR 0.227 (CI 0.078-0.656), P=0.006). Our results indicate that the determination of ESR1 levels by kinetic PCR may be superior to immunohistochemical methods in assessment of biologically relevant levels of ER expression in ovarian carcinoma, and is feasible in routinely used FFPE tissue.

Feasibility and Cardiac Safety of Trastuzumab Emtansine After Anthracycline-Based Chemotherapy As (neo)Adjuvant Therapy for Human Epidermal Growth Factor Receptor 2–Positive Early-Stage Breast Cancer

PURPOSE Trastuzumab emtansine (T-DM1), an antibody-drug conjugate comprising the cytotoxic agent DM1, a stable linker, and trastuzumab, has demonstrated substantial activity in human epidermal growth factor receptor 2 (HER2) -positive metastatic breast cancer, raising interest in evaluating the feasibility and cardiac safety of T-DM1 in early-stage breast cancer (EBC). PATIENTS AND METHODS Patients (N = 153) with HER2-positive EBC and prechemotherapy left ventricular ejection fraction (LVEF) ≥ 55% received (neo)adjuvant doxorubicin plus cyclophosphamide or fluorouracil plus epirubicin plus cyclophosphamide followed by T-DM1 for four cycles. Patients could then receive three to four cycles of optional docetaxel with or without trastuzumab. T-DM1 was then resumed with optional radiotherapy (sequential or concurrent) for 1 year (planned) of HER2-directed therapy. The coprimary end points were rate of prespecified cardiac events and safety. RESULTS Median follow-up was 24.6 months. No prespecified cardiac events or symptomatic congestive heart failures were reported. Four patients (2.7%) had asymptomatic LVEF declines (≥ 10 percentage points from baseline to LVEF < 50%), leading to T-DM1 discontinuation in one patient. Of 148 patients who received ≥ one cycle of T-DM1, 82.4% completed the planned 1-year duration of HER2-directed therapy. During T-DM1 treatment, 38.5% and 2.7% of patients experienced grade 3 and 4 adverse events, respectively. Approximately 95% of patients receiving T-DM1 plus radiotherapy completed ≥ 95% of the planned radiation dose with delay ≤ 5 days. CONCLUSION Use of T-DM1 for approximately 1 year after anthracycline-based chemotherapy was feasible and generally well tolerated by patients with HER2-positive EBC, providing support for phase III trials of T-DM1 in this setting.

Oestrogen receptor 1 mRNA is a prognostic factor in ovarian cancer patients treated with neo-adjuvant chemotherapy: Determination by array and kinetic PCR in fresh tissue biopsies

Oestrogen receptors (ESRs) regulate the growth and differentiation of normal ovarian epithelia. However, to date their role as biomarkers in the clinical setting of ovarian cancer remains unclear. In view of potential endocrine treatment options, we tested the role of ESR1 mRNA expression in ovarian cancer in the context of a neo-adjuvant chemotherapy trial. Study participants had epithelial ovarian or peritoneal carcinoma unsuitable for optimal upfront surgery and were treated with neo-adjuvant platinum-based chemotherapy before surgery. RNA was isolated from frozen tumour biopsies before treatment. RNA expression of ESR1 was determined by microarray and reverse transcriptase kinetic PCR technologies. The prognostic value of ESR1 was tested using univariate and multivariate Cox proportional hazards models, Kaplan-Meier survival statistics and the log-rank test. ESR1 positively correlates with proliferation markers and histopathological grading. ESR1 was a significant predictor of survival as a continuous variable in the univariate Cox regression analysis. In multivariate analysis, elevated baseline ESR1 mRNA levels predicted prolonged progression-free survival (P=0.041) and overall survival (P=0.01) after neo-adjuvant chemotherapy, independently of pathological grade and age. We conclude that pretreatment ESR1 mRNA is associated with tumour growth and is a strong prognostic factor in ovarian cancer, independent of the strongest clinical parameters used in clinical routine. We suggest that ESR1 mRNA status should be considered in order to minimize possible confounding effects in ovarian cancer clinical trials, and that early treatment with anti-hormonal agents based on reliable hormone receptor status determination is worth investigating.

BRCA1/2 mutations associated with progression-free survival in ovarian cancer patients in the AGO-OVAR 16 study

OBJECTIVE AGO-OVAR 16 demonstrated that pazopanib maintenance therapy significantly increased progression-free survival (PFS) in patients with ovarian cancer whose disease had not progressed after first-line therapy. In a sub-study, we evaluated the effect of clinically important germline BRCA1 and BRCA2 mutations on PFS. METHODS Of 940 AGO-OVAR 16 participants, 664 had BRCA1/2 exon sequencing data (pazopanib, n=335; placebo, n=329). A Cox model was used to test the association between genetic variants and PFS. RESULTS Ninety-seven of 664 patients (15%) carried clinically important BRCA1/2 mutations (BRCA1/2 carriers: pazopanib 14%, placebo 16%). Median PFS was longer in BRCA1/2 mutation carriers than in BRCA1/2 non-carriers in the placebo arm (30.3 vs 14.1 months, hazard ratio, 0.48; 95% confidence interval [CI]: 0.29-0.78; P=0.0031); a similar non-significant trend was noted with pazopanib (30.2 vs 17.7 months, hazard ratio, 0.64; 95% CI: 0.40-1.03; P=0.069). Among BRCA1/2 non-carriers, PFS was longer for pazopanib-treated patients than placebo-treated patients (17.7 vs 14.1 months, hazard ratio, 0.77; 95% CI: 0.62-0.97; P=0.024). Among BRCA1/2 carriers, there was no significant PFS difference between treatments, although numbers were small (pazopanib, 46; placebo, 51), resulting in a wide CI (hazard ratio, 1.36; 95% CI: 0.66-2.82). CONCLUSIONS Patients with clinically important BRCA1/2 mutations had better prognosis. BRCA1/2 mutation status might be added as strata in future trials in primary ovarian cancer.