Conservative methotrexate treatment of a scar pregnancy case: adding evidence to the evidence.

A single case of ultrasonographic-guided local injection of methotrexate for managing scar pregnancy is reported. The outcome was successful and had no side effects. The case was reported to increase the evidence supporting this type of management.

Programmed death ligand 1 expression in early stage, resectable non-small cell lung cancer.

INTRODUCTION: For several years non-small cell lung cancer (NSCLC) has been considered non-immunogenic. Recent advances in antitumor immunity brought to the discovery of checkpoints that modulate immune response against cancer. One of them is programmed death receptor 1 (PD-1) and its ligand (PD-L1). Although PD-L1 expression seems predictive of response to anti-PD-1/PD-L1 agents, its prognostic value is unclear. In this study we investigated the prognostic value of PD-L1 expression and its correlation with clinical-pathological characteristics in a cohort of surgically resected NSCLC. MATERIAL AND METHODS: PD-L1 expression was evaluated in 289 surgically resected NSCLC samples by immunohistochemistry. Our cohort included patients not exposed to adjuvant chemotherapy. PD-L1 status was defined as: 1) PD-L1 high (tumor proportion score, TPS≥50%), PD-L1 low (TPS 1-49%), PD-L1 negative (TPS<1%); 2) PD-L1 positive (TPS≥50%) and negative (TPS<50%); 3) as a continuous variable. RESULTS: Patients were mostly males (79%), former or current smokers (81%), with a median age of 67 years, non-squamous histology (67.5%) and high-grade tumors (55%). PD-L1 tumors were 18.7%. There was no significant association with sex, age, smoking status and histology. A strong correlation between high PD-L1 expression and tumor grade was detected. The difference in median OS in the different groups of patients was not statistically significant. CONCLUSION: PD-L1 is not prognostic in surgically resected NSCLC. The association with tumor differentiation suggests that grading could represent an easy-to-assess tool for selecting subjects potentially sensitive to immunotherapy warranting further investigations.

A novel deleterious PTEN mutation in a patient with early-onset bilateral breast cancer.

BACKGROUND: An early age at Breast Cancer (BC) onset may be a hallmark of inherited predisposition, but BRCA1/2 mutations are only found in a minority of younger BC patients. Among the others, a fraction may carry mutations in rarer BC genes, such as TP53, STK11, CDH1 and PTEN. As the identification of women harboring such mutations allows for targeted risk-management, the knowledge of associated manifestations and an accurate clinical and family history evaluation are warranted. CASE PRESENTATION: We describe the case of a woman who developed an infiltrating ductal carcinoma of the right breast at the age of 32, a contralateral BC at age 36 and another BC of the right breast at 40. When she was 39 years-old, during a dermatological examination, mucocutaneous features suggestive of Cowden Syndrome, a disorder associated to germ-line PTEN mutations, were noticed. PTEN genetic testing revealed the novel c.71A > T (p.Asp24Val) mutation, whose deleterious effect, suggested by conservation data and in silico tools, was definitely demonstrated by the incapacity of mutant PTEN to inhibit Akt phosphorylation when used to complement PTEN-null cells. In BC tissue, despite the absence of LOH or somatic mutations of PTEN, Akt phosphorylation was markedly increased in comparison to normal tissue, thus implying additional somatic events into the deregulation of the PI3K/Akt/mTOR pathway and, presumably, into carcinogenesis. Hence, known oncogenic mutations in PIK3CA (exons 10 and 21) and AKT1 (exon 2) were screened in tumor DNA with negative results, which suggests that the responsible somatic event(s) is a different, uncommon one. CONCLUSION: This case stresses the importance of clinical/genetic assessment of early-onset BC patients in order to identify mutation carriers, who are at high risk of new events, so requiring tailored management. Moreover, it revealed a novel PTEN mutation with pathogenic effect, pointing out, however, the need for further efforts to elucidate the molecular steps of PTEN-associated carcinogenesis.

Molecular determinants of outcome in sorafenib-treated patients with hepatocellular carcinoma.

PURPOSE: Preclinical studies show that sorafenib, a multitarget kinase inhibitor, displays anti-proliferative, anti-angiogenic, and pro-apoptotic properties in hepatocellular carcinoma (HCC). However, the determinants of sorafenib sensitivity in vivo remain largely unknown. METHODS: We assessed the expression of Mcl-1, activated/phosphorylated extracellular signal-regulated kinase (pERK) 1/2, and activated/phosphorylated AKT (pAKT) in pretreatment tumor specimens from 44 patients with advanced HCC who received sorafenib. Furthermore, we assessed MYC and MET gene copy numbers (GCN) by fluorescence in situ hybridization. RESULTS: Poorer overall survival (OS) times were correlated with pERK expression [hazard ratio (HR) 1.013; 95 % CI 1.003-1.035] and Mcl-1 expression (HR 1.016; 95 % CI 1.002-1.030) in pretreatment tumor samples. Expression levels of pERK and Mcl-1, however, were not correlated with time to tumor progression (TTP). Increased pERK expression was positively associated with higher Cancer of Liver Italian Program scores (P = 0.012) and was prognostic in patients with scores 2-6 but not in those with scores 0-1. pERK expression was significantly less frequent in specimens sourced from previous surgical procedures compared to biopsy samples (9.6 vs. 92.3 %, respectively; P < 0.0001). Analysis of pAKT expression, MET and MYC GCN, did not indicate any prognostic nor predictive values for these biomarkers in terms of survival. CONCLUSIONS: Expression levels of Mcl-1 and pERK are associated with reduced OS in HCC patients treated with sorafenib and might be useful markers for risk stratification. However, in contrast to previous findings, pERK expression levels, as well as other biomarkers tested, did not affect TTP.

Nogo-A: a useful marker for the diagnosis of oligodendroglioma and for identifying 1p19q codeletion.

The differential diagnosis between oligodendrogliomas and other gliomas remains a critical issue. The aim of this study is to verify the diagnostic value of Olig-2, Nogo-A, and synaptophysin and their role in identifying 1p19q codeletion. A total of 168 cases of brain tumors were studied: 24 oligodendrogliomas, 23 anaplastic oligodendrogliomas, 2 oligoastrocytomas, 2 anaplastic oligoastrocytomas, 30 glioblastoma multiforme, 2 diffuse astrocytomas, 4 anaplastic astrocytomas, 10 pilocytic astrocytomas, 9 ependymomas, 12 anaplastic ependymomas, 10 central neurocytomas, 10 meningiomas, 10 choroid plexus papillomas, 10 dysembryoplastic neuroepithelial tumors, and 10 metastases. All cases were immunostained with Olig-2, Nogo-A, and synaptophysin. In 79 cases, the status of 1p/19q had already been assessed by fluorescence in situ hybridization. Thus, in selected cases, fluorescence in situ hybridization was repeated in areas with numerous Nogo-A-positive neoplastic cells. Nogo-A was positive in 18 (75%) of 24 oligodendrogliomas, 8 (80%) of 10 dysembryoplastic neuroepithelial tumors, 6 (20%) of 30 glioblastoma multiforme, and 2 (20%) of 10 pilocytic astrocytomas. Olig-2 stained 22 (91.6%) of 24 oligodendrogliomas and all dysembryoplastic neuroepithelial tumors but also 24 (80%) of 30 glioblastoma multiforme and 8 (80%) of 10 pilocytic astrocytomas. Finally, synaptophysin stained 13 (54.1%) of 24 oligodendrogliomas, 3 (10%) of 30 glioblastoma multiforme, 1 (10%) of 10 pilocytic astrocytomas, and all neurocytomas. Among the 79 tested cases, original fluorescence in situ hybridization showed 1p/19q codeletion in 12 (52.2%) of 23 oligodendrogliomas, 8 (38%) of 21 anaplastic oligodendrogliomas, and 1 (4%) of 25 glioblastoma multiforme. However, after carrying out the Nogo-A-driven fluorescence in situ hybridization, 1p/19q codeletion was observed in 8 additional cases. Nogo-A is more useful and specific than Olig-2 in differentiating oligodendrogliomas from other gliomas. Furthermore, using a Nogo-A-driven fluorescence in situ hybridization analysis, it is possible to identify a larger number of 1p19q codeletions in gliomas.

Genetic activation of the MET pathway and prognosis of patients with high-risk, radically resected gastric cancer.

PURPOSE: To investigate whether prognosis of patients with high-risk gastric cancer may depend on MET copy number gain (CNG) or an activating truncation within a deoxyadenosine tract element (DATE) in the promoter region of the MET ligand HGF. PATIENTS AND METHODS: A single-institution cohort of 230 patients with stage II/III gastric cancer was studied. Formalin-fixed paraffin-embedded tumor specimens were used for DNA extraction. Quantitative polymerase chain reaction (qPCR) for MET CNG and sequencing for HGF DATE truncation (< 25 deoxyadenosines instead of 30) were used. Results were analyzed for association with disease-free survival (DFS) and overall survival (OS). To assess the reliability of the qPCR measurement, a random sample of cases was reanalyzed using an alternative assay (fluorescent in situ hybridization [FISH]) with calculation of the intracorrelation coefficient (ICC). RESULTS: In 216 assessable patients, MET CNG five or more copies and homozygous HGF-truncated DATE occurred in 21 patients (10%) and 30 patients (13%), respectively. Patients with MET CNG five or more copies (MET-positive) showed significantly worse prognosis with multivariate hazard ratio (HR) of 3.02 (95% CI, 1.71 to 5.33; P < .001) for DFS and multivariate HR of 2.91 (95% CI, 1.65 to 5.11; P < .001) for OS. The agreement between qPCR and FISH was high, with ICC = 0.9% (95% CI, 0.81% to 0.95%; the closer the ICC is to 1, the greater is the agreement). HGF-truncated DATE did not show relevant prognostic effect. CONCLUSION: In this study, qPCR revealed approximately 10% of white patients with gastric cancer harboring MET CNG of five or more copies. This marker was significantly associated with unfavorable prognosis. This information is relevant to the current clinical development of anti-MET compounds.

BRAF(V600E) mutation and expression of proangiogenic molecular markers in papillary thyroid carcinomas.

OBJECTIVE: Tyrosine kinase inhibitors (TKIs) are evaluated for treatment of radioiodine refractory thyroid cancer. Their effects in this setting are based on blockade of proangiogenic signaling mediated by receptors for vascular endothelial growth factors (VEGFs) and platelet-derived growth factors (PDGF). Most TKIs also block other cancer-relevant kinases, such as B-type Raf kinase (BRAF), which are constitutively activated in approximately half of papillary thyroid carcinomas (PTCs), but the impact of these effects is not clear. DESIGN: The aim of our study was to investigate the impact of BRAF(V600E) on proangiogenic gene expression and microvascular features of PTCs. METHODS: mRNA levels for VEGFA, VEGF receptors, and coreceptors (VEGFRs 1, 2, and 3, neuropilin-1), and PDGF receptor ß (PDGFRß or PDGFRB) were measured with real-time PCR in BRAF(V600E) (n=55) and wild-type BRAF (BRAF-wt; n=35) PTCs. VEGF and VEGFR protein expression and microvessel densities (MVD) and lymphatic vessel densities (LVDs) were assessed by immunohistochemistry in 22 of the 90 PTCs (including 11 BRAF(V600E) cases). Angiogenic gene expression was also studied in vitro after induction/silencing of the BRAF(V600E) mutation in thyrocyte lines. RESULTS: Transcript levels of proangiogenic factors were significantly lower in BRAF(V600E) PTCs versus BRAF-wt PTCs (P<0.0001), but MVD and LVDs were not significantly different. VEGFA mRNA levels in thyroid cell lines decreased when BRAF(V600E) mutation was induced (P=0.01) and increased when it was silenced (P=0.01). CONCLUSIONS: Compared with BRAF-wt PTCs, those harboring BRAF(V600E) exhibit downregulated VEGFA, VEGFR, and PDGFRß expression, suggesting that the presence of BRAF mutation does not imply a stronger prediction of response to drugs targeting VEGF and PDGFB signaling pathways.

Immunoproteasome expression is induced in mesial temporal lobe epilepsy.

Immunoproteasome has been associated to neurodegenerative and autoimmune diseases as a marker and regulator of inflammatory mechanisms. Its expression in the brain may occur upon neuroinflammation in different cell types and affect a variety of homeostatic and inflammatory pathways including the oxidized protein clearance and the self-antigen presentation. In the present study we investigated the immunoproteasome expression in hippocampi and cortex of patients affected by different histopathological forms of pharmaco-resistent mesial temporal lobe epilepsy. We identified a pathology-specific pattern of immunoproteasome expression, which could provide insight into the complex neuroinflammatory pathogenic components of this disease.

Frequent and focal FGFR1 amplification associates with therapeutically tractable FGFR1 dependency in squamous cell lung cancer.

Lung cancer remains one of the leading causes of cancer-related death in developed countries. Although lung adenocarcinomas with EGFR mutations or EML4-ALK fusions respond to treatment by epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) inhibition, respectively, squamous cell lung cancer currently lacks therapeutically exploitable genetic alterations. We conducted a systematic search in a set of 232 lung cancer specimens for genetic alterations that were therapeutically amenable and then performed high-resolution gene copy number analyses. We identified frequent and focal fibroblast growth factor receptor 1 (FGFR1) amplification in squamous cell lung cancer (n = 155), but not in other lung cancer subtypes, and, by fluorescence in situ hybridization, confirmed the presence of FGFR1 amplifications in an independent cohort of squamous cell lung cancer samples (22% of cases). Using cell-based screening with the FGFR inhibitor PD173074 in a large (n = 83) panel of lung cancer cell lines, we demonstrated that this compound inhibited growth and induced apoptosis specifically in those lung cancer cells carrying amplified FGFR1. We validated the FGFR1 dependence of FGFR1-amplified cell lines by FGFR1 knockdown and by ectopic expression of an FGFR1-resistant allele (FGFR1(V561M)), which rescued FGFR1-amplified cells from PD173074-mediated cytotoxicity. Finally, we showed that inhibition of FGFR1 with a small molecule led to significant tumor shrinkage in vivo. Thus, focal FGFR1 amplification is common in squamous cell lung cancer and associated with tumor growth and survival, suggesting that FGFR inhibitors may be a viable therapeutic option in this cohort of patients.

p63 short isoforms are found in invasive carcinomas only and not in benign breast conditions.

Two N-terminal isoforms characterize the p63 protein: the transactivating isoform TAp63 and the amino-terminal truncated isoform DeltaNp63. Two further N-terminal isoforms lacking exon 4 (d4TAp63 and DeltaNp73L) have been reported. Purpose of the study was to investigate the molecular expression of N-terminal p63 isoforms in benign and malignant breast tissues. Eighteen randomly selected cases of invasive breast carcinoma (IBC) of luminal type, two cases of in situ duct carcinoma (DCIS/DIN), and 20 specimens of normal and benign breast tissues were studied. All cases were immunostained for p63. Reverse polymerase chain reaction and nested PCR were performed to evaluate p63 N-terminal expression patterns. These isoforms whenever present were validated by sequencing. All cases of normal breast, benign lesions, and the two cases of DCIS/DIN expressed DeltaNp63 and TAp63 isoforms only. The two variants lacking exon 4 (DeltaNp73L and d4TAp63) were not found. All invasive carcinomas expressed the DeltaNp63 and TAp63 isoforms as well as the two short isoforms lacking exon 4 which were found in 11 (d4TAp63) and four (DeltaNp73L) cases. The present cases of luminal-type IBC showed p63 isoforms together with short variants lacking exon 4. These isoforms were not observed in non-neoplastic breast tissue. Presence of p63 in invasive breast carcinomas of luminal type, as seen at molecular level, suggests caution to include p63 as a marker of basal-like carcinomas.

Immunoproteasome LMP2 60HH variant alters MBP epitope generation and reduces the risk to develop multiple sclerosis in Italian female population.

BACKGROUND: Albeit several studies pointed out the pivotal role that CD4+T cells have in Multiple Sclerosis, the CD8+ T cells involvement in the pathology is still in its early phases of investigation. Proteasome degradation is the key step in the production of MHC class I-restricted epitopes and therefore its activity could be an important element in the activation and regulation of autoreactive CD8+ T cells in Multiple Sclerosis. METHODOLOGY/PRINCIPAL FINDINGS: Immunoproteasomes and PA28-alphabeta regulator are present in MS affected brain area and accumulated in plaques. They are expressed in cell types supposed to be involved in MS development such as neurons, endothelial cells, oligodendrocytes, macrophages/macroglia and lymphocytes. Furthermore, in a genetic study on 1262 Italian MS cases and 845 controls we observed that HLA-A*02+ female subjects carrying the immunoproteasome LMP2 codon 60HH variant have a reduced risk to develop MS. Accordingly, immunoproteasomes carrying the LMP2 60H allele produce in vitro a lower amount of the HLA-A*0201 restricted immunodominant epitope MBP(111-119). CONCLUSION/SIGNIFICANCE: The immunoproteasome LMP2 60HH variant reduces the risk to develop MS amongst Italian HLA-A*02+ females. We propose that such an effect is mediated by the altered proteasome-dependent production of a specific MBP epitope presented on the MHC class I. Our observations thereby support the hypothesis of an involvement of immunoproteasome in the MS pathogenesis.

Prospective study of gefitinib in epidermal growth factor receptor fluorescence in situ hybridization-positive/phospho-Akt-positive or never smoker patients with advanced non-small-cell lung cancer: the ONCOBELL trial.

PURPOSE: In non-small-cell lung cancer (NSCLC), clinical and biologic predictors for epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor sensitivity have been identified in retrospective studies, and there is urgent need to validate these results in prospective trials. The ONCOBELL trial is a prospective phase II study evaluating gefitinib sensitivity in NSCLC patients who never smoked or have increased EGFR gene copy number or activation of the antiapoptotic protein Akt. PATIENTS AND METHODS: EGFR gene copy number was evaluated using fluorescence in situ hybridization (FISH), and presence of phospho-Akt was evaluated using immunohistochemistry. Additional tests included immunohistochemistry analysis of EGFR, FISH analysis of HER2, and mutation analysis of EGFR, HER2, and K-ras. RESULTS: From November 2004 to February 2006, 183 patients were screened, and 42 patients were enrolled onto the trial. We observed one complete and 19 partial responses, for an overall response rate (RR) of 47.6% (95% CI, 32.5% to 62.7%). Median duration of response was 6.1 months, median time to progression (TTP) was 6.4 months, 1-year survival rate was 64.3%, and median survival time was not reached. EGFR FISH-positive patients, compared with negative patients, had higher RR (68.0% v 9.1%, respectively; P < .001), longer TTP (7.6 v 2.7 months, respectively; P = .02), and a trend for longer survival (median survival not reached v 7.4 months, respectively; P = .3). Therapy was well tolerated, and there were no drug-related deaths. Median follow-up time was too short for significance tests of differences in survival outcomes. CONCLUSION: Gefitinib is active and well tolerated in patients with trial characteristics, and EGFR FISH analysis is an accurate predictor for such therapy.

EGFR and HER2 gene copy number and response to first-line chemotherapy in patients with advanced non-small cell lung cancer (NSCLC).

BACKGROUND: A critical point in designing clinical trials comparing chemotherapy with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in patients with non-small cell lung cancer (NSCLC) is the expected benefit with standard chemotherapy in presence of biological features indicative of TKI sensitivity. The aim of this study was to assess whether EGFR and HER2 gene copy number and Akt activation are associated with response to first-line chemotherapy. METHODS: Tumor samples from 190 patients with NSCLC were analyzed. EGFR and HER2 gene copy number were evaluated by fluorescence in situ hybridization in 185 and 184 cases, respectively. Akt activation was assessed by immunohistochemistry (n = 176). Additional biomarkers included EGFR DNA sequencing (n = 65), and EGFR immunohistochemistry (n = 185). RESULTS: Response rate was not associated with EGFR, HER2, and P-Akt status, irrespective of the method used for biomarker assessment. Among patients with EGFR gene mutations, response to chemotherapy was observed only in individuals with exon 19 deletion (response rate: 46.6% versus 0%, p = 0.02). Among the 190 patients analyzed, 123 received a treatment with a TKI as second- or third-line therapy. When assessed by fluorescence in situ hybridization or DNA sequencing, EGFR-positive patients seemed to be more sensitive to TKIs than to chemotherapy in terms of response rate and time to progression, whereas in EGFR-negative patients, response rate and time to progression favored chemotherapy. CONCLUSION: This study suggested that EGFR expression and gene copy number, HER2 gene copy number, and P-Akt expression are not associated with response to first-line chemotherapy in NSCLC. Prospective phase III trials should compare standard chemotherapy with a TKI in selected NSCLC.

Immunoproteasome and LMP2 polymorphism in aged and Alzheimer's disease brains.

In this study, we investigated the presence and role of immunoproteasome and its LMP2 subunit polymorphism at codon 60 in Alzheimer's disease (AD). Immunoproteasome was present in brain areas such as hippocampus and cerebellum and localized in neurons, astrocytes and endothelial cells. A higher expression of immunoproteasome was found in brain of AD patients than in brain of non-demented elderly, being its expression in young brain negligible or absent. Furthermore, AD affected regions showed a partial decrease in proteasome trypsin-like activity. The study of LMP2 polymorphism (R/H) showed that it does not influence LMP2 expression (neither the mRNA nor mature protein) in brain tissue. However, control brain areas of AD patients carrying the RR genotype showed an increased proteasome activity in comparison with RH carriers. To test whether this effect of the genotype might be related to AD onset we performed a genetic study, which allowed us to exclude an association of LMP2 codon 60 polymorphism with AD onset, despite its influence on the proteasome activity in human brain.

Akt phosphorylation and gefitinib efficacy in patients with advanced non-small-cell lung cancer.

BACKGROUND: Gefitinib, a specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, has activity against approximately 10% of unselected non-small-cell lung cancer (NSCLC) patients. Phosphatidylinositol 3'-kinase (PI3K)/Akt and Ras/Raf/mitogen-activated protein kinase (MAPK), the two main EGFR-signaling pathways, mediate EGFR effects on proliferation and survival. Because activation of these pathways is dependent on the phosphorylation status of the components, we evaluated the association between phosphorylation status of Akt (P-Akt) and MAPK (P-MAPK) and gefitinib activity in patients with advanced NSCLC. METHODS: Consecutive patients (n = 106) with NSCLC who had progressed or relapsed on standard therapy received gefitinib (250 mg/day) until disease progression, unacceptable toxicity, or patient refusal. P-Akt and P-MAPK positivity was determined with immunohistochemistry using tumor tissues obtained before any anticancer treatment. Association of P-Akt and time to progression was determined by univariable and multivariable analyses. All statistical tests were two-sided. RESULTS: Of the 103 evaluable patients, 51 (49.5%) had tumors that were positive for P-Akt, and 23 (22.3%) had tumors that were positive for P-MAPK. P-Akt-positivity status was statistically significantly associated with being female (P<.001), with never-smoking history (P =.004), and with bronchioloalveolar carcinoma histology (P =.034). Compared with patients whose tumors were negative for P-Akt, patients whose tumors were positive for P-Akt had a better response rate (26.1% versus 3.9%; P =.003), disease control rate (60.9% versus 23.5%; P<.001), and time to progression (5.5 versus 2.8 months; P =.004). Response rate, disease control rate, and time to progression did not differ according to P-MAPK status. The multivariable analysis showed that P-Akt positivity was associated with a reduced risk of disease progression (hazard ratio = 0.58, 95% confidence interval = 0.35 to 0.94). CONCLUSIONS: Patients with P-Akt-positive tumors who received gefitinib had a better response rate, disease control rate, and time to progression than patients with P-Akt-negative tumors, suggesting that gefitinib may be most effective in patients with basal Akt activation.