Maria Grazia Mameli

Phosphoinositide-3-Kinase Catalytic Alpha and KRAS Mutations are Important Predictors of Resistance to Therapy with Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Patients with Advanced Non-small Cell Lung Cancer

Background: Specific mutations of the epidermal growth factor receptor (EGFR) gene are predictive for favorable response to tyrosine kinase inhibitors (TKIs) and are associated with a good prognosis. In contrast, Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation has been shown to predict poor response to such therapy. Nevertheless, tumor that initially responds to EGFR-TKIs almost inevitably becomes resistant later. Other mechanisms of resistance to EGFR inhibitors could involve activating mutations of the other main EGFR effector pathway, i.e., the phosphoinositide-3-kinase/phosphate and tensin homologue deleted from chromosome 10 (PTEN)/alpha serine/threonine protein kinase (AKT) pathway. The aim of this study was to investigate the role of phosphoinositide-3-kinase catalytic alpha (PIK3CA), EGFR, and KRAS gene mutations in predicting response and survival in patients with non-small cell lung cancer (NSCLC) treated with EGFR-TKIs. Patients and Methods: A total of 166 patients with advanced NSCLC treated with EGFR-TKI with available archival tissue specimens were included. PIK3CA, EGFR, and KRAS mutations were analyzed using polymerase chain reaction-based sequencing. Results: EGFR mutation was detected in 25.3% of patients, PIK3CA mutation in 4.1%, and KRAS mutation in 6.7%. PIK3CA mutation correlated with shorter median time to progression (TTP) (p = 0.01) and worse overall survival (OS) (p < 0.001). EGFR mutation (p < 0.0001) correlated with favorable response to TKIs treatment and longer TTP (p < 0.0001). KRAS mutation correlated with progressive disease (p = 0.05) and shorter median TTP (p = 0.003) but not with OS. Cox multivariate analysis including histology and performance status showed that PIK3CA mutation was an independent factor to predict worse OS (p = 0.0001) and shorter TTP (p = 0.03), while KRAS mutation to predict shorter TTP (p = 0.01). Conclusion: PIK3CA and KRAS mutations seem to be indicators of resistance and poor survival in patients with NSCLC treated with EGFR-TKIs.

High coexpression of both insulin-like growth factor receptor-1 (IGFR-1) and epidermal growth factor receptor (EGFR) is associated with shorter disease-free survival in resected non-small-cell lung cancer patients

BACKGROUND Insulin-like growth factor receptor-1 (IGFR-1) represents a novel molecular target in non-small-cell lung cancer (NSCLC). IGFR-1 and epidermal growth factor receptor (EGFR) activation is essential to mediate tumor cell survival, proliferation and invasion. We explored the correlation between IGFR-1 and EGFR, their relationship with clinicopathological parameters and their impact on outcome in resected stage I-III NSCLC patients. PATIENTS AND METHODS Tumors from 125 surgical NSCLC patients were evaluated for IGFR-1 and EGFR expression by immunohistochemistry. Kaplan-Meier estimates of survival and time to recurrence were calculated for clinical variables and biologic markers using the Cox model for multivariate analysis. RESULTS IGFR-1 protein overexpression was detected in 36.0% of NSCLC patients and was associated with larger tumor size (P = 0.04) but not with other clinical or biological characteristics. EGFR protein overexpression was observed in 55.2% of NSCLC, more frequently in squamous cell carcinoma (SCC) than non-SCC (63.7% versus 36.3%, chi(2) = 9.8, P = 0.001). IGFR-1 protein expression was associated with EGFR protein expression (P = 0.03). At the multivariate analysis, high coexpression of both IGFR-1 and EGFR was a significant prognostic factor of worse disease-free survival (DFS) (hazard ratio 2.51, P = 0.01). CONCLUSION A statistically significant association was observed between high coexpression of both IGFR-1 and EGFR and worse DFS in early NSCLC patients.

EGFR, pMAPK, pAkt and PTEN status by immunohistochemistry : correlation with clinical outcome in HER2-positive metastatic breast cancer patients treated with trastuzumab

BACKGROUND In an attempt to identify markers of resistance to trastuzumab, we evaluated both the profiling of human epidermal growth factor receptor 2 (HER2)-positive tumor cells measuring the relative levels of EGFR, pMAPK, pAkt and PTEN and their correlations with clinical outcome in HER2-positive metastatic breast cancer patients treated with trastuzumab. PATIENTS AND METHODS Tumor tissues for this retrospective analysis were available from 45 out of 76 patients with metastatic breast cancer treated from April 1999 to March 2006 with trastuzumab-based therapy at our Institution. Evaluations of EGFR, pMAPK, pAkt and PTEN status by immunohistochemistry (IHC) were carried out on all 45 tissue samples and their correlations with response to trastuzumab, incidence of central nervous system (CNS) metastases, time to progression (TTP), overall survival from diagnosis of breast cancer (OS1), from diagnosis of metastatic disease (OS2) and from the start of trastuzumab (OS3) were analyzed. RESULTS We observed that TTP (P = 0.001) and median OS2 and OS3 were significantly longer in patients responsive to trastuzumab-based regimen compared with nonresponsive patients. EGFR, pMAPK, pAkt and PTEN status by IHC were not significantly associated with response to trastuzumab, TTP, overall survival (OS1, OS2, OS3) and CNS metastases incidence. A trend for shorter OS3 was observed for pMAPK-positive patients compared with pMAPK-negative patients (22.8 versus 31.2 months; P = 0.076). Median OS1 resulted shorter in 22 pAkt-positive patients (69.8 months) compared with 23 pAkt-negative patients (108.2 months); P = 0.091. It is likely that high expression of pMAPK (pMAPK-positive status) or pAkt (pAkt-positive status) could identify a subgroup of HER2-positive tumors with high activity of proliferation and survival pathways and with resistance to trastuzumab. CONCLUSIONS In HER2-positive metastatic breast cancers, EGFR, pMAPK, pAkt and PTEN status evaluated by IHC was not significantly associated with response to trastuzumab, TTP, OS and CNS metastases incidence. However, HER2 status determined by IHC and/or FISH assays may not be sufficient to predict response to trastuzumab-based therapy.

Long Glucocorticoid-induced Leucine Zipper (L-GILZ) Protein Interacts with Ras Protein Pathway and Contributes to Spermatogenesis Control

Background: Understanding how spermatogenesis occurs in mammals is not yet fully understood. Results: L-GILZ deficiency in germ cells leads to complete loss of germ cell lineage resulting in male sterility. Conclusion: Our study identifies L-GILZ as an important factor for spermatogenesis. Significance: Identification of genes critical for maintenance of spermatogenesis is pivotal for diagnosis and treatment of male infertility. Correct function of spermatogonia is critical for the maintenance of spermatogenesis throughout life, but the cellular pathways regulating undifferentiated spermatogonia proliferation, differentiation, and survival are only partially known. We show here that long glucocorticoid-induced leucine zipper (L-GILZ) is highly expressed in spermatogonia and primary spermatocytes and controls spermatogenesis. Gilz deficiency in knock-out (gilz KO) mice leads to a complete loss of germ cell lineage within first cycles of spermatogenesis, resulting in male sterility. Spermatogenesis failure is intrinsic to germ cells and is associated with increased proliferation and aberrant differentiation of undifferentiated spermatogonia and with hyperactivity of Ras signaling pathway as indicated by an increase of ERK and Akt phosphorylation. Spermatogonia differentiation does not proceed beyond the prophase of the first meiotic division due to massive apoptosis associated with accumulation of unrepaired chromosomal damage. These results identify L-GILZ as a novel important factor for undifferentiated spermatogonia function and spermatogenesis.

Indoleamine 2,3-Dioxygenase 1 (IDO1) Is Up-Regulated in Thyroid Carcinoma and Drives the Development of an Immunosuppressant Tumor Microenvironment

CONTEXT Indoleamine 2,3-dioxygenase 1 (IDO1) is a single chain oxidoreductase that catalyzes tryptophan degradation to kynurenine. In cancer, it appears to exert an immunosuppressive function as part of an acquired mechanism of immune escape mediated by the inhibition of lymphocyte proliferation and survival and by the induction of FoxP3+ T regulatory cells. OBJECTIVE The objective of the study was to evaluate IDO1 expression in thyroid carcinoma and demonstrate its immunosuppressive function in the context of thyroid tumors. SETTING IDO1 expression was evaluated by quantitative PCR in 105 papillary thyroid carcinomas (PTCs), 11 medullary thyroid carcinomas, six anaplastic thyroid carcinomas, and five thyroid carcinoma cell lines (TCCLs), by immunohistochemistry in 55 PTCs and by Western blotting in five TCCLs. FoxP3+ Treg lymphocyte density was evaluated by immunohistochemistry in 29 PTCs. IDO1 inhibitory effect on lymphocyte proliferation was tested in coculture experiments of TCCLs and activated lymphocytes. RESULTS IDO1 mRNA expression resulted significantly higher in all the analyzed thyroid carcinoma histotypes compared with normal thyroid. Interestingly, an increase of IDO1 mRNA expression magnitude could be observed with gain of aggressiveness (PTCs and medullary thyroid carcinomas ≪ anaplastic thyroid carcinomas). In PTCs, IDO1 mRNA expression magnitude correlated with IDO1 immunostaining intensity in cancer cells and with FoxP3+ Treg lymphocyte density in the tumor microenvironment. IDO1 was expressed in human thyroid cancer cell lines in vitro, and FTC-133 cells showed high kynurenine concentration in the conditioned medium and a strong suppressive action on the proliferation of activated lymphocytes in coculture experiments. CONCLUSIONS For the first time, this study demonstrates a pivotal role of IDO1 in the suppression of lymphocyte function in thyroid carcinoma microenvironment.

Primary signet-ring cell carcinoma of the urinary bladder: a clinicopathologic and immunohistochemical study of 5 cases.

Primary signet-ring cell carcinoma of the urinary bladder is a rare histologic variant of adenocarcinoma. Generally, this neoplasm occurs in middle age and the clinical presentation does not differ from the most frequent transitional cell carcinomas. The prognosis is frequently poor as at diagnosis it is often in an advanced phase. It is essential to distinguish this carcinoma from metastases, as different therapeutic strategies are often necessary. We present 5 cases of primary signet-ring cell carcinoma of the urinary bladder and we used a panel of histochemical and immunohistochemical markers for differential diagnosis from secondary carcinoma in an attempt to elucidate the histogenetic derivation of this neoplasia.

Amniotic membrane: separation of amniotic mesoderm from amniotic epithelium and isolation of their respective mesenchymal stromal and epithelial cells.

The human amniotic membrane (hAM) or amnion contains two principal types of cells: amniotic epithelial cells (hAECs) and amniotic mesenchymal stromal cells (hAMSCs), located in two distinct regions: the epithelium and the stromal layer. Emerging evidence suggests that both of them retain multipotent/pluripotent characteristics, making the amniotic membrane a promising and very attractive source of cells for regenerative medicine. Therefore, the isolation of hAECs and hAMSCs has recently received great interest; they can be released by differential enzymatic digestion and various procedures have been reported; however, significant contamination of hAMCs with hAECs and vice versa frequently occurs. This unit describes an efficient and rapid method to separate, mechanically, amniotic mesoderm from amniotic epithelium in order to obtain, after subsequent enzymatic digestions, purified population of hAMCs and hAECs. In this way, the cells can be cultured or investigated for other aims avoiding additional procedures related to their purification.

HER FAMILY RECEPTORS EXPRESSION IN SQUAMOUS CELL CARCINOMA OF THE TONGUE: STUDY OF THE POSSIBLE PROGNOSTIC AND BIOLOGICAL SIGNIFICANCE

OBJECTIVES The squamous cell carcinoma of the tongue (SCCT) is biologically and epidemiologically distinct from other oral cavity cancers and is associated with lower overall survival rates. The role of HER family members (HER-1, HER-2/neu, HER-3 and HER-4) in the pathogenesis and progression of head and neck squamous cell carcinomas has been demonstrated but no report have focused on SCCT. This study investigated, the expression of all members of the HER family, in a series of SCCT and studied the possible prognostic value and correlation with various clinico-pathological parameters. METHODS HER-1, HER-2/neu, HER-3 and HER-4 expression was analysed by semi-quantitative immunohistochemical staining on paraffin embedded tissue specimens from 40 patients who underwent surgery for SCCT between 1996 and 2006. RESULTS HER-1 was overexpressed in 26 cases (65%), HER-2/neu in two (5%), HER-3 in 19 (48%) and HER-4 in three cases (8%). No significant correlation was found between clinicopathological variables and expression of HER-1 and HER-2/neu. HER-3 overexpression was significantly related to nodal stage, age (>or=64 years) and decreased overall survival (P <or= 0.05). HER-4 overexpression was significantly associated with low histological grade including when it was coexpressed with HER-3 but in this case the prognosis was worse (P < 0.05). CONCLUSIONS These results suggest that HER-1 and HER-2/neu when determined with stringent criteria are not useful indicators of prognosis in SCCT. Only HER-3 overexpression may help in identifying SCCT with greater malignant potential also when it is coexpressed with HER-4. Instead, as in other malignancies, HER-4 could play a protective role in SCCT.

Epithelial cyst of the spleen with foci of ectopic liver

Epithelial cysts account for about 20% of all splenic cysts. Their pathogenesis is unclear, and different authors have proposed many hypotheses. It has been suggested that they are derived from embryonal epithelial inclusions during splenic development, from invagination of capsular surface mesothelium in splenic sulci with subsequent metaplasia, or from trauma. Moreover, a congenital, genetic, or teratomatous origin has also been hypothesized. We describe an unusual case of epithelial splenic cyst with mature liver foci in its wall. This finding supports its possible dysontogenetic origin.

Endometriosis-associated skeletal muscle regeneration: a hitherto undescribed entity and a potential diagnostic pitfall.

Skeletal muscle undergoes regeneration generally after an injury and in some cases it may mimic a malignant process. We observed these aspects in association with abdominal wall endometriosis and as no similar conditions were found in the literature this prompted us to study the main clinicopathologic and immunohistochemical profile of this phenomenon. Thirteen cases of abdominal wall endometriosis were retrieved from the files of our Institute. All original slides were reviewed to reveal the presence of skeletal muscle and 8 cases were enrolled for morphologic and immunohistochemical studies as follows: vimentin, desmin, myoglobin, myogenin, myoD1, CD56, S100, and p21. Histologically, in 4 of the 8 cases in the skeletal muscle adjacent to the endometriotic foci there was a proliferation of round cells with the typical appearance of maturing myoblasts. More peripherally, myotubes and early myocytes were present. This proliferation was florid in 1 case and focal in 3 cases. At immunohistochemical investigation, the less differentiated cells reacted with vimentin, desmin, S100, CD56, myoD1, and myogenin but not with myoglobin or p21. On the contrary, intermediately differentiated cells showed a progressive loss of vimentin, CD56, and myoD1 whereas they were positive for desmin, S100, myogenin, myoglobin, and p21. Terminally differentiated cells reacted only with desmin and myoglobin. This peculiar immunohistochemical profile was consistent with the immunophenotype of maturing myoblasts, confirmed the regenerative nature of the phenomenon and allowed differential diagnosis with other proliferations sharing a similar morphology. The expression of early differentiation markers was greatest in the islands of cells nearest to endometriosis, whereas in the more distant areas the markers of late differentiation prevailed. This gradient of expression suggests that muscle cells are stimulated by growth factors or other signals produced by the cycling endometrioid foci. In conclusion, we report a hitherto undescribed entity that may mimic a malignant process, especially when the reaction is florid or when endometriotic glands and stroma are not clearly evident, as during the examination of small biopsies, frozen sections, or cytology samples. Therefore, although the histologic diagnosis of endometriosis is usually straightforward, pathologists should be aware of the concomitant regenerative effects on skeletal muscle, which may represent a possible diagnostic pitfall.