Expression of nuclear survivin in normal skin and squamous cell carcinoma: a possible role in tumour invasion.

BACKGROUND: Survivin is detected in few adult normal cells and it is highly expressed in cancer. Nuclear survivin facilitates cell cycle entry, whereas the mitochondrial pool protects cells from apoptosis. Survivin is overexpressed in keratinocyte stem cells (KSCs) and protects them from apoptosis. METHODS: As KSCs are at the origin of squamous cell carcinoma (SCC), we evaluated survivin expression in normal and cancerous skin in vivo by immunohistochemistry and western blotting. HaCaT cells overexpressing survivin and wound-healing assay are used. Analysis of variance and Student's T-tests are used for statistical analysis. RESULTS: Survivin is localised in both the cytoplasm and nucleus of normal adult and young keratinocytes. Nuclear survivin is detected in one every 10 of 11 basal keratinocytes. When present in suprabasal cells, nuclear survivin is coexpressed with K10 but not with K15 or p75-neurotrophin receptor (p75NTR), a transit amplifying cell marker. Nuclear, but not cytoplasmic, survivin expression markedly increases in actinic keratosis and in SCC in situ, as compared with normal epidermis, and it is highest in poorly differentiated SCC. In SCC tumours, nuclear survivin-positive cells are mainly K10/p75NTR-negative and K15-positive. In poorly differentiated tumours, survivin mostly localises in the deep infiltrating areas. When overexpressed in keratinocytes, survivin increases cell migration. CONCLUSION: High survivin expression and the subcellular localisation of survivin correlate with keratinocyte differentiation and are associated with undifferentiated and more invasive SCC phenotype.

[External quality controls for allergy analysis in the Quality Control Center Switzerland from 2006 to 2008]. / Les contrôles de qualité externes pour le laboratoire d'allergologie auprès du Centre suisse de contrôle de qualité de 2006 à 2008.

External assessment of analytical performance is part of the quality assurance in medical laboratory. These external controls are mandatory in Switzerland since 2006 for IgE analysis. The Swiss Society for Immunology and Allergy and the Swiss external quality centers had launched a program for total IgE, IgE specific for cat epithelium, birch pollen and peanut, and multi-specific IgE. They have set up criteria for proficiency assessment. Analysis of data obtained from 2006 to 2008 in the Quality Control Center Switzerland shows that results are very good for all the methods used and that a large number of participants fulfill the requirements to obtain the certificate of QUALAB conformity.

Chemokine receptors in advanced breast cancer: differential expression in metastatic disease sites with diagnostic and therapeutic implications.

BACKGROUND: We investigated the expression of CXCR4, CCR7, estrogen receptor (ER), progesterone receptor (PR) and HER2-neu in human metastatic breast cancers to determine whether these biological biomarkers were preferentially expressed in any organ-specific metastases. MATERIALS AND METHODS: CXCR4, CCR7, ER, PR and HER2-neu expression levels were evaluated by immunohistochemical staining using paraffin-embedded tissue sections of metastatic breast cancers (n = 41) obtained by either diagnostic biopsy or surgical resection. RESULTS: The metastatic sites included the following: bone (n = 15), brain (n = 14), lung (n = 6), liver (n = 2), and omental metastases (n = 2). CXCR4 was expressed in 41% of cases, CCR7 expression was demonstrated in 10%, and HER2-neu overexpression was present in 27%. CXCR4 was more likely to be expressed in bone metastases than visceral metastases (67% versus 26%, P = 0.020). Visceral sites demonstrated a lower rate of CXCR4 positivity (33% and 23%, respectively, for lung and brain metastases). Similarly, CCR7 was more likely to be found in bone metastases than visceral sites (27% versus 0%, P = 0.037). CONCLUSIONS: These results indicate that CXCR4 can contribute to the homing of breast cancer cells to the bone. This finding might have important clinical implications since patients with metastatic bone disease may achieve the highest benefit from a CXCR4-targeted therapy.

Imatinib mesylate (Gleevec) in advanced breast cancer-expressing C-Kit or PDGFR-beta: clinical activity and biological correlations.

BACKGROUND: Novel molecular therapies for metastatic breast cancer (MBC) are necessary to improve the dismal prognosis of this condition. Imatinib mesylate (Gleevec) inhibits several protein tyrosine kinases, including platelet-derived growth factor receptor (PDGFR) and c-kit, which are preferentially expressed in tumor cells. We tested the activity of imatinib mesylate in MBC with overexpression of PDGFR or c-kit. Additionally, we sought to determine the biological correlates and immunomodulatory effects. PATIENTS AND METHODS: Thirteen patients were treated with Imatinib administered orally at 400 mg p.o. b.i.d. (800 mg/day), until disease progression. All patients demonstrated PDGFR-beta overexpression and none showed c-kit expression. RESULTS: No objective responses were observed among the 13 patients treated in an intention-to-treat analysis. All patients experienced disease progression, with a median time to progression of 1.2 months. Twelve patients have died, and the median overall survival was 7.7 months. No patient had a serious adverse event. Imatinib therapy had no effect on the plasma levels of the angiogenesis-related cytokines, vascular endothelial growth factor, PDGF, b-fibroblast growth factor, and E-selectin. Immune studies showed imatinib inhibits interferon-gamma production by TCR-activated CD4(+) T cells. CONCLUSION: Imatinib as a single agent has no clinical activity in PDGFR-overexpressing MBC and has potential immunosuppressive effects.

Expression of growth factor and chemokine receptors: new insights in the biology of inflammatory breast cancer.

PURPOSE: Recent studies have indicated that expression of chemokine receptors CXCR4 and CCR7 could be an indicator of the metastatic potential of breast cancer. Expression of CXCR4 and CCR7 along with the biomarkers HER2-neu and epidermal growth factor receptor (EGFR) was investigated in inflammatory breast cancer (IBC) to evaluate their prognostic implications. EXPERIMENTAL DESIGN: CXCR4, CCR7, and EGFR were evaluated by immunohistochemical staining (IHC) of paraffin-embedded tissue sections. HER2-neu amplification was assessed by FISH and/or IHC. All patients received chemotherapy, surgery, and radiation. RESULTS: Forty-four cases diagnosed with IBC from 1994 to 2002 were included in the study. In all, 18 (40.9%) patients had positive CXCR4, 10 (22.7%) had positive CCR7, 21 (47.7%) had positive HER2-neu, and EGFR was positive in 12 of 40 patients (30%). The 5-year overall survival (OS) was 24.8% for CXCR4-positive disease versus 42.3% for CXCR4-negative patients (P = 0.53) and 20.0% for CCR7-positive disease versus 41.9% for CCR7-negative patients (P = 0.24). EGFR-positive disease had significantly worse OS compared with EGFR-negative disease (P = 0.01). CONCLUSIONS: These data demonstrate the expression of growth factor and chemokine receptors in IBC. The expression of these receptors is associated with increased risk of recurrence and death, and thus, they may represent potential therapeutic targets in IBC.

Cardiac toxicity of high-dose chemotherapy.

Cardiac toxicity is an uncommon but potentially serious complication of high-dose (HD) chemotherapy and little is known about incidence, severity and underlying mechanisms. We have systematically reviewed the literature of the last 30 years to summarize and appraise the published evidence on cardiac toxicity associated with HD chemotherapy. HD cyclophosphamide-containing regimens have been most commonly associated with cardiac toxicity, with a progressively decreasing incidence over time. Dosage, application regimens and coadministration of other chemotherapeutic agents emerged as risk factors. While cardiac toxicity has been rarely associated with other cytotoxic drugs, an unexpected incidence of severe cardiotoxicity resulted from reduced-intensity conditioning regimens containing melphalan and fludarabine. Predictive value of cardiologic examination of patients is limited, and patients with a slight depression of cardiac performance could tolerate HD chemotherapy. Clinical examination, resting electrocardiography and dosage adjustment in overweight patients remain the mainstay of prevention, with bidimensional echocardiography (2D echo) for patients with a history of anthracycline exposure. Strategies to decrease the long-term negative impact of anthracycline administration on cardiac performance are being investigated. New 2D echo-based techniques and circulating markers of cardiac function hold promise for allowing identification of patients at high risk for and early diagnosis of cardiac toxicity.

Treatment of advanced mycosis fungoides by allogeneic stem-cell transplantation with a nonmyeloablative regimen.

SUMMARY: Given the poor prognosis of patients with advanced cutaneous T-cell lymphoma and the high transplant-related mortality associated with conventional allogeneic bone marrow transplantation, we performed nonmyeloablative transplantation of allogeneic stem cells (ASCT) from HLA-identical siblings in three patients with this disease. All patients achieved full donor engraftment, clearance of clonal T cells leading to durable complete remissions but experienced high incidence of infections, which proved fatal in one case. These results suggest that nonmyeloablative ASCT is a novel and potentially curative therapy for patients with advanced T-cell lymphomas who have a histocompatible sibling.

Serum cardiac troponin I levels and ECG/Echo monitoring in breast cancer patients undergoing high-dose (7 g/m(2)) cyclophosphamide.

High-dose cyclophosphamide (HD-CTX) is largely employed in high-dose chemotherapy (HD-CHT) protocols. HD-CTX dose-limiting toxicity expresses itself as cardiac toxicity which is fatal in a minority of patients. The pathophysiology of HD-CTX-associated cardiotoxicity is still poorly understood. Autopsy studies in patients who died from acute HD-CTX-induced cardiac toxicity revealed hemorrhagic myocardial cell death and interstitial edema. Recently troponins, in particular troponin I (cTnI), have been found to represent a uniquely sensitive and specific marker of myocyte membrane integrity and therefore to increase in response to minimal myocardial cell damage in different settings, including doxorubicin-induced cardiotoxicity. We performed a multiparametric cardiologic monitoring in 16 consecutive breast cancer patients undergoing HD-CTX by means of serial ECG registrations and cardiac enzymes (CPK, CPK-MB and cTnI) determinations plus echocardiography in order to clarify acute cardiac events following HD-CTX administration. Neither overt cardiac toxicity nor cardiac enzymes elevation were recorded. Serial ECGs revealed in six cases little and reversible reduction of QRS voltage and/or ST abnormalities. Echo monitoring showed in four cases mild and transient increase of LV diastolic/systolic diameter/volume without decrease of FS% or EF% below normal values: in two of them abnormalities of diastolic function (E/A mitral doppler ratio) were also recorded. We conclude that our protocol of HD-CTX administration does not cause myocardial cell damage as analyzed by serum cTnI levels, thus suggesting that myocyte membrane injury may not be the first direct mechanism of HD-CTX cardiotoxicity. ECG (ie QRS voltages ) and Echo (ie E/A ratio) monitoring leads us to hypothesize that slight interstitial edema with reduction of LV diastolic compliance may be initial signs of cardiac dysfunction in this clinical setting.

[Assessment of cardiotoxicity of high dose cyclophosphamide with electrocardiographic, echocardiographic, and troponin I monitoring in patients with breast tumors]. / Analisi della cardiotossicità da ciclofosfamide ad alte dosi mediante monitoraggio elettrocardiografico, ecocardiografico e della troponina I in pazienti con tumore della mammella.

BACKGROUND: High-dose cyclophosphamide is the nucleus for virtually all high-dose chemotherapy protocols. Non-hematologic dose-limiting toxicity is represented by acute cardiomyopathy, even fatal in a minority of patients. The pathophysiology of such a cardiotoxicity is still poorly understood. Postmortem studies revealed hemorrhagic myocardial cell death, endothelial damage, and interstitial edema. Recently troponins, in particular troponin I, have been found to represent uniquely sensitive and specific markers of myocyte membrane integrity, thus to increase in response to myocardial cell damage in different clinical settings. METHODS: We performed a multiparametric monitoring in 16 consecutive breast cancer patients undergoing cyclophosphamide, by means of serial ECGs, cardiac enzymes determinations (creatine phosphokinase, MB mass and troponin I) through 0 to 72 hours, and echocardiography at baseline and after 48 hours. RESULTS: Neither overt cardiac failure nor enzyme elevation were recorded. Serial ECGs revealed a reduction in QRS voltage and/or ST segment abnormalities in 6 cases. Echocardiography showed an increase in left ventricular diastolic and/or systolic diameters and volumes in 4 cases but without any decrease in fractional shortening and ejection fraction under normal values: in 2 of them abnormalities of diastolic function (E/A mitral Doppler ratio, isovolumic relaxation time and deceleration time) were also recorded. CONCLUSIONS: Our protocol of cyclophosphamide administration did not cause cardiac toxicity by myocardial cell damage, as analyzed by troponin I levels, thus suggesting that myocyte membrane injury is not the first mechanism of it. ECG (i.e. QRS voltages) and echo-Doppler (i.e. E/A ratio) monitoring lead to hypothesize that endothelial injury and interstitial edema with subsequent reduction in left ventricular diastolic compliance may be the first signs of cardiac dysfunction in this clinical setting.

Glutamate synthase: identification of the NADPH-binding site by site-directed mutagenesis.

To contribute to the understanding of glutamate synthase and of beta subunit-like proteins, which have been detected by sequence analyses, we identified the NADPH-binding site out of the two potential ADP-binding regions found in the beta subunit. The substitution of an alanyl residue for G298 of the beta subunit of Azospirillum brasilense glutamate synthase (the second glycine in the GXGXXA fingerprint of the postulated NADPH-binding site) yielded a protein species in which the flavin environment and properties are unaltered. On the contrary, the binding of the pyridine nucleotide substrate is significantly perturbed demonstrating that the C-terminal potential ADP-binding fold of the beta subunit is indeed the NADPH-binding site of the enzyme. The major effect of the G298A substitution in the GltS beta subunit consists of an approximately 10-fold decrease of the affinity of the enzyme for pyridine nucleotides with little or no effect on the rate of the enzyme reduction by NADPH. By combining kinetic measurements and absorbance-monitored equilibrium titrations of the G298A-beta subunit mutant, we conclude that also the positioning of its nicotinamide portion into the active site is altered thus preventing the formation of a stable charge-transfer complex between reduced FAD and NADP(+). During the course of this work, the Azospirillum DNA regions flanking the gltD and gltB genes, the genes encoding the GltS beta and alpha subunits, respectively, were sequenced and analyzed. Although the Azospirillum GltS is similar to the enzyme of other bacteria, it appears that the corresponding genes differ with respect to their arrangement in the chromosome and to the composition of the glt operon: no genes corresponding to E. coli and Klebsiella aerogenes gltF or to Bacillus subtilis gltC, encoding regulatory proteins, are found in the DNA regions adjacent to that containing gltD and gltB genes in Azospirillum. Further studies are needed to determine if these findings also imply differences in the regulation of the glt genes expression in Azospirillum (a nitrogen-fixing bacterium) with respect to enteric bacteria.

Three new active cisplatin-containing combinations in the neoadjuvant treatment of locally advanced and locally recurrent breast carcinoma: a randomized phase II trial.

We designed three new four-drug cisplatin-containing combinations and evaluated their activity in a randomized phase II study including patients with locally advanced (stage III) and locally recurrent breast carcinoma. All combinations included methotrexate (M) on day 1 and cisplatin (P) on day 2 (MVAC-like combinations) and differed from one another by the addition of Epirubicin (Epi), Vincristine (V), Etoposide (E), Mitomycin (Mi). Based on the administered agents, they were named MPEMi, MPEpiE, MPEpiV. The combinations were randomly assigned to 101 patients, 57 with locally advanced and 44 with locally recurrent breast carcinoma. Response was evaluated after 4 cycles. The complete response (CR) rates were 7% and 43% and the CR plus partial response (PR) rates were 84% and 89% in locally advanced and in locally recurrent disease, respectively. In locally advanced disease, a pathologic CR (pCR) was assessed in seven of 57 patients (12%). There were no significant differences among the three combinations. The toxicities were at times severe, but generally tolerable, as demonstrated by the high cumulative doses of the drugs received by the patients. In conclusion, these three innovative chemotherapy regimens induced high CR plus PR rates in the neoadjuvant treatment of stage III and of locally recurrent breast carcinoma, and a high rate of pCR in stage III disease. These regimens warrant testing in phase III trials.

Detection of the breakpoint cluster region-ABL fusion in chronic myeloid leukemia with variant Philadelphia chromosome translocations by in situ hybridization.

Fluorescence in situ hybridization (FISH) technique has been successfully used to detect the BCR-ABL gene fusion in chronic myeloid leukemia (CML) with the classic form of the Philadelphia chromosome (Ph). We applied FISH to study three CML patients showing variant Ph chromosome (either complex or simple type). The results demonstrate that the use of a yeast artificial chromosome (YAC)-derived probe (D107F9) and a cosmid probe (cos-abl 8), specific for BCR and ABL genes respectively, allows also the detection of the BCR-ABL fusion in CML patients with variant Ph.

Epirubicin + G-CSF as peripheral blood progenitor cells (PBPC) mobilising agents in breast cancer patients.

BACKGROUND: In an attempt to mobilise peripheral blood progenitor cells (PBPC) from patients with breast cancer, Epirubicin supported with G-CSF was tested. Another aim of the study was also to optimize the procedure so that the number of leukapheresis procedures could be reduced. These cells were subsequently reinfused as hematologic rescue after high-dose chemotherapy programs. PATIENTS AND METHODS: Twenty-nine patients received Epirubicin 150 mg/sqm + G-CSF at the dose of 5 micro/kg/bw s.c. daily, starting 24 hours after chemotherapy. Twelve had metastatic, eight inflammatory or locally advanced disease, and nine were treated in an adjuvant setting. RESULTS: The median numbers of CD34+ cells and CFU-GM collected were 12.9 x 106/kg/bw and 111.7 x 10(4)/kg/bw, respectively. The mean number of leukapheresis procedures per patient was 1.8 +/- 0.3 (range 1-3), and the mean day of the first procedure was the tenth +/- 1 (range 8-13) after Epirubicin. The minimum required target for one high-dose procedure was collected in a single leukapheresis in 13 patients. Moreover, in 9 cases one procedure was adequate for two high-dose courses (i.e. > or = 10 x 10(6)/kg/bw CD34+ cells). Response to Epirubicin was evaluable in 14/20 cases, with a response rate of 50%. CONCLUSIONS: Epirubicin delivered at 150 mg/sqm is a very effective mobilising agent for breast cancer patients; to ameliorate the response rate other active drug(s) should be added.

Gastric dysplasia. A follow-up study.

Gastric dysplasia is generally accepted as a precancerous lesion. Ninety-nine patients with an initial diagnosis of gastric dysplasia, based on examination of endoscopic biopsies taken because of symptoms of dyspepsia, were followed to define the magnitude of the neoplastic risk. The degree of dysplasia in the initial biopsy was mild in 73 cases, moderate in 16, and severe in 10. Mild dysplasia was no longer detected in 74% of patients, persisted in 19%, and progressed in 7% (in four cases, to carcinoma). Moderate dysplasia regressed to mild dysplasia in 31% of cases, it was no longer found in 56%, and progressed to cancer in 13%. Our data show that both lesions can progress slowly, although in most instances they remain stable or regress. Thus, annual endoscopic and histologic controls appear to be advisable. Severe dysplasia was no longer detected in 20% of cases, regressed to moderate in 10%, persisted in 10%, and progressed to cancer in 60%; in half of these patients, carcinoma was detected within 3 months. Thus, severe dysplasia indicates a high risk of cancer, often a synchronous one, and it requires gastrectomy when it persists in repeated biopsies.

Ciliated bodies in ovarian cyst aspirates.

Ciliated bodies, or detached ciliary tufts, are ciliated fragments of cytoplasm from columnar epithelial cells. In gynecologic cytology they have been observed in cervicovaginal smears and peritoneal washings and very occasionally in fluids from the pouch of Douglas. They occur frequently in the fluids of ovarian cysts. The authors detected ciliated bodies in 30 of 326 ovarian cyst fluid samples (9.20% of cases) taken from 27 patients aged 14-68 years. Ciliated bodies were found in the fluids from both bilateral cysts in one patient and in slides from both former and recurrent cysts in two. Surgery was performed in 14 of the cases, with histologic examination revealing 5 simple serous cysts, 3 paraovarian cysts, 3 serous papillary cystoadenofibromas, 1 serous cystoadenoma and 2 mature cystic teratomas. In the remaining cases the pertinent clinical data, including ultrasound ecography, revealed benign paraovarian and ovarian cysts. The existence of ciliated bodies in the fluids of ovarian cysts indicated the presence of ciliated columnar epithelial cells on the wall of the cysts, which would exclude cysts of follicular origin.

Allogeneic bone marrow transplantation in children from other than HLA-identical sibling donor.

Optimal allogeneic bone marrow transplantation (BMT) presupposes the use of a HLA-identical sibling as donor. Unfortunately, only about 30% of patients have an HLA-matched donor, so that the use of alternative donors has been increasingly used. We report an analysis of 13 children transplanted using an HLA-partially matched donor as source of haemopoietic stem cells. They suffered of ALL (3 pts), ANLL (1 pt), SAA (2 pts), Osteopetrosis (1 pt), Wiskott-Aldrich Syndrome (2 pts), Severe Combined Immunodeficiency Disease (2 pts) and Familial Haemophagocitic Lymphohistiocytosis (2 pts). Full engraftment was obtained in all 11 of the patients who survived longer than 14 days and, globally, a moderate incidence of acute GvHD (grade II-IV) was observed in the evaluable patients (3 out of 11 with a percentage of 27%); only a patient of the six survivors more than one hundred days after BMT had severe chronic GvHD (16.6%). Four pts (31%) are actually alive and well (mean follow-up 358 days) with a mean Karnofsky score of 95%. Our data suggest that BMT from HLA-partially matched donors could represent a possible alternative therapeutic strategy in children when a compatible donor is not available. This is especially due to the reduced severity of GvHD in childhood and because of T-cell depleted marrow transplants could obtain more satisfactory results when employed in typical pediatric non-malignant disorders (i.e. immunodeficiencies) rather than in leukemia.