Diagnostic and prognostic value of low percentage of glycosylated ferritin in acquired hemophagocytic lymphohistiocytosis: A single-center study.

INTRODUCTION: Hemophagocytic lymphohistiocytosis (HLH) is an aggressive and life-threatening syndrome characterized by an excessive immune activation. Glycosylated ferritin (GF) level has been proposed as highly specific of HLH. METHODS: We have studied 12 subjects with HLH according to the HLH-04 trial criteria and 11 patients with a clinical and laboratoristic suspicion of HLH. The percentage of GF was measured by an in-house assay. RESULTS: The only biomarkers that were significantly different in the two groups were fraction of GF (P<.001) and the presence of hemophagocytosis in bone marrow (P=.006). Subjects with HLH had significantly lower percentage of GF than patients with other inflammatory conditions mimicking HLH. A fraction of GF ≤20% was strongly consistent with a diagnosis of HLH. CONCLUSIONS: Fraction of GF is useful to identify subjects at high risk for early death and therefore in need of early treatment.

Polyclonal and monoclonal B lymphocytes response in HCV-infected patients treated with direct-acting antiviral agents.

Hepatitis C virus (HCV) chronic infection can be associated with extrahepatic manifestations such as mixed cryoglobulinaemia and lymphoproliferative disorders that are endowed with increased rates of morbidity and all-cause mortality. In this study, we used flow cytometry to evaluate the effect of interferon-free antiviral treatment on peripheral blood lymphocytes in HCV-infected patients with or without associated lymphoproliferative disorders. Flow cytometry analysis of peripheral blood lymphocytes was performed at baseline and at the end of treatment. In HCV-infected patients with lymphoproliferative disorders, we evaluated immunoglobulin (Ig) light chain κ/λ ratio variations as a measure of monoclonal B-cell response to antiviral therapy. Healthy volunteers were enrolled as controls. A total of 29 patients were included, nine with and 20 without lymphoproliferative disorders. Sustained virological response was achieved in 29 of 29 patients. We observed a significant reduction in the B-cell compartment (39% global reduction) in eight of nine HCV-infected patients with lymphoproliferative disorders after viral clearance. We recognized the same trend, even if less pronounced, in HCV-infected patients without lymphoproliferative disorders (9% global reduction). Among HCV-infected patients with lymphoproliferative disorders, three showed an improvement/normalization of the immunoglobulin light chain ratio, whereas in the remaining six patients monoclonal B cells persisted to be clonally restricted even 1 year after the end of treatment. Our data show that DAAs treatment can be effective in reducing the frequency of pathological B cells in the peripheral blood of HCV-infected patients affected by HCV-associated lymphoproliferative disorders; however, monoclonal populations can persist after viral eradication.

Definition of the upper reference limit for thyroglobulin antibodies according to the National Academy of Clinical Biochemistry guidelines: comparison of eleven different automated methods.

PURPOSE: In the last two decades, thyroglobulin autoantibodies (TgAb) measurement has progressively switched from marker of thyroid autoimmunity to test associated with thyroglobulin (Tg) to verify the presence or absence of TgAb interference in the follow-up of patients with differentiated thyroid cancer. Of note, TgAb measurement is cumbersome: despite standardization against the International Reference Preparation MRC 65/93, several studies demonstrated high inter-method variability and wide variation in limits of detection and in reference intervals. Taking into account the above considerations, the main aim of the present study was the determination of TgAb upper reference limit (URL), according to the National Academy of Clinical Biochemistry guidelines, through the comparison of eleven commercial automated immunoassay platforms. METHODS: The sera of 120 healthy males, selected from a population survey in the province of Verona, Italy, were tested for TgAb concentration using eleven IMA applied on as many automated analyzers: AIA-2000 (AIA) and AIA-CL2400 (CL2), Tosoh Bioscience; Architect (ARC), Abbott Diagnostics; Advia Centaur XP (CEN) and Immulite 2000 XPi (IMM), Siemens Healthineers; Cobas 6000 (COB), Roche Diagnostics; Kryptor (KRY), Thermo Fisher Scientific BRAHMS, Liaison XL (LIA), Diasorin; Lumipulse G (LUM), Fujirebio; Maglumi 2000 Plus (MAG), Snibe and Phadia 250 (PHA), Phadia AB, Thermo Fisher Scientific. All assays were performed according to manufacturers' instructions in six different laboratories in Friuli-Venezia Giulia and Veneto regions of Italy [Lab 1 (AIA), Lab 2 (CL2), Lab 3 (ARC, COB and LUM), Lab 4 (CEN, IMM, KRY and MAG), Lab 5 (LIA) and Lab 6 (PHA)]. Since TgAb values were not normally distributed, the experimental URL (e-URL) was established at 97.5 percentile according to the non-parametric method. RESULTS: TgAb e-URLs showed a significant inter-method variability. Considering the same method, e-URL was much lower than that suggested by manufacturers (m-URL), except for ARC and MAG. Correlation and linear regression were unsatisfactory. Consequently, the agreement between methods was poor, with significant bias in Bland-Altman plot. CONCLUSIONS: Despite the efforts for harmonization, TgAb methods cannot be used interchangeably. Therefore, additional effort is required to improve analytical performance taking into consideration approved protocols and guidelines. Moreover, TgAb URL should be used with caution in the management of differentiated thyroid carcinoma patients since the presence and/or the degree of TgAb interference in Tg measurement has not yet been well defined.

First-line screening tests for Cushing's syndrome in patients with adrenal incidentaloma: the role of urinary free cortisol measured by LC-MS/MS.

INTRODUCTION AND AIM: Patients with adrenal incidentaloma present a wide range of cortisol secretion, which is not always properly defined by first-line screening tests recommended to rule out Cushing's syndrome (CS), such as 1-mg dexamethasone suppression test (1-mg DST), late night salivary cortisol (LNSC), or 24-h urinary free cortisol (UFC). Therefore, we examined the diagnostic performance of each screening test in patients with adrenal incidentaloma. MATERIALS AND METHODS: In a series of 164 consecutive patients with adrenal incidentaloma, we measured serum cortisol after 1-mg DST, LNSC, and UFC (with LC-MS/MS). Medical history was investigated for cardiovascular events (CVE) in a subgroup of 93 patients with at least 2 years of follow-up. RESULTS: Serum cortisol <50 nmol/L after 1-mg DST presented the highest sensitivity (100%) to rule out CS, despite a low specificity (62%). UFC > 170 nmol/24 h achieved the highest diagnostic accuracy (sensitivity 98%, specificity 91%, and negative/positive likelihood ratios of 0.02/10.83, respectively). The prevalence of CVE was higher in patients with non-suppressed cortisol after 1-mg DST and high UFC levels (p = 0.018). Traditional cardiovascular risk factors (hypertension, diabetes mellitus, dyslipidemia, BMI > 30 kg/m2, smoke or high gender-based waist circumference) were not associated with CVE. CONCLUSIONS: The 1-mg DST at its lowest threshold presented high sensitivity in identifying CS, but its low specificity encourages us to consider UFC levels, measured with LC-MS/MS, to reduce false-positive test results. High UFC levels could also be considered as markers to stratify cardiovascular risk in patients with adrenal incidentaloma.

[Gender differences in the use of tumour markers]. / Diferencias de género en el uso de marcadores tumorales.

INTRODUCTION AND OBJECTIVE: Gender is one of the factors that can influence the use of health resources. The use of tumour markers is widespread, due to the importance of these in monitoring cancer development. The aim of this study is to analyse the influence of gender on the use of tumour markers, and to investigate whether there are differences in their use. MATERIAL AND METHODS: A longitudinal, retrospective and descriptive study, with a 2-year follow-up, was conducted in the catchment area of the University Hospital of Padua. An analysis was performed on 23,059 analytical requests for tumour markers. A descriptive and frequency analysis was performed on all variables. The statistical analysis was performed using Chi squared, Student t and Mann-Whitney U to test for significance. RESULTS: The number of requests for women (1.5) was lower than men (1.6). In patients with tumour pathology, the number of requests was higher than in patients without tumour disease. In the analysis by disease and gender, the difference remained significant. As regards the number of tumour markers per request, the difference between genders was also significant: 2.13 in males versus 2.85 in women. Similar results were obtained when requests for tumour markers linked to gender-related diseases were eliminated. CONCLUSIONS: There are differences in the use of tumour markers by gender with the number of requests for male patients being higher than for females. However, the number of tumour markers per request is greater in women than in men.

Is HE4 a useful endometrioma marker?

PURPOSE OF INVESTIGATION: By the comparison between most used tumor marker trend (cancer antigen 125: CA 125 and human epididymal secretory protein E4: HE4) before and after laparoscopic surgery, the aim of the present study was to assess HE4 usefulness in ovarian benign cyst and endometrioma diagnosis. MATERIALS AND METHODS: Thirty-eight patients were enrolled in this prospective study: 25 women underwent unilateral endometriosis ovarian cyst excision, 13 underwent benign ovarian cyst incision, and 26 were healthy controls. CA 125 and HE4 serum levels were estimated before surgery (in the early proliferative phase of the cycle) and one month after surgery. RESULTS: A statistically significant decrease of CA 125 serum level was found after an endometrioma surgical excision but no decreases in HE4 serum level. CONCLUSION: In patients with endometrioma, no alteration was found in HE4 serum levels before and after surgery, while CA125 serum levels decreased after surgery. HE4 may better distinguish a malign cyst from benign one, but it is not useful in the diagnosis of low risk endometrioma.

Impact of hypertension on vascular remodeling in patients with psoriatic arthritis.

We studied the impact of hypertension along with traditional and new cardiovascular risk factors on the structural and functional properties of arteries in psoriatic arthritis (PsA) patients. We examined 42 PsA subjects (aged 51±9 years) stratified according to hypertensive status (19 normotensive, PsA-NT and 23 hypertensives, PsA-HT). Thirty-eight normotensive subjects (C-NT) and 23 hypertensives (C-HT) comparable by age and sex served as controls. Mean carotid intima-media thickness (mean-IMT) and mean of the maximum IMT (M-Max) were evaluated by ultrasound in carotid artery segment bilaterally. Post-occlusion flow-mediated dilation (FMD) of the brachial artery was evaluated by ultrasonography. These parameters were correlated with risk factors, markers of inflammation and disease activity. Values of mean-IMT were higher in both groups of PsA patients compared with C-NT (0.68 mm in PsA-NT and 0.75 mm in PsA-HT versus 0.61 mm in C-NT). PsA-HT displayed higher M-Max (0.95 mm) versus both C-HT (0.71 mm) and PsA-NT (0.79 mm). FMD was impaired in PsA subjects compared with C-NT (5.7% in PsA-NT and 6.0% PsA-HT versus 9.3% in C-NT), whereas there was no difference among PsA-HT, PsA-NT, and C-HT groups. Values of carotid IMT were directly related to tumor necrosis factor (TNF)-α, osteoprotegerin (OPG), blood pressure and lipid profile levels. FMD showed an inverse relationship with TNF-α and blood pressure, but no correlation with lipids. In conclusion, PsA per se implies a pro-atherogenic remodeling, which is enhanced by the hypertensive status. TNF-α and OPG may have an independent role in the development of such vascular damage.

Quality indicators to detect pre-analytical errors in laboratory testing.

The identification of reliable quality indicators (QIs) is a crucial step in enabling users to quantify the quality of laboratory services. The current lack of attention to extra-laboratory factors is in stark contrast to the body of evidence pointing to the multitude of errors that continue to occur, particularly in the pre-analytical phase. The ISO 15189: 2012 standard for laboratory accreditation defines the pre-analytical phase, and recognizes the need to evaluate, monitor and improve all the procedures and processes in the initial phase of the testing cycle, including those performed in the phase of requesting tests and collecting samples, the so-called "pre-pre-analytical phase". Therefore, QIs should allow the identification of errors and non-conformities that can occur in all steps of the pre-analytical phase. Traditionally, pre-analytical errors are grouped into identification and sample problems. However, appropriate test requesting and complete request forms are now recognized as fundamental components in providing valuable laboratory services. The model of QIs developed by the Working Group of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) includes indicators related to both identification and sample problems as well as all other pre-analytical defects, including those in test requesting and request forms. It, moreover, provides the framework (with objective criteria) necessary for promoting the harmonization of available QIs in the pre-analytical phase.

Tryptophan hydroxylase autoantibodies as markers of a distinct autoimmune gastrointestinal component of autoimmune polyendocrine syndrome type 1.

BACKGROUND: Autoantibodies to tryptophan hydroxylase (TPHAbs) directed against serotonin-producing enterochromaffin cells (EC) have been reported in autoimmune-polyendocrine-syndrome type 1 (APS-1) patients with gastrointestinal dysfunction (GID). Serotonin plays a critical role in enteric function and its peripheral blood levels reflect serotonin release from the gastrointestinal tract. AIMS: We test the hypothesis that TPHAbs mark a distinct autoimmune component of APS-1 characterized by an autoimmune attack toward EC, which results in clinical GID. METHODS: TPHAbs were measured in 64 APS-1 patients. Endoscopy with gastric (antrum/body) and duodenal biopsy was carried in 16 TPHAbs+ patients (8 with and 8 without GID) and in 2 TPHAbs- patients (without GID). Immunohistochemistry of biopsy specimens was carried out using antibodies to serotonin, chromogranin-A, CD3, CD4, CD8, and CD20. Serotonin serum levels were measured in TPHAbs+ and TPHAbs- patients who had endoscopy. RESULTS: Thirty-seven of 64 patients were TPHAbs+ (11/12 with GID and 26/52 without GID; P < .001). Gastric and duodenal biopsies in all 8 TPHAb+ patients with GID showed lymphocytic infiltration with increased CD3+CD8+ intraepithelial lymphocytes and absence of EC. Furthermore, mean serotonin serum levels were below the normal range in TPHAb+ patients with GID (P < .01). In 8 TPHAb+ patients without GID gastric and duodenal biopsies showed different grades of inflammatory infiltration and reduced number of EC. Mean serotonin serum levels were near the lower limit of the normal range. In all TPHAbs+ patients the biopsies showed a reduced number of chromogranin-A positive cells consistent with enteroendocrine cells depletion. TPHAbs- patients without GID showed normal gastrointestinal mucosa and serotonin serum levels. CONCLUSIONS: TPHAbs appear to be markers of a distinct autoimmune component of APS-1. Progressive involvement of the gastrointestinal EC leads to the transition from preclinical to clinical disease, characterized by GID and reduced serotonin serum levels.

Altered bone status in unilateral testicular cancer survivors: Role of CYP2R1 and its luteinizing hormone-dependency.

BACKGROUND: Recent data suggest a potential role of testis in vitamin D activation, where Leydig cells could represent key players in this process since they express the highest amount of CYP2R1, a key enzyme involved in vitamin D 25 hydroxylation. AIM: To evaluate bone status in unilateral orchiectomy and to assess in vivo and in vitro LH-dependency of Vitamin D 25 hydroxylation. SUBJECTS AND METHODS: 125 normotestosteronemic patients with testicular cancer (TC), featured by unilateral orchiectomy and 41 age-matched healthy male controls were studied in the Center for Human Reproduction Pathology at the University of Padova. To evaluate LH-dependency of Vitamin D 25 hydroxylation in vitro, Leydig cell cultures were stimulated with hCG and assessed for CYP2R1 expression, whereas in vivo 10 hypogonadotropic hypogonadal (HH) patients were evaluated before and after treatment with gonadotropins for bone metabolism markers. Hormonal pattern and bone metabolism markers were measured in all subjects, whereas 105 patients and 41 controls underwent bone densitometry by DEXA. RESULTS: In TC patients 25-hydroxyvitamin D levels were significantly lower compared to controls. Furthermore, 23.8% of patients with TC displayed low bone density (Z-score <-2 SD). None of the 41 control subjects showed any significant alteration of BMD. In vitro and in vivo studies revealed that CYP2R1 expression in Leydig cells appeared to be hCG dependent. CONCLUSION: Our data show an association between TC and alteration of the bone status, despite unvaried androgen and estrogen levels, suggesting the evaluation of bone status and possible vitamin D deficiency in TC survivors.

The trend of VEGF-A and PlGF in pregnant patients: a perspective case-control study on 214 women.

OBJECTIVE: The aim of this study was to measure plasmatic concentrations of vascular endothelial growth factor-A (VEGF-A) and placental growth factor (PIGF) in pregnant women, and to evaluate their relationship with age, hormonal status, gestational age, and different diseases of pregnancy. METHODS: We selected a control group of 163 patients (96 fertile and 67 in menopause) and a group of 214 pregnant patients during the whole gestational period. VEGF-A and PlGF were assayed by ELISA and EIA methods, respectively. Statistical analysis was performed using the Mann-Whitney test. RESULTS: The control group showed mean VEGF-A and PlGF values of 89.87 pg/ml and 10.22 pg/ml, respectively; PlGF showed the highest values in menopausal patients. The group of pregnant patients showed VEGF-A values of 27.05 pg/ml and PlGF values of 231.36 pg/ml respectively, with lower (for the VEGF-A) and higher (for the PlGF) statistical significance. These values were not influenced by biological age, but were related to gestational age: VEGF-A showed a decrease and PlGF an increase particularly after the 20th gestational week. PlGF showed a statistically significant decrease compared to physiological gestation in spontaneous and threatened abortions (p < 0.0001) and in ectopic pregnancies (p < 0.0001), an increase in ultrasound and CTG alterations (p < 0.05), and threatened premature delivery and uterine hypercontractility (p < 0.01); on the other hand VEGF-A showed a statistically significant increase in ectopic pregnancies (p < 0.05). CONCLUSIONS: VEGF-A and PlGF may play a diagnostic and prognostic role in pregnancy. Further studies are required to better understand the meaning of variability of their values.

A comparative study of serum and synovial fluid lipoprotein levels in patients with various arthritides.

UNLABELLED: The aim of this study was to investigate apolipoprotein (apo) A-I, apo B, lipoprotein (Lp) (a), HDL-cholesterol (C), LDL-C, triglycerides (TG) and total cholesterol (TC) values in the serum and synovial fluid (SF) of untreated rheumatoid arthritis (RA), psoriatic arthritis (PsA), and osteoarthritis (OA) patients. METHODS: Paired SF and serum samples were collected simultaneously from 14 patients with RA, 14 with PsA, and 16 with OA and tested for apo A-I, apo B, HDL-C, LDL-C, Lp(a), TC and TG. Serum C reactive protein (CRP) and amyloid A (SAA) levels were also determined. RESULTS: The inflammatory arthritis patients had higher SF lipid levels with the exception of HDL. Reflecting increased synovial permeability, the lipid SF/serum ratio was always higher in RA and PsA with respect to OA patients. The positive correlation between serum and SF apo A-I, apo B, HDL-C, TG, and Lp(a) levels confirmed that there is lipoprotein diffusion into the SF. RA and PsA patients had lower concentrations of all serum lipids except for Lp(a) with respect to OA patients. The levels in the RA patients were similar to those in healthy matched controls, while the PsA patients had significantly lower apo A-I and HDL levels and higher apo B and LDL values. CONCLUSIONS: Lipid diffusion into the joint cavity, which largely depends on the degree of inflammation, may contribute to modulating local inflammatory processes.

HE4 in ovarian cancer: from discovery to clinical application.

Despite the relatively low prevalence, ovarian cancer is the fifth leading cause of death from cancer among women. As such, an early diagnosis for establishing a timely surgical and/or chemotherapeutic treatment is essential for improving the outcome. The most reliable, but not always straightforward, approach to diagnose ovarian cancer relies on multiple, time-consuming and expensive investigative tools. These typically include clinical presentation (i.e., pelvic or abdominal pain, urinary frequency or urgency, increased abdominal size or bloating) with pelvic examination, transvaginal ultrasonography (US), and measurement of carbohydrate antigen 125 (CA125). Although the conventional pathway to develop and market a clinically useful biomarker is challenging, recent advances in genomic and proteomic technologies have led to the identification of previously unknown candidate markers of ovarian cancer. Some of these are currently under clinical validation. The human epididymis protein 4 (HE4) has recently been approved by the Food and Drug Administration for monitoring recurrence or progression of epithelial ovarian cancer. Nevertheless, reliable clinical evidence demonstrates that HE4, used alone or in combination with CA125, substantially improves the accuracy of screening and/or disease monitoring. This chapter will review the current knowledge on biologic and clinical applications of ovarian cancer biomarkers, with particular emphasis on the newly proposed marker, HE4.

Efficacy of tamoxifen based on cytochrome P450 2D6, CYP2C19 and SULT1A1 genotype in the Italian Tamoxifen Prevention Trial.

The role of pharmacogenomics and tamoxifen was investigated by analyzing several polymorphisms of cytochrome P450 and SULT1A1 gene in a nested case control study from the Italian Tamoxifen Prevention Trial. This study included 182 Caucasian subjects, 47 breast cancer (BC) cases and 135 matched controls. We used the AmpliChip CYP450 Test to screen 33 alleles of CYP2D6 and 3 of CYP2C19. One more variant for CYP2C19*17 and two single-nucleotide polymorphisms for the gene SULT1A1 were also performed. By using the AmpliChip CYP450 Test, out of 182 subjects, we identified 8 poor metabolizer (PM), 17 intermediate metabolizer (IM), 151 extensive metabolizer (EM) and 3 ultrarapid metabolizer (UM). PM women allocated to the tamoxifen arm showed a higher risk of developing BC compared to the remaining phenotypes (P=0.035). In an exploratory analysis, among 58 women with a CYP2D6*2A allele, 9 BCs were diagnosed in the placebo arm and only 1 in the tamoxifen arm (P=0.0001). CYP2C19 and SULT1A1 polymorphisms did not show any correlation with tamoxifen efficacy. Tamoxifen showed reduced efficacy in CYP2D6 PMs in the chemoprevention setting. Conversely, the CYP2D6*2A allele may be associated with increased efficacy of tamoxifen. These findings support the relevance of pharmaco-genomics in tailoring tamoxifen treatment.

Chronic anemia due to mitomycin C is drug dose-dependent, normocytic, progressive, related to erythropoietin levels and quantitatively predictable: implications for radiochemotherapy.

Mitomycin C (MC) is used as therapy against solid tumors, also combined with other chemotherapeutic agents or radiotherapy. It may cause acute, subacute, or chronic anemia capable of modifying the results of chemo- and radiotherapy. Erythropoietin may be lowered by cancer itself or because of chemoradiotherapy. There are few studies investigating the relationship between erythropoietin and chronic anemia.We prospectively analyzed the chronic anemia and erythropoietin in 38 patients with solid cancer. Patients were 40 to 82 years of age. MC was randomly given every 3 weeks as a single drug at 10 or 20 mg/m². When myelotoxicity occurred the next therapy cycle was delayed until recovery. RBC indices, hemolysis, erythropoietin, liver and kidney function were studied. MC cycles were 136 (3.6 ± 1.4 per pt), 32 being delayed because of myelotoxicity.Hematocrit, hemoglobin and RBC were inversely related to the cumulative dose (r = 0.70 to 0.86; p 0.03 to 0.01) of MC. Other tests remained stable. Anemia occurred almost twofold earlier in the 20 mg/m² group (p=0.049). basal erythropoietin, already lower than in age and sex watched 81 non cancerous subjects (p<0.001), decreased during MC therapy (p<0.01). For each given MC mg/m² a 0.0372 Hb mg/dl reduction occurred. Chronic anemia due to MC is accompanied by erythropoietin reduction. These results can help in designing chemoradiotherapy.

Gastritis OLGA-staging and gastric cancer risk: a twelve-year clinico-pathological follow-up study.

BACKGROUND: Intestinal-type gastric cancer (GC) still ranks among the high-incidence, highly lethal malignancies. Atrophic gastritis is the cancerization field in which GC develops. The current histological reporting formats for gastritis do not include any (atrophy-based) ranking of GC risk. AIM: To test the gastritis OLGA-staging (Operative Link for Gastritis Assessment) in prognosticating neoplastic progression. METHODS: Ninety-three Italian patients were followed up for more than 12 years (range: 144-204 months). Clinical examinations, pepsinogen serology, endoscopy and histology (also assessing Helicobacter pylori status) were performed both at enrolment (T1) and at the end of the follow-up (T2). RESULTS: All invasive or intra-epithelial gastric neoplasia were consistently associated with high-risk (III/IV) OLGA stages. There was a significant inverse correlation between the mean pepsinogen ratio and the OLGA stage (test for trend; P < 0.001). OLGA-staging at T1 predicted both the OLGA stage (Kaplan-Maier log-rank test, P = 0.001) and the neoplasia at T2 (Kaplan-Maier log-rank test, P = 0.001). CONCLUSIONS: This long-term follow-up study provides the first evidence that gastritis OLGA-staging conveys relevant information on the clinico-pathological outcome of gastritis and therefore for patient management. According to OLGA-staging and H. pylori-status, gastritis patients could be confidently stratified and managed according to their different cancer risks.

Heat-induced transcription of diphtheria toxin A or its variants, CRM176 and CRM197: implications for pancreatic cancer gene therapy.

Vectors combining the heat shock proteins (HSPs) promoter with the catalytic subunit A of the diphtheria toxin (DTA) or its variants, cross-reacting material (CRM) 176 and 197, were engineered to investigate the effect of bacterial toxins on pancreatic cancer (PC) cells. Three heat-inducible enhanced green fluorescent protein (eGFP)-expression vectors were obtained: V1 (91% homology to HSPA6), V2 (five heat shock elements upstream the minimal HSPA6 promoter) and V3 (V1 and V2 combined). The highest eGFP transcription and translation levels were found in V3 transfected PC cells. The V3 promoter was used to control DTA, CRM176 and CRM197 expression, treatment response being investigated in four PC cell lines. DTAwt or CRM176 transfected cell growth was completely arrested after heat shock. CRM197 toxin presumed to be inactive, caused mild distress at 37 degrees C and induced a 25-50% reduction in cell growth after heat shock. Preliminary in vivo findings showed that heat treatment arrests tumor growth in DTA197 stably transfected PSN1 cells. In conclusion, the efficient HSP promoter identified in this study may be extremely useful in controlling the transcription of toxins such as CRM197, which have lethal dose-related effects, and may thus be a promising tool in PC gene therapy in vivo.