New biomarkers to predict the evolution of in situ breast cancers.

BACKGROUND: Genomic studies have shown that gene expression profiles are similar in in situ (CIS) and invasive breast cancers, suggesting that several biofunctional modifications of the transformation process occur before or during the development of CIS lesion. METHODS: We investigated 3 biomarkers in 44 patients with CIS: TG2 (transglutaminase 2), HJURP (Holliday junction recognition protein), and HIF-1α (hypoxia inducible factor-1 alpha). RESULTS: TG2 was more highly expressed than the other two markers and significantly more so in stromal than in tumor cells. HIF-1α evaluation showed a higher expression in both tumor and stromal cells in patients with relapsed G3 tumors, indicating a potential role of this marker in CIS evolution. A greater than sevenfold higher risk of relapse (P = 0.050) was observed in patients highly expressing HJURP in stroma and a tenfold higher recurrence risk (P = 0.026) was seen in those with a higher stromal HIF-1α expression. An important increase in risk accuracy (AUC 0.80) was obtained when HIF-1α and HJURP were evaluated together. CONCLUSIONS: Despite the limited number of relapsed patients, we formulated some hypotheses on the factors responsible for malignant evolution and recurrence which are now being tested in a large case series with a longer follow-up.

Combining cytology, TRAP assay, and FISH analysis for the detection of bladder cancer in symptomatic patients.

BACKGROUND: There is a need to improve the performance of urine cytology in bladder cancer diagnosis. We assessed the diagnostic performance of (i) telomerase activity detected by telomeric repeat amplification protocol (TRAP) assay, (ii) cytology and TRAP assay in parallel, (iii) cytology in parallel with the in-series combination of TRAP assay and FISH analysis, and (iv) the in-series combination of TRAP assay and FISH analysis. PATIENTS AND METHODS: Cross-sectional study of 289 consecutive patients who presented with urinary symptoms at a north Italian hospital between 2007 and 2008. All underwent cystoscopy and cytology evaluation, and conclusive results were available for TRAP assay and FISH analysis. RESULTS: Sensitivity and specificity were 0.39 and 0.83, respectively, for cytology; 0.66 and 0.72 for TRAP; 0.78 and 0.60 for the combination of cytology and TRAP; 0.78 and 0.78 for the combination of cytology, TRAP, and FISH; and 0.65 and 0.93 for the combination of TRAP and FISH. All differences versus cytology alone were significant (P ≤ 0.011). CONCLUSION: Compared with cytology alone, the combination of cytology, TRAP, and FISH provided the best trade-off between increase in sensitivity and loss in specificity, especially among non-bleeding patients, low-grade cancers, and early-stage cancers.

In vitro and in vivo antitumor efficacy of docetaxel and sorafenib combination in human pancreatic cancer cells.

The response of pancreatic cancer to treatments remains unsatisfactory, highlighting the need for more effective therapeutic regimens. Sorafenib, an orally available multikinase inhibitor, is active against different tumors, including pancreatic cancer. We studied the antitumor efficacy of sorafenib in combination with different antitumor drugs currently used in clinical practice in in vitro and in vivo experimental models of human pancreatic cancer. The cytotoxic effect of sorafenib and conventional antitumor drug combinations was evaluated in vitro in human pancreatic cancer cell lines and the efficacy of the most active combination was tested on tumor-bearing mice. Flow cytometric, Western blot and immunohistochemistry analyses were performed to investigate the mechanisms involved in the activity of single drugs and in their interaction when used in combination. Sorafenib showed a strong sequence-dependent synergistic interaction in vitro with docetaxel, which was highly dependent on the drug sequence employed. In vivo, human pancreatic cancer-xenografted mice treated with docetaxel followed by sorafenib reduced and delayed tumor growth, with complete tumor regression observed in half of the mice. This marked antitumor effect resulted in an overall increase in mouse survival of about 70% and in a complete cure in 3 of the 8 treated mice. The strong activity was also accompanied by marked apoptosis induction, inhibition of tumor angiogenesis and downregulation of ERK signalling. Our results show that the docetaxel and sorafenib combination exerts high therapeutic efficacy in experimental models of human pancreatic cancer, indicating a promising antitumor strategy for clinical use.

Activity of endovesical gemcitabine in BCG-refractory bladder cancer patients: a translational study.

Intravesical gemcitabine (Gem) has shown promising activity against transitional cell carcinomas (TCC) of the bladder, with moderate urinary toxicity and low systemic absorption. The present phase II study evaluated the activity of biweekly intravesical treatment with Gem using a scheme directly derived from in vitro preclinical studies. Patients with Bacille Calmette-Guérin (BCG) -refractory Ta G3, T1 G1-3 TCC underwent transurethral bladder resection and then intravesical instillation with 2000 mg Gem diluted in 50 ml saline solution on days 1 and 3 for 6 consecutive weeks. Thirty-eight (95%) of the 40 patients showed persistent negative post-treatment cystoscopy and cytology 6 months after Gem treatment, while the remaining 2 patients relapsed at 5 and 6 months. At a median follow-up of 28 months, recurrences had occurred in 14 patients. Among these, four had downstaged (T) disease, three had a lower grade (G) lesion and three had a reduction in both T and G. Urinary and systemic toxicity was very low, with no alterations in biochemical profiles. In conclusion, biweekly instillation of Gem proved active in BCG-refractory Ta G3, T1 G1-3 TCC. Our results highlight the importance of preclinical studies using in vitro systems that adequately reproduce the conditions of intravesical clinical treatment to define the best therapeutic schedule.

Procalcitonin: a marker of bacteraemia in SIRS.

A number of European studies have documented the ability of procalcitonin (PCT), a novel inflammatory marker, to discriminate patients with sepsis from those with other causes of systemic inflammatory response syndrome (SIRS). The aim of this study was to assess procalcitonin's performance in an Australian intensive care unit (ICU) setting to examine whether it could discriminate between these two conditions. One hundred and twenty-three consecutive adult ICU patients fulfilling criteria for SIRS were enlisted in the study. Over a period of five days, daily serum PCT and C-reactive protein (CRP) levels were measured. At least two sets of cultures were taken of blood, sputum/broncho-alveolar lavage (BAL) and urine. Other cultures were taken as clinically indicated. Questionnaires to ascertain clinical suspicion of sepsis were prospectively answered by the ICU senior registrars. PCT values were ten times higher in patients with positive blood cultures; CRP values were also significantly higher in the bacteraemic patients. Both PCT and CRP had a good ability to discriminate bacteraemia from non-infectious SIRS, with the area under receiver operating characteristics (ROC) curves for PCT being 0.8 and for CRP being 0.82. However neither PCT or CRP was able to discriminate patients with localized sepsis from those without. Utilizing both tests resulted in a more sensitive screen than either one alone, while PCT was a more accurate diagnostic test for bacteraemia than CRP. The PCT value also differed between those who died in hospital and those who survived. Measurement of PCT alone or in combination with CRP can aid discrimination of septicaemia/bacteriemia with associated SIRS from non-infectious SIRS in an Australian ICU setting.

Quality control for biomarker determination in oncology: the experience of the Italian Network for Quality Assessment of Tumor Biomarkers (INQAT).

Biomarker analysis and evaluation in oncology is the product of a number of processes (including managerial, technical and interpretation steps) which need to be monitored and controlled to prevent and correct errors and guarantee a satisfactory level of quality. Several biomarkers have recently moved to clinical validation studies and successively to clinical practice without any definition of standard procedures and/or quality control (QC) schemes necessary to guarantee the reproducibility of the laboratory information. In Italy several national scientific societies and single researchers have activated -- often on a pilot level -- specific external quality assessment protocols, thereby potentially jeopardizing the clinical reality even further. In view of the seriousness of the problem, in 1998 the Italian Ministry of Health sponsored a National Survey Project to coordinate and standardize the procedures and to develop QC programs for the analysis of cancer biomarkers of potential clinical relevance. Twelve QC programs focused on biomarkers and concerning morphological, immunohistochemical, biochemical, molecular, and immunoenzymatic assays were coordinated and implemented. Specifically, external QC programs for the analytical phase of immunohistochemical p53, Bcl-2, c-erb-2/neu/HER2, and microvessel density determination, of morphological evaluation of tumor differentiation grade, and of molecular p53 analysis were activated for the first time within the project. Several hundreds of Italian laboratories took part in these QC programs, the results of which are available on the web site of the Network (www.cqlaboncologico.it). Financial support from the Italian Government and the National Research Council (CNR) will guarantee the pursuit of activities that will be extended to new biomarkers, to preanalytical phases of the assays, and to revision of the criteria of clinical usefulness for evaluating the cost/benefit ratio.

Prognostic and predictive role of proliferation indices in adjuvant therapy of breast cancer.

In breast cancer, proliferative activity represents one of the biologic processes most thoroughly investigated for its association with tumor progression. In addition to the mitotic activity component of pathologic grading systems, several proliferation indices have provided independent information on prognosis and response to specific treatments in large retrospective studies. Recently, results from treatment protocols prospectively planned to test the clinical utility of proliferative activity have indicated that tumor cell proliferation markers identify two subsets among patients with lymph node-negative cancers: 1) those at a very low risk of relapse and 2) those who will benefit from regimens including antimetabolites. Future efforts should compare the prognostic accuracy of different proliferation markers, confirm preliminary evidence of a relationship between proliferation and response to specific systemic treatments, and standardize assay techniques to facilitate their transfer to general oncology practice.

Cell proliferation and outcome following doxorubicin plus CMF regimens in node-positive breast cancer.

At the Istituto Nazionale Tumori of Milan, a randomised adjuvant chemotherapy trial was carried out from 1982 to 1990 to compare alternating with sequential regimens of doxorubicin and CMF in 403 patients with more than 3 positive axillary nodes. Tumour proliferative activity was determined in 71% (285 cases) of women entering the clinical study. We investigated the relation between proliferative rate, determined as the [(3)H]thymidine labelling index (TLI) on tumour specimens obtained at diagnostic surgery, and clinical outcome following the 2 regimens, in which the same drugs were administered at the same dose intensity but with a different schedule. A high TLI was significantly associated with 12-year overall relapse (P = 0.009), distant metastasis (P = 0.001), and death (P = 0.002), even in the presence of information provided by tumour size, lymph node involvement, oestrogen receptors, and treatment regimen. The highest relapse-free survival (RFS) probability (45%, 95% CI 34-55%) was observed for patients with tumour TLI <5% and subjected to the sequential treatment. The lowest RFS probability (11%, 95% CI 0-26%) was observed for patients with tumour TLI >9% following the alternating regimen. Intermediate RFS probabilities, ranging from 23% to 34%, were observed for the other kinetic subgroups following the 2 treatment regimens. The benefit of sequential administration of doxorubicin and CMF was evident mainly in patients with tumours at low to intermediate proliferation.

Biomarkers and outcome after tamoxifen treatment in node-positive breast cancers from elderly women.

The predictive role of tumour proliferative rate and expression of p53, bcl-2 and bax proteins, alone and in association with tumour size, nodal involvement and oestrogen receptors (ER), was analysed on 145 elderly patients (> or =70 years of age) with histologically assessed node-positive breast cancers treated with radical or conservative surgery plus radiotherapy followed by adjuvant tamoxifen for at least 1 year. The 7-year probability of relapse was significantly higher for patients with tumours rapidly proliferating (hazard ratio (HR) = 2.0, P = 0.01), overexpressing p53 (HR = 4.4, P = 0.0001), weakly or not exhibiting bcl-2 (HR = 1.9, P = 0.02), without ERs (HR = 3.4, P = 0.0001) or with > or = 4 positive lymph nodes (HR = 2.3, P = 0.003) than for patients with tumours expressing the opposite patho-biological profile. Conversely, tumour size and bax expression failed to influence relapse-free survival. Adjustment for the duration of tamoxifen treatment did not change these findings. Oestrogen receptors, cell proliferation, p53 accumulation and bcl-2 expression were also predictive for overall survival. Within ER-positive tumours, cell proliferation, p53 accumulation, bcl-2 expression and lymph node involvement provided significant and independent information for relapse and, in association, identified subgroups of patients with relapse probabilities of 20% (low-risk group, exhibiting only one unfavourable factor) to 90% (high-risk group, exhibiting three unfavourable factors). Such data could represent the initial framework for a biologically tailored therapy even for elderly patients and highlight the importance of a patho-biological characterization of their breast cancers.

Biological markers as indicators of response to primary and adjuvant chemotherapy in breast cancer.

Interest in translational studies on breast cancer is presently devoted to identify biological predictors of treatment response. In patients with operable breast cancer, subjected to primary and adjuvant chemotherapy, we analyzed the predictivity on objective clinical response and relapse-free survival of biological markers related to different cellular aspects and functions. Tumour proliferative rate (evaluated as the (3)H-thymidine-labelling index, TLI), oestrogen and progesterone receptors (ER and PgR, evaluated by the dextran-coated-charcoal method), nuclear DNA ploidy and the immunocytochemical expression of p53, bcl-2 and bax proteins were determined before primary treatment, at the time of diagnosis, and after primary chemotherapy, at surgery. Objective clinical response was significantly related only to pre-treatment p53 expression or PgR status, with a higher rate for tumours not expressing than for those expressing p53 (94% vs. 72%), as well as for PgR-negative (PgR(-)) than for PgR-positive (PgR(+)) tumours (86% vs. 68%). In the overall series, 8-year clinical outcome was significantly related only to post-treatment steroid receptors. In particular, higher 8-year relapse-free survival rate was observed for patients with ER(-) or PgR(-) than for those with ER(+) (64% vs. 38%) or PgR(+) (53% vs. 37%) tumours. Such findings held true even within the sub-set of patients who received adjuvant post-operative chemotherapy, i.e., those with node-positive (N(+)) or ER(-)/node-negative (N(-)) tumours, among whom also rapid proliferation or the presence of apoptosis-favouring markers (bcl-2(-) or bax(+), singly and in association) on surgical specimens identified a sub-set of women who benefited from systemic treatment. The different biological markers were variously indicative of clinical outcome, with a predictivity on tumour shrinkage for p53 and PgR, detected before primary chemotherapy, and on long-term follow-up for ER, PgR and, to a lesser extent, TLI and apoptosis-modulating markers. Int. J. Cancer (Pred. Oncol.) 84:580-586, 1999.

Atypical C-ANCA following high dose intravenous immunoglobulin.

AIMS: (1) To assess a range of intravenous immunoglobulin products for atypical classical antineutrophil cytoplasmic antibody (C-ANCA) staining and to determine if this is present in patients treated with high dose intravenous immunoglobulin (2 g/kg/month) and replacement doses (200 mg/kg fortnightly); (2) using the United Kingdom national external quality assessment scheme (NEQAS), to determine if laboratories could differentiate this pattern from classical ANCA. METHODS: ANCA testing was performed on 30 batches of intravenous immunoglobulin from several manufacturers. Six patients treated with high dose intravenous immunoglobulin and 11 receiving replacement doses of immunoglobulin for hypogammaglobulinaemia were tested for ANCA by indirect immunofluorescence on cytospin preparations of ethanol fixed neutrophils and by enzyme linked immunosorbent assay (ELISA). One of the positive immunoglobulin batches was tested blindly by 125 laboratories involved in NEQAS by indirect immunofluorescence and by ELISA in some laboratories. RESULTS: 16 of 31 batches of intravenous immunoglobulin from six different manufacturers were atypical C-ANCA positive. Three of six patients receiving high dose intravenous immunoglobulin and none of 11 patients on replacement doses were atypical C-ANCA positive. The results of the NEQAS assessment by indirect immunofluorescence were 68% C-ANCA positive, 17% negative, 9% atypical C-ANCA, and 6% P-ANCA. CONCLUSIONS: Some but not all intravenous immunoglobulin products yield a positive atypical cANCA by indirect immunofluorescence. An identical pattern may be observed in patients receiving high dose intravenous immunoglobulin but not in those on replacement doses. Of laboratories participating in NEQAS, 68% reported this pattern as cANCA. This reinforces the importance of reporting only "classical ANCA," defined by international ANCA workshops, to maintain the specificity of ANCA immunofluorescence and its close disease associations.

Diagnostic value of classical and atypical antineutrophil cytoplasmic antibody (ANCA) immunofluorescence patterns.

BACKGROUND: The "classical" antineutrophil cytoplasmic antibody (C-ANCA) pattern seen on indirect immunofluorescence (IIF) is characterised by granular cytoplasmic staining showing central or interlobular accentuation, and is strongly associated with antiproteinase-3 antibodies (PR3-ANCA) and Wegener's granulomatosis. However, many laboratories report C-ANCA in the presence of any cytoplasmic IIF staining, regardless of pattern, which risks reducing the diagnostic value of this pattern. AIMS: To classify different cytoplasmic ANCA patterns and thus determine whether stringent application of the classical criteria for C-ANCA would produce better correlation between C-ANCA and (1) PR3-ANCA enzyme linked immunosorbent assay (ELISA) results; (2) a diagnosis of systemic vasculitis (including Wegener's granulomatosis). METHODS: 72 sera with cytoplasmic IIF collected over a two year period were analysed by IIF and a commercial PR3-ANCA ELISA kit. RESULTS: Three IIF patterns were defined: "classical/true" C-ANCA as described above (n = 27 (37.5%)); "flat" ANCA with homogeneous cytoplasmic staining (n = 21 (29%)); and "atypical" ANCA which included all other cytoplasmic patterns (n = 24 (33.5%)). Twenty five of the 27 true C-ANCA sera (92.5%) contained PR3-ANCA (p < 0.0001), but none of the 21 with flat ANCA and only one of the 24 with atypical ANCA. From clinical data on 23 of the 27 true C-ANCA positive patients, 20 (87%) had evidence of Wegener's granulomatosis or systemic vasculitis (p < 0.0001 v the other two patterns). However, none of 19 sera with flat ANCA and clinical data had evidence of systemic vasculitis. CONCLUSIONS: Restricting the term "c-ANCA" to the "classical" description of central/interlobular accentuation on IIF, will improve its correlation with PR3-ANCA positivity and a diagnosis of systemic vasculitis.

Clinical utility of anticardiolipin antibody assays: high inter-laboratory variation and limited consensus by participants of external quality assurance programs signals a cautious approach.

Antibodies that bind phospholipid (anti-phospholipid antibodies [APA]) are a focus of major interest to both clinical and laboratory personnel across a variety of disciplines because of the assortment of disorders with which they are reportedly associated. A solid phase assay using cardiolipin as the test phospholipid (anti-cardiolipin antibody assay [ACA]) has now become a laboratory standard for the detection of APA. In the current report, data from two separate external quality assurance programs (QAP) and collected over the past four years, have been evaluated to assess the utility of the ACA. Despite attempted standardizations, exceedingly high inter-laboratory variation and a general lack of test result consensus would signal the adoption of a cautious clinical approach towards laboratory findings. For example, for a total of 21 cross-laboratory tested serum samples (tested for both IgG and IgM for 41 quantitative estimations), inter-laboratory variation for both ACA-IgG and ACA-IgM was higher than 50% in 20 of 41 testing cases (48.8%). The situation with regard to testing consensus was equally concerning. Total consensus (i.e., 100% of participating laboratories agreed that a given serum sample gave an ACA result of either negative or positive) occurred in less than 20% of cases for ACA-IgG. More importantly, in about 50% of serum testing occasions there was no general consensus in returned laboratory data (i.e., more than 80% of labs could not agree on whether a serum sample tested was either ACA-positive or ACA-negative). Thus, despite attempted international standardizations, exceedingly high inter-laboratory variation and a general lack of test result consensus would argue that the assay has limited utility. We conclude that single point laboratory results must be used with considerable caution before accepting that ACA activity is present in patient serum or not. We agree with recommendations indicating that laboratory tests should be repeated at least once prior to making a clinical diagnosis of any APS-like disorder.

International Consensus Statement on Testing and Reporting of Antineutrophil Cytoplasmic Antibodies (ANCA)

Antineutrophil cytoplasmic antibody (ANCA) tests are used to diagnose and monitor inflammatory activity in the primary systemic small vessel vasculitides. ANCA is best demonstrated in these diseases by using a combination of indirect immunofluorescence (IIF) of normal peripheral blood neutrophils and enzyme-linked immunosorbent assays (ELISAs) that detect ANCA specific for proteinase 3 (PR3) or myeloperoxidase (MPO). For ANCA testing in "new" patients, IIF must be performed on all serum samples. Serum samples containing ANCA, any other cytoplasmic fluorescence, or an antinuclear antibody (ANA) that results in homogeneous or peripheral nuclear fluorescence then should be tested in ELISAs for PR3-ANCA and MPO-ANCA. Optimally, ELISAs for PR3-ANCA and MPO-ANCA should be performed on all serum samples. Inclusion of the most recent positive sample in the IIF or ELISA may help demonstrate a change in antibody level. Reports should use recommended terms. Any report of positive neutrophil fluorescence issued before the ELISA results are available should indicate that positive fluorescence alone is not specific for the diagnosis of Wegener granulomatosis or microscopic polyangiitis and that decisions about treatment should not be based solely on the ANCA results.

Clinical studies of Bcl-2 and treatment benefit in breast cancer patients.

Interest in translational studies aimed at investigating the role of biologic markers in predicting clinical outcome of breast cancer patients and, in particular, response to specific treatments, has progressively increased. Among biologic variables presently under investigation, apoptosis markers, in particular Bcl-2 and Bax expression, are receiving much attention for their relationship with the cellular response to genotoxic damage in experimental tumors. Retrospective, independent studies were carried out by several research groups on about 5000 patients with breast cancer at different stages and with an adequate follow-up. The outcome of separate analyses as a function of treatment generally demonstrated that Bcl-2 overexpression, which correlates with biologic features of a differentiated phenotype (slow proliferation, high steroid receptor levels, absence of p53 and c-erB-2 expression), is associated with a favorable outcome. Such a finding is mainly evident following surgery as well as endocrine treatment. Conversely, no or weak Bcl-2 expression, alone or in association with bax overexpression, appears indicative of a radiation response, and preliminary emerging evidence supports the involvement of such an association of apoptosis-related markers even as predictors of long-term response to neoadjuvant cytotoxic treatment. Although the findings of an involvement of Bcl-2 and Bax as determinants of treatment response should be confirmed within the context of randomized clinical trials, they indicate a combined consideration of proteins that negatively and positively regulate apoptosis in translational studies on the effect of chemical and physical agents at a cellular level.