Surface and composition effects on the biphasic cytotoxicity of nanocomposites synthesized using leaf extracts.

The Malus sylvestris L. (LE1), Pinus sylvestris L. (LE2), and Sorbus aucuparia L. (LE3) leaves` extracts were used for the synthesis of silver (Ag) nanocomposites containing different amounts of silver chloride (AgCl), silver metal (Agmet), and silver phosphate (Ag3PO4). These nanocomposites were capped with the organic functional groups in the leaf extract. Notably, the nanocomposites caused biphasic cytotoxic response on cells; first attributed to the inhibition of cell growth and second to cell death. The nanocomposites were biocompatible with normal embryonic kidney (HEK293) cells in the cytotoxic range for cancer cells. [25(±1) °C synthesis] nanocomposites exhibited the highest cytotoxicity towards HeLa (lethal concentration- LC50 value of 11.4 µg mL-1) and A549 (LC50 value of 14.7 µg mL-1) after 24-h incubation and its efficiency was shown also for the more resistant MCF-7 and MDA-MB-231, however, their respective LC50 values were larger. For the HeLa cell line, this designed nanocomposite exhibited an LC50 value similar to the effective concentration (EC50) value of Cisplatin and about 3 times larger than Doxorubicin. nanocomposite contained Ag3PO4 in the composite and P on the surface, higher AgCl content, smaller crystallite size of all nanoparticle phases, and carbon-rich oxygen-deficient surface compared to all other nanocomposites.

Percutaneous tracheostomy: Comparison of three different methods with respect to tracheal cartilage injury in cadavers-Randomized controlled study.

Background: Performing tracheostomy improves patient comfort and success rate of weaning from prolonged invasive mechanical ventilation. Data suggest that patients have more benefit of percutaneous technique than the surgical procedure, however, there is no consensus on the percutaneous method of choice regarding severe complications such as late tracheal stenosis. Aim of this study was comparing incidences of cartilage injury caused by different percutaneous dilatation techniques (PDT), including Single Dilator, Griggs' and modified (bidirectional) Griggs' method. Materials and methods: Randomized observational study was conducted on 150 cadavers underwent post-mortem percutaneous tracheostomy. Data of cadavers including age, gender and time elapsed from death until the intervention (more or less than 72 h) were collected and recorded. Primary and secondary outcomes were: rate of cartilage injury and cannula malposition respectively. Results: Statistical analysis revealed that method of intervention was significantly associated with occurrence of cartilage injury, as comparing either standard Griggs' with Single Dilator (p = 0.002; OR: 4.903; 95% CI: 1.834-13.105) or modified Griggs' with Single Dilator (p < 0.001; OR: 6.559; 95% CI: 2.472-17.404), however, no statistical difference was observed between standard and modified Griggs' techniques (p = 0.583; OR: 0.748; 95% CI: 0.347-1.610). We found no statistical difference in the occurrence of cartilage injury between the early- and late post-mortem group (p = 0.630). Neither gender (p = 0.913), nor age (p = 0.529) influenced the rate of cartilage fracture. There was no statistical difference between the applied PDT techniques regarding the cannula misplacement/malposition. Conclusion: In this cadaver study both standard and modified Griggs' forceps dilatational methods were safer than Single dilator in respect of cartilage injury.

T cell immunity ameliorates COVID-19 disease severity and provides post-exposure prophylaxis after peptide-vaccination, in Syrian hamsters.

Background: The emergence of novel SARS-CoV-2 variants that resist neutralizing antibodies drew the attention to cellular immunity and calls for the development of alternative vaccination strategies to combat the pandemic. Here, we have assessed the kinetics of T cell responses and protective efficacy against severe COVID-19 in pre- and post-exposure settings, elicited by PolyPEPI-SCoV-2, a peptide based T cell vaccine. Methods: 75 Syrian hamsters were immunized subcutaneously with PolyPEPI-SCoV-2 on D0 and D14. On D42, hamsters were intranasally challenged with 102 TCID50 of the virus. To analyze immunogenicity by IFN-γ ELISPOT and antibody secretion, lymphoid tissues were collected both before (D0, D14, D28, D42) and after challenge (D44, D46, D49). To measure vaccine efficacy, lung tissue, throat swabs and nasal turbinate samples were assessed for viral load and histopathological changes. Further, body weight was monitored on D0, D28, D42 and every day after challenge. Results: The vaccine induced robust activation of T cells against all SARS-CoV-2 structural proteins that were rapidly boosted after virus challenge compared to control animals (~4-fold, p<0.05). A single dose of PolyPEPI-SCoV-2 administered one day after challenge also resulted in elevated T cell response (p<0.01). The vaccination did not induce virus-specific antibodies and viral load reduction. Still, peptide vaccination significantly reduced body weight loss (p<0.001), relative lung weight (p<0.05) and lung lesions (p<0.05), in both settings. Conclusion: Our study provides first proof of concept data on the contribution of T cell immunity on disease course and provide rationale for the use of T cell-based peptide vaccines against both novel SARS-CoV-2 variants and supports post-exposure prophylaxis as alternative vaccination strategy against COVID-19.

Safety and Activity of PolyPEPI1018 Combined with Maintenance Therapy in Metastatic Colorectal Cancer: an Open-Label, Multicenter, Phase Ib Study.

PURPOSE: Although chemotherapy is standard of care for metastatic colorectal cancer (mCRC), immunotherapy has no role in microsatellite stable (MSS) mCRC, a "cold" tumor. PolyPEPI1018 is an off-the-shelf, multi-peptide vaccine derived from 7 tumor-associated antigens (TAA) frequently expressed in mCRC. This study assessed PolyPEPI1018 combined with first-line maintenance therapy in patients with MSS mCRC. PATIENTS AND METHODS: Eleven patients with MSS mCRC received PolyPEPI1018 and Montanide ISA51VG adjuvant subcutaneously, combined with fluoropyrimidine/biologic following first-line induction with chemotherapy and a biologic (NCT03391232). In Part A of the study, 5 patients received a single dose; in Part B, 6 patients received up to three doses of PolyPEPI1018 every 12 weeks. The primary objective was safety; secondary objectives were preliminary efficacy, immunogenicity at peripheral and tumor level, and immune correlates. RESULTS: PolyPEPI1018 vaccination was safe and well tolerated. No vaccine-related serious adverse event occurred. Eighty percent of patients had CD8+ T-cell responses against ≥3 TAAs. Increased density of tumor-infiltrating lymphocytes were detected post-treatment for 3 of 4 patients' liver biopsies, combined with increased expression of immune-related gene signatures. Three patients had objective response according to RECISTv1.1, and 2 patients qualified for curative surgery. Longer median progression-free survival for patients receiving multiple doses compared with a single dose (12.5 vs. 4.6 months; P = 0.017) suggested a dose-efficacy correlation. The host HLA genotype predicted multi-antigen-specific T-cell responses (P = 0.01) indicative of clinical outcome. CONCLUSIONS: PolyPEPI1018 added to maintenance chemotherapy for patients with unresectable, MSS mCRC was safe and associated with specific immune responses and antitumor activity warranting further confirmation in a randomized, controlled setting.

Valorizing the Unexplored Filtration Waste of Brewing Industry for Green Silver Nanocomposite Synthesis.

The brewing industry generates a substantial amount of by-products rich in polyphenols, carbohydrates, sugars, sulfates, nitrogen compounds, organic carbon, and several elements, including chlorine, magnesium, and phosphorus. Although limited quantities of these by-products are used in fertilizers and composts, a large amount is discarded as waste. Therefore, it is crucial to identify different ways of valorizing the by-products. Research regarding the valorization of the brewery by-products is still in its nascent stage; therefore, it still has high potential. Herein, we report the valorization of the brewery by-product from the filtration stage of the brewing process (BW9) to synthesize silver nanocomposites as this waste has remained largely unexplored. The BW9 nanocomposites have been compared to those obtained from the brewery product B. The chemical composition analysis of BW9 and B revealed several organic moieties capable of reducing metal salts and capping the formed nanoparticles. Therefore, the brewery waste from stage 9 was valorized as a precursor and added to silver-based precursor at various temperatures (25, 50, and 80 °C) and for various time periods (10, 30, and 120 min) to synthesize silver nanocomposites. The nanocomposites obtained using BW9 were compared to those obtained using the main product of the brewing industry, beer (B). Synthesized nanocomposites composed of AgCl as a major phase and silver metal (Agmet) was incorporated in minor quantities. In addition, Ag3PO4 was also found in B nanocomposites in minor quantities (up to 34 wt.%). The surface morphology depicted globular nanoparticles with layered structures. Small ball-like aggregates on the layer representative of Ag3PO4 were observed in B nanocomposites. The surface of nanocomposites was capped with organic content and functional groups present in the brewery products. The nanocomposites demonstrated high antibacterial activity against Escherichia coli (E. coli), with BW9 nanocomposites exhibiting a higher activity than B nanocomposites.

In Silico Model Estimates the Clinical Trial Outcome of Cancer Vaccines.

Over 30 years after the first cancer vaccine clinical trial (CT), scientists still search the missing link between immunogenicity and clinical responses. A predictor able to estimate the outcome of cancer vaccine CTs would greatly benefit vaccine development. Published results of 94 CTs with 64 therapeutic vaccines were collected. We found that preselection of CT subjects based on a single matching HLA allele does not increase immune response rates (IRR) compared with non-preselected CTs (median 60% vs. 57%, p = 0.4490). A representative in silico model population (MP) comprising HLA-genotyped subjects was used to retrospectively calculate in silico IRRs of CTs based on the percentage of MP-subjects having epitope(s) predicted to bind ≥ 1-4 autologous HLA allele(s). We found that in vitro measured IRRs correlated with the frequency of predicted multiple autologous allele-binding epitopes (AUC 0.63-0.79). Subgroup analysis of multi-antigen targeting vaccine CTs revealed correlation between clinical response rates (CRRs) and predicted multi-epitope IRRs when HLA threshold was ≥ 3 (r = 0.7463, p = 0.0004) but not for single HLA allele-binding epitopes (r = 0.2865, p = 0.2491). Our results suggest that CRR depends on the induction of broad T-cell responses and both IRR and CRR can be predicted when epitopes binding to multiple autologous HLAs are considered.

A Peptide Vaccine Candidate Tailored to Individuals' Genetics Mimics the Multi-Targeted T Cell Immunity of COVID-19 Convalescent Subjects.

Long-term immunity to coronaviruses likely stems from T cell activity. We present here a novel approach for the selection of immunoprevalent SARS-CoV-2-derived T cell epitopes using an in silico cohort of HLA-genotyped individuals with different ethnicities. Nine 30-mer peptides derived from the four major structural proteins of SARS-CoV-2 were selected and included in a peptide vaccine candidate to recapitulate the broad virus-specific T cell responses observed in natural infection. PolyPEPI-SCoV-2-specific, polyfunctional CD8+ and CD4+ T cells were detected in each of the 17 asymptomatic/mild COVID-19 convalescents' blood against on average seven different vaccine peptides. Furthermore, convalescents' complete HLA-genotype predicted their T cell responses to SARS-CoV-2-derived peptides with 84% accuracy. Computational extrapolation of this relationship to a cohort of 16,000 HLA-genotyped individuals with 16 different ethnicities suggest that PolyPEPI-SCoV-2 vaccination will likely elicit multi-antigenic T cell responses in 98% of individuals, independent of ethnicity. PolyPEPI-SCoV-2 administered with Montanide ISA 51 VG generated robust, Th1-biased CD8+, and CD4+ T cell responses against all represented proteins, as well as binding antibodies upon subcutaneous injection into BALB/c and hCD34+ transgenic mice modeling human immune system. These results have implications for the development of global, highly immunogenic, T cell-focused vaccines against various pathogens and diseases.

Challenging tumour immunological techniques that help to track cancer stem cells in malignant melanomas and other solid tumours.

AIM OF THE STUDY: The arsenal of questions and answers about the minor cancer initiating cancer stem cell (CSC) population put responsible for cancer invasiveness and metastases, has left with an unsolved puzzle. Specific aims of a complex project were partly focused on revealing new biomarkers of cancer. We designed and set up novel techniques to facilitate the detection of cancerous cells. MATERIALS AND METHODS: As a novel approach, we investigated B cells infiltrating breast carcinomas and melanomas (TIL-B) in terms of their tumour antigen binding potential. By developing the TIL-B phage display technology we provide here a new technology for the specific detection of highly tumour-associated antigens. Single chain Fv (scFv) antibody fragment phage ELISA, immunofluorescence (IF) FACS analysis, chamber slide technique with IF confocal laser microscopy and immunohistochemistry (IHC) in paraffin-embedded tissue sections were set up and standardized. RESULTS: We showed strong tumour-associated disialylated glycosphingolipid expression levels on various cancer cells using scFv antibody fragments, generated previously by uniquely invasive breast carcinoma TIL-B phage display library technology. CONCLUSIONS: We report herein a novel strategy to obtain antibody fragments of human origin that recognise tumour-associated ganglioside antigens. Our investigations have the power to detect privileged molecules in cancer progression, invasiveness, and metastases. The technical achievements of this study are being harnessed for early diagnostics and effective cancer therapeutics.

[Detection of leptospira by culture of vitreous humor and detection of antibodies against leptospira in vitreous humor and serum of 225 horses with equine recurrent uveitis]. / Kultureller Nachweis von Leptospiren in Glaskörperflüssigkeit und Antikörpernachweis gegen Leptospiren in Glaskörperflüssigkeit und Serum von 225 Pferden mit equiner rezidivierender Uveitis (ERU).

In the ongoing discussion regarding the aetiopathogenesis of equine recurrent uveitis (ERU) it was the aim of the present study to elucidate the relationship of leptospira infection and ERU. In a population of 225 horses leptospira were examined in vitreous humor by culture and leptospira antibody were detected in vitreous humor and serum samples. Preoperative serum samples were collected from 221/225 ERU patients of different age, gender and breed. Undiluted vitreous humor was aseptically taken from 198/225 patients that underwent pars plana vitrectomy at the beginning of surgery and from 27/225 patients' eyeball after enucleation: Serum and vitreous humor were tested for specific leptospiral antibodies by microscopic agglutination test (MAT). Furthermore, vitreous humor was examined by culture. 20 patients which were euthanized due to a live-threatening disease other than ERU served as a control group. A total of 127/221 (57.5%) horses had serum antibodies (≥ 1:100). Most frequently antibodies against L. interrogans serovar Grippotyphosa were detected (79/127), followed by L. interrogans serovar lcterohaemorrhagiae (34/127) and L. interrogans serovar Bratislava (29/127). Only 79/225 horses (35.1%) had leptospiral antibodies in vitreous humor, in which L. interrogans serovar Grippotyphosa (67/79) was identified most frequently followed by L. interrogans serovar Pomona (18/79) and L. interrogans serovar lcterohaemorrhagiae (8/79) which was identified as single or multiple reaction. Isolation of leptospira from vitreous humor was positive in 34/212 horses (16%). 10/20 control horses had a positive antibody titer against leptospira in serum and 2/20 horses in vitreous humor, whereas there was no leptospira detected in culture. The result of 84% negative cultures from vitreous humor of 212 ERU patients is decisive for the diagnosis and therapy of ERU.

[Genetic information from tumor-infiltrating B lymphocytes as a driver tool ("GPS") for anti-tumor T cell CARs]. / A tumorban felhalmozódó B-limfociták genetikai információja, mint az immun T-sejt vezette tumorellenes "autó" (CAR, kiméra antigénreceptor) célba irányító "GPS"-e.

The rapidly growing field of gene therapy techniques to modify T cells with chimeric antigen receptors (CARs) for cancer care solutions, reached considerable achievements. However, there is an urgent need of reliable, well tolerable tumor-associated antigen specific antibodies. Tumor-infiltrating B (TIL-B) cell originated single chain Fv (scFv) gene regions could be selected with tumor specificity. DNA sequences of these antibody variable regions were subjects to get engineered into new CAR constructs. Our novel strategy harnesses tumor-infiltrating B cells' unique capacity to reveal highly tumor-associated disialylated glycosphingolipids (GD3 gangliosides). We used these human antibody fragments for generating GD3 ganglioside specific CAR gene constructs for potential usage in solid tumors.

BRET-monitoring of the dynamic changes of inositol lipid pools in living cells reveals a PKC-dependent PtdIns4P increase upon EGF and M3 receptor activation.

Deciphering many roles played by inositol lipids in signal transduction and membrane function demands experimental approaches that can detect their dynamic accumulation with subcellular accuracy and exquisite sensitivity. The former criterion is met by imaging of fluorescence biosensors in living cells, whereas the latter is facilitated by biochemical measurements from populations. Here, we introduce BRET-based biosensors able to detect rapid changes in inositol lipids in cell populations with both high sensitivity and subcellular resolution in a single, convenient assay. We demonstrate robust and sensitive measurements of PtdIns4P, PtdIns(4,5)P2 and PtdIns(3,4,5)P3 dynamics, as well as changes in cytoplasmic Ins(1,4,5)P3 levels. Measurements were made during either experimental activation of lipid degradation, or PI 3-kinase and phospholipase C mediated signal transduction. Our results reveal a previously unappreciated synthesis of PtdIns4P that accompanies moderate activation of phospholipase C signaling downstream of both EGF and muscarinic M3 receptor activation. This signaling-induced PtdIns4P synthesis relies on protein kinase C, and implicates a feedback mechanism in the control of inositol lipid metabolism during signal transduction.

The novel panel assay to define tumor-associated antigen-binding antibodies in patients with metastatic melanomas may have diagnostic value.

We aim to harness the natural humoral immune response by various technologies to get novel biomarkers. A complex antibody analysis in sera and in the tumor microenvironment leads to reveal tumor-specific antibodies. More strategies were introduced to select the most effective one to identify potential tumor antigen-binding capacity of the host. Epstein-Barr virus transformation and cloning with limiting dilution assay, magnetic cell sorting and antibody phage display with further methodological improvements were used in epithelial and neuroectodermal cancers. Column-purified sera of patient with melanoma were tested by immunofluorescence assay, while sera of further melanoma patients were processed for membrane-binding enzyme-linked immunosorbent assay. Some supernatants of selected B cell clones and purified antibodies showed considerable cancer cell binding capacity by immunofluorescence FACS analysis and confocal laser microscopy. Our native tumor cell membrane preparations helped to test soluble scFv and patients' sera for tumor binder antibodies. A complex tumor immunological study was introduced for patients with melanoma (ethical permission: ETT TUKEB 16462-02/2010); peripheral blood (n = 57) and surgically removed primary or metastatic tumors (n = 44) were gathered and processed at cellular immunological level. The technological developments proved to be important steps forward to the next antibody profile analyses at DNA sequence level. Cancer cell binding of patient-derived antibodies and natural immunoglobulin preparations of pooled plasma product intravenous immunoglobulins support the importance of natural human antibodies. Important cancer diagnostics and novel anticancer strategies are going to be built on these tools.

Long-term psychological and sexual outcomes of severe penile hypospadias repair.

INTRODUCTION: Hypospadias is the most common penis malformation, and there exist a variety of surgical approaches used to correct the abnormal position of the meatus. Although the long-term outcomes of surgery are considered important for psychosexual development, only a few attempts have been made to evaluate patient satisfaction. AIM: The aim of our study was to evaluate surgical results and psychosocial adaptations in a homogeneous group of subjects with severe penile hypospadias who underwent the same types of surgical repairs during childhood and compare the results to data obtained from age-matched healthy controls. METHODS: In this cross-sectional study, 104 men (between 24 and 42 years old) who underwent an uncomplicated two-stage hypospadias repair in their childhood and 63 age-matched healthy men without genital malformations completed the questionnaire. MAIN OUTCOME MEASURES: Difference in self-perception assessed by a 15-item questionnaire regarding psychosexual well-being and penile appearance between subjects with corrected hypospadias and healthy participants. RESULTS: On average, subjects with a hypospadias repair were less satisfied with their genital appearance; however, they were more satisfied with their sex lives compared to healthy controls. The meatus distance was approximately 1.5 cm from the tip of the penis after surgical correction. None of the postoperative surgical results correlated with patient satisfaction. Furthermore, the small percentage of patients (11%) who were very unsatisfied with their surgical outcomes had no significant differences in surgical outcomes compared to satisfied patients. However, there was a significant difference between the two groups in almost all psychological outcome measures. CONCLUSIONS: In adults who underwent an uncomplicated ventral repair of a severe penile hypospadias 20-30 years earlier, healthy psychosexual development was achieved despite the lack of a glanular meatus. Early identification of unsatisfied patients is important for appropriate long-term follow-up and counseling.

Comparison of mastectomy with breast-conserving surgery in invasive lobular carcinoma: 15-Year results.

BACKGROUND: Invasive lobular cancer (ILC) is biologically distinct from invasive ductal cancer and there is disagreement regarding appropriate local management of this disease. AIM: The current study reports long term results comparing mastectomy with breast-conserving surgery (BCS) in the treatment of ILC. MATERIAL AND METHODS: Study includes 235 women with ILC treated between 1983 and 1987. All of them underwent axillary dissection and either mastectomy (n = 163) or BCS (n = 72). 50 Gy adjuvant radiotherapy (RT) was given for 53 BCS and 81 mastectomy patients. The BCS group was compared with the mastectomy group. RESULTS: Patients treated with mastectomy or BCS had a similar outcome at 15 years with regard to distant metastasis-free (62% vs. 70%; p, 0.2017) and breast cancer-specific (62% vs. 70%; p, 0.1728) survival. In the BCS group the actuarial rate of ipsilateral in breast recurrences was 10% with and 53% without RT at 15 years (relative risk [RR], 0.10; p < 0.0001). In the mastectomy group the actuarial rate of chest wall recurrences was 16% with and 13% without RT at 15 years (RR, 1.45; p, 0.3965). Isolated ipsilateral in breast recurrence did not (RR, 1.73; p, 0.2767) but isolated chest-wall recurrence did (RR, 2.65; p, 0.0089) adversely affect cause-specific survival. CONCLUSION: Breast cancer specific survival is not affected by the type of surgical treatment. BCS and RT is a safe option to control local disease in patients with ILC.

[The effect of oxygenation on the biological behaviour of tumours]. / Az oxigenizáció hatása a daganatok biológiai viselkedésére.

Malignant tumours often display hypoxic tissue areas where the oxygen tension is < 7 mm Hg. Studies in this field have proved that the hypoxic state boosts tumour progression and aggressive behaviour. In tissue culture experiments "in vitro" oxygenation was found to inhibit in itself the proliferation of cells of healthy tissues as well as benign and malignant tumours. It is a very important observation from oncotherapeutic point of view that in the presence of partial oxygen pressure < 2.5 mm Hg the radiosensitivity decreases (intrinsic radioresistance). Most of the anticancer drugs (cytostatics) are also ineffective in hypoxic tumours (chemoresistance). The same is true for photodynamic treatments in oxygen deficiency or hypoxia. From time to time attempts based on these experimental and clinical observations are made to use oxygenation either as an adjuvant or an independent treatment in tumour patients. The most frequent treatment forms are: inhalation of oxygen gas (hyperbaric oxygen therapy), use of oxygen saturated water either in water or drinking cure. Recent international studies unanimously confirm the beneficial effect of oxygen intake on therapy, radio- and chemosensitization. The widespread erythropoietin treatment underlines the significance of oxygenation in tumour therapy. It seems reasonable to extend the preliminary studies on the tumour inhibitory, radio- and chemosensitizing effect of oxygenation to large study populations in major medical institutes in Hungary.

[Paradigm shift in surgical pathology of cancer: molecular diagnostics, prognosticators and predictive pathology]. / Paradigmaváltás a daganatok patológiai diagnosztikájában: molekuláris alapú differenciáldiagnosztika, prognosztikus és prediktív patológia.

Classical methodology of surgical pathology extended first with ultrastructural methods, then with immunohistochemistry and more recently with molecular/ genomic techniques. These changes added new dimensions to the classical tissue-and cellular levels of diagnostics: the molecular level. This change is the primary motor of the development of the diagnostic as well as the prognostic and predictive pathology. It is now possible to identify an etiological factor (HPV) or reclassify an entire entity (soft tissue tumors). Prognosis of cancer relies more and more on molecular/genetic parameters such as microsatellite instability, gene amplifications etc. Targeted therapy of cancer develops parallel with the molecular predictive pathology such as the anti-HER2 or anti-EGFR therapies. In conclusion, it is fair to say that molecular pathology contributes significantly to the development of clinical oncology.

HER-2/neu genotype of breast cancer may change in bone metastasis.

The genotype of breast cancer (BRC) is considered to be relatively stable during tumor progression, accordingly, determination of the estrogen receptor and HER-2/neu status is currently based on the primary tumor. However, recent data suggest that the gene expression profile of the metastatic lesion can be different compared to that of the primary BRC. Accordingly, it is possible that the HER-2/neu status is different in the metastatic lesion and the primary BRC. Since the bone is the most frequent metastatic site during the progression of BRC, we have analyzed the HER-2/neu status of 48 bone metastatic BRC cases by immunohistochemistry and fluorescent in situ hybridization, and it was possible to compare it to the primary site in 23 cases. The frequency of HER-2/neu amplification of BRC in the primary tumors was found to be 17.4% compared to 10.5% in bone metastases. Half of BRC cases with HER-2/neu amplification lost this genotype in bone metastases (4/23 versus 2/23, respectively) and even in the 2 cases where HER-2/neu amplification was retained in the metastases, the copy number was found to be decreased compared to the primary tumor. Based on our data and previous reports in the literature, we suggest to perform HER-2/neu testing both on primary tumor and samples obtained from BRC metastases, at least in case of primary tumors with HER-2/neu amplification, before introduction of HER-2/neu-targeting therapy.

Microvascular density of breast cancer in bone metastasis: influence of therapy.

BACKGROUND: Bone is the most frequent site of systemic progression of breast cancer (BRC). Angiosuppressive therapy has now entered the management of progressing cancers, therefore it is clinically important to obtain information on vascular endothelial growth factor (VEGF) expression and microvessel density (MVD) of bone metastasis of BRC. MATERIALS AND METHODS: VEGF expression and MVD were evaluated in bone metastases of BRC immunohistochemically in paraffin samples of 18 patients and compared to their primary tumors. MVD was determined by using the hot-spot method and the endothelial marker, CD34. RESULTS: Chemo- and/or endocrine therapy-naïve BRC cases progressed to the bone with a concomitant increase in their angiogenic potential, suggesting that this is the "natural history" of BRC progression. On the other hand, this study revealed that vascularization of the bone metastases of BRC patients that had received adjuvant (chemo- and/or endocrine) therapy was significantly decreased compared to the corresponding primary tumors, also supported by a decreased VEGF expression in metastases, both suggesting that the treatment significantly affected the angiogenic phenotype of the progressing disease. CONCLUSION: Angiosuppressive therapy is a new approach to cancer management including BRC and is frequently applied in the advanced stage of disease. Tumors with a prominent angiogenic phenotype (high MVD and VEGF) are primary candidates for such regimes. The fact that chemo-endocrine adjuvant therapy of BRC resulted in a weaker angiogenic phenotype in bone metastases compared to non-treated cases may suggest that these latter patients are better candidates for angiosuppressive interventions.

Novel ganglioside antigen identified by B cells in human medullary breast carcinomas: the proof of principle concerning the tumor-infiltrating B lymphocytes.

The potential tumor-recognizing capacity of B cells infiltrating human breast carcinoma is an important aspect of breast cancer biology. As an experimental system, we used human medullary breast carcinoma because of its heavy B lymphocytic infiltration paralleled to a relatively better prognosis. Ig-rearranged V region V(H)-J(H), Vkappa-Jkappa, and Vlambda-Jlambda genes, amplified by RT-PCR of the infiltrating B cells, were cloned, sequenced, and subjected to a comparative DNA analysis. A combinatorial single-chain variable fragment Ab minilibrary was constructed out of randomly selected V(H) and Vkappa clones and tested for binding activity. Our data analysis revealed that some of the V(H)-J(H), Vkappa-Jkappa, and Vlambda-Jlambda region sequences were being assigned to clusters with oligoclonal predominance, while other characteristics of the Ab repertoire were defined also. A tumor-restricted binder clone could be selected out of the single-chain variable fragment kappa minilibrary tested against membrane fractions of primary breast tumor cells and tumor cell lines, the V(H) of which proved to be the overexpressed V(H)3-1 cluster. The specific binding was confirmed by FACS analysis with primary breast carcinoma cells and MDA-MB 231 cell line. ELISA and thin layer chromatography dot-blot experiments showed this target Ag to be a ganglioside D3 (GD3). Our results are a proof of principle about the capacity of B cells infiltrating breast carcinomas to reveal key cancer-related Ags, such as the GD3. GD3-specific Abs may influence tumor cell progression and could be used for further development of diagnostic and/or therapeutic purposes.

Tumor-infiltrating B cell immunoglobulin variable region gene usage in invasive ductal breast carcinoma.

A major focus of tumor immunology is to reveal the potential role and capacity of immunocompetent cells found in different solid tumor tissues. The most abundant infiltrating cells (TIL), the T lymphocytes have been investigated in details concerning T-cell receptor usage and specificity. However, B cells have hardly been investigated in this respect, although high cellular B-cell infiltration has been correlated with improved patients' survival in some breast carcinomas. This led to our objectives to study variable region gene usage of the tumor-infiltrating B cells in different breast carcinoma types. By defining the immunoglobulin repertoire of the tumor-infiltrating B lymphocytes in the most common invasive ductal carcinoma (IDC) of the breast we compared it to the rare medullary breast carcinoma (MBC). After phenotyping infiltrating ductal carcinomas, B cells were obtained from tumor tissue by microdissection technique. Numerous rearranged TIL-B immunoglobulin heavy chain V genes (VH) were amplified, cloned, sequenced, and comparatively analyzed. Some characteristics were found for both breast carcinoma types. The immunoglobulins produced by TIL-B in ductal carcinoma are highly matured and oligoclonal. We conclude that Ig variable region gene usage reveals similar and distinguishable characteristics of TIL-B immunoglobulin repertoires, which are representative of the nature of the immune responses in invasive ductal and medullary breast carcinomas.