bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular markers expression in mediastinal nodes from resected stage I non-small cell lung cancer (NSCLC): Prognostic impact and potential role as markers of occult micrometastasis
Authors: Massuti B. , Galbis J. , Alenda C. , Peiró F. M. , Martí J. , Baswitch B. , Rodriguez-Paniagua J. M. , Rosell R. , Taron M. , Benlloch S.
Published: March 2020

21016

Background: 5-y surv in surgically resected St I NSCLC is 60–70% whereas in cases with affected lymph nodes (LN) it is < 50%. The main risk factor for recurrence is nodal disease. Current histopathologic analysis can miss occult micrometastases in nodal tissues at initial diagnosis. Detection of micromets with a more sensitive technique would be useful to define a high-risk population selecting patients (p) for postop treatment. We assessed the role of several genes mRNA expression in pathological negative LN from resected St I NSCLC p as markers of occult micromets and correlated the results with relapse, and survival. Methods: Paired tumor and histological negative LN (n=344) obtained by systematic mediastinal lymphadenectomy from 38 surgically resected St I NSCLC p were analyzed for the presence of 12 genes mRNA expression using RT-Q-PCR in an ABIPRISM 7500. RNA was extracted using ABIPRISM 6100. Specifically designed primers and probes were purchased from Applied Biosystems as Assay-on-demand; GADPH was used as an endogenous control. Samples were also analyzed by ICH for LN staging. Results: 38 NSCLC p; 12 adenoca, 16 squamous cell, 10 undifferentiated . From the 12 tested genes CEA and PLUNC were found with high expression in lung tissue and low or null expression in normal LN. We consider molecular + LN those in which expression of CEA or PLUNC was detected. In the 344 pathological negative LN, 13% (44/344) were positive for CEA, 16% (54/344) for PLUNC. The expression patterns were similar for both markers. At a median follow-up of 24 mo (9–46) 11 p had died from NSCLC and 1 had died without recurrence. None of the living p had tumor recurrence. For the prognostic assessment, molecular + LN were classified as N1 and N2. Median DFS was 15 ± 11.74 mo in p with N2 molecular + nodes was and has not yet reached in cases of molecular (-) LN (p=0.028). Median survival of p with N2 molecular positive nodes was 17.3 ± 5.7 mo and has not yet reached (p= 0.0083) for molecular (-) LN. Conclusions: CEA and PLUNC mRNA expression could be used as molecular markers of occult micrometastases in mediastinal LN showing a prognostic effect . CEA and PLUNC expression provides a tool for selecting high-risk p considered for adjuvant therapies

No significant financial relationships to disclose.
Concordance of local and central laboratory hormone and HER2 receptor status in ECOG 2197
Authors: Badve S. S. , Baehner F. L. , Gray R. , Childs B. , Maddala T. , Rowley S. , Shak S. , Davidson N. , Goldstein L. J. , Sparano J.
Published: March 2020

21022

Background: Central and local laboratory concordance for hormone and HER2 receptor measurement is of national interest. This study compares ER/PR/HER2 by local laboratories using immunohistochemistry (IHC) and central laboratories (IHC & quantitative RT-PCR). Methods: Of 2952 patients in E2197, a case-cohort sample of 776 patients who either did (N=179) or did not recur was studied. Central IHC for ER/PR/HER2 was performed using single 0.6 mm microarrays; Allred score (AS) was used for ER/PR (AS>2 = positive). Positive HER2 was 3+ staining in >10% cells for Central IHC and 2+ or 3+ for Local IHC. RT-PCR analysis by Oncotype DX™ for ER/PR/HER2 was performed using pre-defined cutoffs of 6.5, 5.5 and 11.5 units, respectively. Hormone receptor (HR) pos was defined as ER &/or PR pos. Results: Results from Local IHC (ER/PR in 776 & HER2 in 517 pts) were compared with Central IHC (760 pts) and RT-PCR results (776 pts). The discordance between HR positivity by Local IHC and RT-PCR was very low. However, 12% of HR neg pts by Local IHC (38/321) & Central IHC (39/326) were HR pos by RT-PCR. The relationship between ER and recurrence as a function of AS was examined. Patients with AS of 3–4 were found to be closer to the AS=2 group than to the AS>4 group Patients with AS of 3–4 were found to be closer to the AS ÿ 2 group than to the AS > 4 group (Est.HR for ER 0.97 for AS 3–4 vs. 0–2 and 0.46 for AS 5–8 vs. 0–2, and for PR were 0.84 for AS 3–4 vs. 0–2 and 0.41 for AS 5–8 vs. 0–2). Conclusions: There is a high degree of overall concordance among Local IHC, Central IHC, and Central RT-PCR for ER and PR. The degree of concordance is even greater for HR compared to ER or PR alone. Although the concordance with local labs for HER2 testing was poor, the concordance between Central IHC and RT-PCR was very high. The relatively high incidence (12%) of IHC HR neg pts who are HR pos by RT- PCR is notable.

[Table: see text]

[Table: see text]
Detection of DNA methylation in inflammatory breast tumours by a multiplex nested methylation-specific PCR
Authors: Van der Auwera I. , Van den Bosch S. , Van Laere S. , Van den Eynden G. , van Dam P. , Colpaert C. , van Engeland M. , Van Marck E. , Vermeulen P. , Dirix L.
Published: March 2020

21027

Background: Hypermethylation of gene promoters is a common mechanism of loss of gene function in cancer cells. Few data on epigenetic alterations in inflammatory breast cancer (IBC) cells are available. Microarray studies have shown that IBC is characterised by a distinct gene expression profile when compared to non-IBC. We sought to determine whether the profiles of gene hypermethylation differ between IBC and non-IBC by using a multiplex nested methylation-specific PCR (MSP). We selected genes for which evidence exists for their involvement in breast carcinogenesis by means of methylation. Methods: The SLμCut system (MMI) was used for laser capture microdissection of paraffin-embedded tissues. 10-μm-thick sections from 10 patients with IBC and 12 patients with non-IBC were used. Genomic DNA from the microdissected tissue was extracted and treated with sodium bisulfite. The methylation status of the promoter regions of APC, E- cadherin, THBSP-1, DAPK, GSTP1, RASSF1A and BRCA1 were investigated using a multiplex nested MSP method. DNA isolated from normal peripheral lymphocytes from healthy individuals served as a negative methylation control. In vitro methylated DNA served as the positive methylation control. MSP products were analyzed on agarose gel electrophoresis, and were determined to have methylation if a visible band was observed in the methylation reaction. Results: Methylation of the APC gene promoter was observed in 33.3% (4/12) of non-IBC samples and 70% (7/10) of IBC samples. Methylation of the BRCA1 gene promoter was observed in 16.7% (2/12) of non-IBC samples and 30% (3/10) of IBC samples. Methylation of the DAPK gene promoter was observed in 16.7% (2/12) of non-IBC samples and 10% (1/10) of IBC samples. Methylation of the GSTP1 gene promoter was observed in 33.3% (4/12) of non-IBC samples and 30% (3/10) of IBC samples. No methylation of the E-cadherin and THBSP-1 gene promoters was observed in non-IBC samples or IBC samples. Conclusions: Also in this study, the multiplex nested MSP approach was proven to be very suitable for the detection of methylation in samples harbouring small amounts of poor quality DNA. In the near future, the methylation status of other genes will be determined and the study population will be enlarged.

No significant financial relationships to disclose.
EBV-positive lymphoma patients have a selective deficiency in EBV immunity
Authors: Heller K. N. , Steinherz P. G. , Portlock C. S. , Münz C.
Published: March 2020

21032

Background: Epstein-Barr virus (EBV) asymptomatically establishes persistent infections in more than 90% of the adult population. However, due to effective immune control, only a minority of infected carriers develops spontaneous EBV-associated lymphomas. Since EBV nuclear antigen-1 (EBNA1) is the only protein expressed in all proliferating EBV infected cells we hypothesize that EBNA1 specific immune response is critical in preventing EBV-positive lymphomas. Methods: After informed consent, peripheral blood from healthy volunteers and lymphoma patients (prior to therapy- no evidence of cytopenia) were stimulated (ex vivo) with overlapping peptides covering the immunogenic EBNA1 (aa400–641) sequence. Frequency of EBNA1-specific T-cells were assessed by intracellular cytokine staining and flow cytometric proliferation assays. Cytokine pattern, surface marker phenotype and functional reactivity against EBV specific and control antigens were analyzed. Results: Patient and volunteer immune responses to control antigens and other viruses were assessed and statistically indistinguishable. EBNA1 specific CD4+ T cell responses were detected among 18 of 20 healthy carriers, and among 10 of 16 patients with EBV-negative lymphoma (relative to healthy volunteers p=0.145 via paired student T test). None of the patients with EBV-positive lymphomas (n=8) had a detectable EBNA1-specific CD4+ T-cell response (p<0.003 relative to healthy volunteers and patients with EBV-negative lymphomas). Conclusions: Healthy volunteers and patients with EBV-negative lymphoma have statistically similar EBNA1-specific CD4+ T cell responses. Although patients with EBV-positive lymphoma have intact immune responses to common viruses and antigens, they selectively lack an EBNA1-specific CD4+ T cell response. An intact EBNA1 specific immune response among patients with EBV-negaitve lymphoma implies that lymphoma is not a cause of a selective immune deficiency. On the contrary, these findings suggest that EBNA1-specific CD4+ T cells are critical in the prevention of EBV mediated lymphomas, and a defect in EBNA1 specific immunity may leave EBV carriers suseptible to EBV-positive lymphomas. EBNA1- specific CD4+ T cell function may be a new target for therapies of EBV-associated malignancies.

No significant financial relationships to disclose.
Proliferation and apoptosis before and after primary systemic therapy for operable breast cancer
Authors: Guarneri V. , Piacentini F. , Frassoldati A. , Ficarra G. , Jovic G. , Puglisi F. , Michelotti A. , Bisagni G. , Berardi R. , Conte P.
Published: March 2020

21045

Background: The identification of biological markers able to predict tumor sensitivity to primary systemic therapy (PST) may have an important clinical output. Moreover, the effect of treatment on these biomarkers might have prognostic relevance. Aims of this analysis: to evaluate the predictive role of proliferation (Ki-67), apoptotic index (AI) and growth index (GI) before PST, the effect of PST on these parameters and their relation with tumor response and patients outcome. Methods: Ki-67, AI (Tunel Test), and GI were assessed at baseline and at surgery in 90 stage II-IIIB breast cancer patients enrolled in a phase II randomized trial of PST with 4 cycles of epirubicin-paclitaxel ± gefitinib. Pathologic response was defined as tumor regression grade (TRG) (criteria of Miller and Payne). Results: patients characteristics were as follows: median age, 51 yrs (range 29–69); stage IIA 41%, IIB-IIIA 59%. A significant correlation between Ki-67 and AI was found at baseline (p=0.001). A significantly higher Ki-67 and AI were observed in ER- vs ER+ tumors (median Ki-67: 62% vs 25%, p<0.0001; median AI: 1.33 vs 0.85, p=0.009). A significantly higher Ki-67 and GI were observed in EGFR+ vs EGFR- tumors (median Ki-67: 60% vs 25%, p=0.0006; median GI: 48 vs 30.7, p=0.03). No significant differences were observed according to HER2 expression. After PST, 84% of pts was classified as having a TRG 4 or 5, and 16% as having a TRG 1–3. No significant correlation among baseline Ki-67, AI and GI and TRG was observed. After PST, Ki 67 and AI were significantly reduced from baseline. Lower Ki 67 and lower AI were significantly correlated with higher TRG (p=0.008 and 0.01 respectively). We observed 1 cancer death and 5 relapses only, therefore correlation with patient outcome has been planned for the time of the meeting Conclusions: ER- and EGFR+ tumors show more aggressive behaviour. PST significantly reduces Ki-67 and AI, and this reduction is significantly correlated with TRG. In particular, the reduced AI at surgery was not found to be a marker of resistance, more probably reflecting the reduced Ki-67 of responding tumors.

No significant financial relationships to disclose.
EGFR expression is associated with increased cell turnover and poor histologic grade in human colon carcinomas
Authors: Rego R. L. , Smyrk T. C. , Foster N. R. , Sargent D. J. , Kabat B. F. , French A. J. , Thibodeau S. N. , O'Connell M. J. , Windschitl H. , Sinicrope F. A.
Published: March 2020

21046

Background: The Epidermal Growth Factor Receptor (EGFR) is a transmembrane receptor tyrosine kinase that is important for colon cancer cell growth and represents a therapeutic target. We studied EGFR expression in relation to rates of apoptosis and cell proliferation. Methods: TNM stage II and III colon carcinomas (n= 361) from patients treated in a 5-FU-based adjuvant trial were studied. EGFR expression (EGFR pharmDx™) and extent were analyzed and their relationship to rates of cell turnover, histologic grade, mismatch repair (MMR) status (defective if instability at BAT 26 locus plus loss of hMLH1, hMSH2 or hMSH6 proteins) and clinical outcome were determined. Results: EGFR plasma membrane staining was detected in 214 (59.3 %) colon carcinomas. Increased EGFR intensity (2+, 3+) was associated with a higher number of caspase-3- (p= 0.02) and Ki-67- (p< 0.001)positive tumor cells. Increased EGFR intensity (p= 0.02) and extent (p= 0.03) were associated with poor differentiation, and did not depend upon MMR status (Breslow-Day test p ≥ 0.46). Diffuse (vs. focal) EGFR extent was increased in tumors with defective MMR [22 of 32 (69%)] compared to those with intact MMR [136 of 271 (50%)] (p= 0.047). EGFR intensity was unrelated to MMR status. Neither EGFR intensity nor extent was significantly associated with patient survival. Conclusions: Stage II and III colon cancers with EGFR overexpression show adverse prognostic variables, i.e., increased rates of cell turnover and poor histologic grade, suggesting that EGFR may be an important target for adjuvant therapy. *Chi-square test for categorical data, Wilcoxin rank-sum test for continuous data

[Table: see text]

No significant financial relationships to disclose.
Role of MSI and DCC status to predict response to FOLFOX in metastatic colorectal cancer
Authors: Bennamoun M. , Schischmanoff O. , Martin A. , Mariani P. , Ah Koon L. , Morere J. , Des Guetz G.
Published: March 2020

21056

Background: Microsatellite instability (MSI) and lack of Deleted Colon Cancer (DCC) protein expression have been observed respectively in ∼15% and 70% of colon cancer cases and are considered as prognostic factors in colorectal cancer (CRC). In adjuvant setting these markers could be considered to decide treatment. We know that MSI colon cancer do not benefit from 5Fluorouracyl (5FU)-based chemotherapy. A current treatment of reference for colon cancer is a combination of 5FU and Oxaliplatin (FOLFOX). Our aim was to determine whether MSI status or DCC expression are predictive markers of FOLFOX efficiency in patients with metastatic CRC. Methods: Tumour specimens were collected from patients with metastatic colon cancer treated with FOLFOX. The FOLFOX regimen was evaluated in first line treatment according to the WHO criteria. The microsatellite instability status was assessed by measurement of the length of five monomorphic mononucleotide markers. DCC protein expression were evaluated by immunohistochemistry. Results: Forty patients (22 men, 18 women), median age 63.5 years (27–83) were treated with FOLFOX. Nine patients had tumours exhibiting high microsatellite instability (microsatellite instability group, MSI group) and 31 patients had tumours exhibiting microsatellite stability (microsatellite stable group, MSS group). In MSS group, we observed 11 partial responses (36%) and only two in MSI group (22%) (not significant difference). Both disease free survival and overall survival were not significantly different for MSI and MSS groups. Determination of DCC status is under investigation. Conclusions: There was no significant difference in chemosensitivity to FOLFOX for MSI and MSS metastatic patients. Therefore FOLFOX regimen could be proposed to metastatic patients irrespective of MSI status. The study of the relationship between DCC expression and response to FOLFOX is ongoing.

No significant financial relationships to disclose.
Differential gene expression profile in breast cancer-derived stromal fibroblasts
Authors: Singer C. F. , Gschwantler-Kaulich D. , Fink-Retter A. , Hudelist G. , Haas C. , Czerwenka K. , Kubista E. , Kubista E.
Published: March 2020

21075

Background: Breast cancer is chatacterized by malignant transformation of epithelial cells, but stromal cells also play an important role in tumorigenesis. While tumoral fibroblasts display unique phenotypical properties, it is unclear whether they also represent are a specific subpopulation. Materials and Methods: Stromal fibroblasts deriving from malignant tissue of 10 women with invasive breast cancer, and from normal breast tissue of 10 women with benign breast disorders, were subjected to differential complementary DNA Microarray Analysis by using a 2400 gene cDNA array. Gene expression results were validated by real-time PCR and by immunohistochemistry. Results: In a cDNA Array that allows to analyze the differential gene expression of more than 2400 genes, the mRNA expression of 135 genes were increased more than 2 fold in fibroblasts from malignant breast tumors. The majority of these genes encode tumor-promoting cytokines, transcription factors and cell-matrix associated proteins. The mRNA expression of 110 genes decreased to less than 0.5 fold. The remaining 2155 genes were not significantly altered. Immunohistochemistry for selected proteins performed on biopsies from breast cancer and normal breast tissues confirmed the clinical relevance of our findings. Conclusion: Breast cancer-derived stromal fibroblasts show a distinctive gene expression pattern that differentiates them from normal breast stroma. Our observation of increased expression of tumor promotion-associated genes even in the absence of adjacent malignant epithelium suggests that tumor stroma is comprised of a fibroblastic subpopulation that provides for a microenvironment which supports tumor growth and invasion.

No significant financial relationships to disclose.
Pattern of expression of calpain subunits (large and small) in human breast cancer and the prognostic significance
Authors: Chhabra A. , Fernando H. , Mansel R. E. , Jiang W. G. ,   Metastasis & Angiogenesis Research Group
Published: March 2020

21078

Background: Calpains belongs to family of non-lysosomal calcium dependent cysteine proteases which are known to regulate cellular migration, apoptosis and cell cycle progression in normal and tumour cells. Calpain has also been indicated in the metastatic process of certain tumours like RCC and identified as a potential tumor suppressor for gastric cancer. Little is known about the expression pattern of calpain in human breast cancer. The current study investigated the expression pattern of the large (calpainL) and small (calpainS) subunits of calpain which are encoded by different genes, in human breast cancer and attempted to correlate the expression with clinical outcome. Methods: RNA was extracted from frozen sections of breast tissue (n=120, median clinical follow up of the patients - 72 months) for gene amplification. The expression of both subunits of calpain was determined by using RT-PCR and quantitative RT-PCR. Statistical analysis was carried out using Mann- Whitney U test and the Kruskal- Wallis test. Results: We found significantly higher level of both calpain subunits in tumour tissue (n=120) as compared to normal non-neoplastic mammary tissues (calpainL, p=0.022 and calpainS, p=0.039). The expression of calpainL was significantly lower in patients with poor clinical outcome (with metastasis, p=0.024, with local recurrence, p=0.024 and who died of breast cancer, p=0.028), than those who were disease free. In contrast, the levels of calpainS were high in patients with poor prognosis, metastasis and who died of breast cancer but were statistically not significant. The expression of calpainL was marginally lower in node positive tumours than the node negative tumours. CalpainS transcripts were higher in node positive than node negative tumours (p=0.23). Similarly, calpainL showed a low level in high grade tumours whereas calpainS displayed a contrasting high level in these tumours. Conclusions: The large and small subunits of calpain have a distinct pattern of expression in human breast cancer. A decrease expression of calpainL but an increase expression of calpainS may be associated with poor prognostic tumours, thus indicating the intimate nature of the molecular pair in the disease progression of human breast cancer.

No significant financial relationships to disclose.
Identification of a new subset of myeloid suppressor cells in peripheral blood of melanoma patients and modulation by GM-CSF-based anti-tumor vaccine
Authors: Filipazzi P. , Valenti R. , Huber V. , Iero M. , Pilla L. , Parmiani G. , Santinami M. , Rivoltini L.
Published: March 2020

21082

Background: Phenotypic and functional features of myeloid suppressor cells (MSC), known to serve as critical regulators of anti-tumor T cell responses in tumor-bearing mice, are still poorly defined in human cancers. Here we analyzed myeloid subsets with suppressive activity present in peripheral blood of metastatic melanoma patients (MM) and evaluated their modulation by a GM-CSF-based anti- tumor vaccine. Methods: Stage IV AJCC MM patients (n=16) vaccinated with autologous tumor-derived heat-shock protein peptide complexes gp96 (HSPPC-96) and low dose GM-CSF provided pre- and post-treatment whole blood samples. Peripheral blood mononuclear cells (PBMC) were analyzed by flow cytometry, separated into cellular subsets and used for in vitro proliferation assays. PBMC from stage- matched melanoma patients (n=12) treated with non-GM-CSF-based vaccines (i.e. HSPPC-96 alone or IFNa/melanoma-derived peptides) or gender and age-matched healthy donors (n=16) were also analyzed for comparison. Results: The lack or low levels of HLA-DR expression was found to identify a CD14+ cell subset with high suppressive activity on lymphocyte proliferation and functions. CD14+HLA-DR-/lo cells were significantly expanded in all MM patients, while undetectable in healthy donors. Suppressive activity was mediated by TGFβ, as suggested by functional experiments with neutralizing specific antibodies. In contrast, no involvement of arginase and iNOS pathways could be detected. CD14+HLA-DR-/lo cells, as well as spontaneous ex- vivo release and plasma levels of TGFβ, were augmented after administration of the HSPPC-96/GM-CSF vaccine. Interestingly, the expansion of suppressive CD14+ monocytes was associated to the inability to mount a significant CD8-mediated T cell response upon vaccination. On the other hand, no quantitative or qualitative enhancement of the CD14+HLA-DR-/lo suppressive cell population was observed in patients receiving a non-GM-CSF based vaccine. Conclusions: CD14+HLA-DR- /lo cells exerting TGFβ-mediated immune suppression may represent a new subset of myeloid suppressive cells, potentially expandable by the administration of GM-CSF-based vaccines in melanoma patients.

[Table: see text]
Overexpression of VEGF-C and -D may predict unfavorable outcome in resected non-small cell lung cancer
Authors: Ko Y. , Jung C. , Lee M. , Kang J. , Hong Y. , Lee K.
Published: March 2020

21114

Background: Correlation of Vascular endothelial growth factor (VEGF)-C, VEGF-D and/or VEGF receptor 3 (VEGFR-3) with lymphatic spread, tissue invasion and poor clinical outcomes has been observed in various cancers. Nodal metastasis is known to one of poor prognostic factors in non-small cell lung cancer (NSCLC). We investigated the correlation of VEGF-C,-VEGF-D and VEGFR-3 with clinicopathologic parameters and patient survival in NSCLC. Methods: Using immunohistochemial staining, we analyzed the protein expressions of VEGF-C, VEGFD and VEGFR-3 on the tissue array specimens from 180 patients with completely resected NSCLC. A 0 to +1 immunohistochemical staining (IHCS) of the cancer cell was defined as negative, +2 to +3 IHCS was as positive. Results: The expressions of VEGF-C, VEGF-D and VEGFR-3 were observed in 28.9%, 32.8% and 30.6% of cases, respectively. In the Cox regression-based multivariate analysis, it was proved that VEGF-C and D proved to be an independent prognostic factors as well as known prognostic factors, such as tumor size and lymph node metastases (VEGF-C, p=0.001; VEGF-D, p=0.014). VEGF-C expression showed statistically significant correlation with the expression of VEGFR-3 (p=0.02). Conclusion: To be taken, the present study revealed that VEGF-C and D expression may predict poor prognosis in resected NSCLC patients. Therefore, these results seems to be worth developing promising targeting inhibitors on lymphangiogenesis which plays major role in locoregional recurrence for surgically NSCLC patinets.

No significant financial relationships to disclose.
Steroid hormone status and HER2/neu expression in pregnancy-associated breast cancer
Authors: Litwiniuk M. , Breborowicz E. , Breborowicz D. , Filas V. , Breborowicz J.
Published: March 2020

21115

Background: Breast cancer occurring during pregnancy or within the first year after delivery is considered to be a pregnancy-associated breast cancer. Some studies have shown a decreased estrogen receptor-positive and progesterone receptor-positive status in pregnant patients with breast cancer, others, however, have not. Moreover, little information is available about the expression of HER2/neu and about the other human estrogen receptor, estrogen receptor beta (ERbeta) in pregnancy-associated breast cancers. The aim of this work was to determine the extent of the expression of ER alpha and beta, progesterone receptor (PgR) and HER2/neu in pregnancy- associated breast cancer. Material and Methods: Formalin-fixed, paraffin embedded tissues from 16 patients with pregnancy- associated breast cancer were used in this study. Immunostaining for ERalpha, ERbeta and PgR was performed using monoclonal antibodies against ERalpha, PgR (DakoCytomation) and against ERbeta (CHEMICON). The EnVision detection system was applied. Tumors were considered to be expressing receptors if a positive reaction (regardless of intensity) could be identified in at least 10% of cells. The HER-2 status was analyzed using HercepTest TM (IHC), and IHC 2+ results were confirmed with FISH test. Results: 44% of the tumors (7/16) were ERalpha and PgR positive. The expression of ERbeta protein was observed in 94% of pregnancy-associated breast cancers. As many as 50% of ERbeta positive tumors showed no expression of ERalpha. Further, 31% (5 of 16) of breast cancers were HER2/neu positive. Conclusions: This frequent expression of ERbeta in pregnancy-associated breast cancer may result from their prodifferentiative functions which increase during pregnancy and lactation.

No significant financial relationships to disclose.
Noninvasive diagnostic test in breast cancer; utopia or option with serum proteomics?
Authors: de Noo M. , van der Werff M. , Gelderblom H. , Deelder A. , Tollenaar R.
Published: March 2020

21118

Background: With a lifetime risk currently estimated 1 in 9, breast cancer is among the most common diagnosed malignancies. Proteomic expression profiling generated by mass spectrometry has been suggested as a potential tool for the early diagnosis of cancer. The objective of our study was to assess and validate the feasibility of this approach for the detection of breast cancer. Methods: In a randomized block design pre-operative serum samples obtained from 63 breast cancer patients and 73 controls were used to generate high-resolution MALDI-TOF protein profiles as a calibration set. The median age of the patient and control group was respectively, 52 (20–81) and 57 years (39–87). The MALDI-TOF spectra generated using WCX magnetic beads assisted mass spectrometry (Ultraflex) were smoothed, binned and normalized after baseline correction. After pre-processing of the spectra, linear discriminant analysis with double cross-validation, based on principal component analysis, was used to classify the protein profiles. Consequently, the classifier constructed on the first 2 plates was applied on the spectra of an independent validation set. This validation set consisted of serum samples from 29 breast cancer patients and 38 controls. The median age was 59 years (26–87) and 57 years (24–71) for the patient and control group respectively. Results: Double cross-validatory analysis carried out on the protein spectra of the calibration set yielded a total recognition rate of 86%, a sensitivity of 88% and a specificity of 84% for the detection of breast cancer within the calibration set. The AUC of this classifier was 90.3%. When this classifier was applied on the spectra of the independent validation set a total recognition rate of 80.9%, a sensitivity of 72% and a specificity of 89% were found. Conclusions: The use of a randomized block design, but mainly an independent validation set proves that discriminating protein profiles can be detected between breast cancer patients and healthy controls. Although further validation in larger series and identification of the discriminating proteins must be achieved, the high sensitivity and specificity indicate that serum protein profiles could be an option for the detection of breast cancer.

No significant financial relationships to disclose.
Oncogenotoxicology: A new approach to individual therapy and to the follow-up of patients
Authors: Gamulin M. , Grgic M. , Bonassi S. , Lucas J. N. , Fucic A.
Published: March 2020

21131

Background: The growing awareness of differences in inter-individual response to radiochemical exposure calls for a new approach in the therapy and follow-up of cancer patients. Results obtained by genotoxicological methods (Bonassi, 2006) showed that surrogate tissue could be used to predict cancer risk. Chromosome aberration (CA) and micronucleus (MN) assay enables:determination of radio- and chemosensitivity level in a patient, adjustment of therapy and detection of chromosomal rearrangements possibly related to the development of secondary cancer using fluorescent in situ hybridization (FISH) Methods: Ten women with breast cancer (BP) and 10 men with oropharinx cancer (OP) were analyzed before, during and after radiotherapy by CA and MN assay. Confounding parameters (age, diagnostics, smoking, drinking) were monitored and matched. Results were compared with 20 matched control subjects. The BP group received an irradiation dose of 60 Gy and the OP group of 62 Gy Results: Before therapy, the OP patients had a significantly higher MN frequency than BP and controls (23.2±4.9‰ versus 6.3±1.6 ‰ and 6.7±3.7‰). On measurements taken 6 and 12 months after the therapy, OP patients continued to have a significantly higher MN frequency than BP patients (60.2±11.4‰ vs. 8.4±2.4‰ and 30.7±4.7‰ vs.5.11±2.5‰, respectively). CA frequency was similar in both groups before therapy (2.5±1.1% in BC and 3.4±1.3% in OP; controls 1.2%). Twelve months after the therapy, the elimination of CA in OP patients was significantly slower than in BC patients (17.7±10.9% vs. 8±5.4%, respectively). As OP patients consumed more than 120 g/day of pure alcohol, it is possible that alcohol could have caused additional genome damage (Maffei, 2002). The highest inter-individual difference in genome damage was observed in BP patients Conclusions: The results show that the CA and MN assay are sensitive methods which can be used to introduce a new concept, oncogenotoxicology, whose goal is to improve the therapy results by evaluating inter-individual differences in response to radiochemical treatment and to improve the evaluation of risk of secondary cancer development. FISH in suspension (Wu, 2005) will allow automation of CA detection, and facilitate a more accurate prospective study of the same population.

No significant financial relationships to disclose.
Intraoperative radiotherapy (IORT) impairs surgical wound-stimulated breast cancer cell invasion
Authors: Baldassarre G. , Belletti B. , Vaidya J. S. , D'Andrea S. , Roncadin M. , Perin T. , Trova M. G. , Candiani E. , Veronesi A. , Colombatti A. , Massarut S.
Published: March 2020

21139

Background: Risk of local recurrence after complete excision of breast cancer is higher in the area around the original tumor, suggesting that wound healing may be implicated. Methods: We collected blood samples (BS) before surgery and wound fluid (WF) in the 24 hours after breast conserving surgery from 50 patients. Twenty five of these patients were also treated with TARGeted Intraoperative radioTherapy (TARGIT, 20Gy to tumor bed surface in one session), immediately after the surgical excision. The ability of the BS and WF to stimulate growth and motility of a panel of normal and mammary carcinoma cells was studied. A proteomic approach was used to analyze the expression pattern of WF and BS. Results: We assayed five cell lines in a transwell based assay using individual patient BS and wound fluids WF as potential chemo-attractants. WF strongly attracted cells from all 5 types of breast cancer cell lines that we tested including MDA- MB 231, MCF-7, MDA-MB 453, T47D and SKBR-3. The WF attracted the cells better than both the respective BS. Importantly, in all tested cell lines TARGIT significantly impaired the ability of WF to attract cancer cells (p=0.03 MDA-MB 231 and MCF-7. p=0.01 for MDA-MB 453, and SKBR-3). Moreover, while BS did not stimulate 3D motility over the control WF strongly stimulated 3D movement of MDA-MB 231 and MDA-MB- 453 cells. This stimulatory effect was abrogated in the WF taken from patients who had received TARGIT (p=0.01 for MDA-MB 231 and p<0.0001 for MDA-MB 453). Similar results were obtained when cell proliferation was evaluated using the same cell lines and BS or WF samples. Finally, proteomics analyses demonstrated that TARGIT modifies the expression levels of several key proteins involved in tumor cell growth and dissemination. Conclusion: TARGIT delivered to the tumor bed alters the cytokines and growth factors expression patterns in the surgical wound and abrogates its stimulatory effect on cancer cell growth and motility. This novel mechanism of action of radiotherapy could partly explain the very low recurrence rates found in large pilot studies of this technique and open new avenues for peri-operative therapies.

No significant financial relationships to disclose.
Pathologic complete remission of cytologically proven axillary node metastases may be a strong prognostic factor in HER2-positive breast cancer
Authors: Sakakibara M. , Nagashima T. , Nakamura R. , Fujimoto H. , Arai M. , Kadowaki M. , Miyazaki M.
Published: March 2020

21149

Background: Preoperative chemotherapy has become one of the standard modes of treatment in patients with breast cancer, and pathologic complete remission (pCR) thus obtained has become an important prognostic factor. However, the number of cells in the main lesion is enormous and changes over time in a heterogeneous manner. Thus, time and individual differences in sensitivity influence the acquisition of pCR, and questions about the coincidence with the effects of metastatic lesions also remain. On the other hand, although pCR of axillary lymph node metastasis lesions with relatively lower numbers of the cells is considered one of the best surrogate prognostic markers, evaluation criteria have not yet been established because definitive preoperative diagnosis is difficult. In our department, preoperative axillary cytodiagnosis has been conducted in all surgical breast cancer cases. In this study, we investigated pCR of the axillary lymph nodes (ALN- pCR) in cytologically proven axillary node metastasis cases. Subjects and Methods: ALN-pCR, DFS, and OS were investigated in 52 surgical breast cancer cases (observation period: 15–52 months; medial level: 28 months) positive for cytological axillary metastasis and received preoperative chemotherapy (AC-Paclitaxel) during the period from 2002 to 2005. Results: ALN-pCR was found in 21 of 52 cases (40.4%). Rates of disease-free survival (DFS) at 36 months in ALN-pCR and non-ALN-pCR cases were 85.0% and 42.3% (P=0.031) and those of overall survival (OS; 48 months) were 100% and 87.9% (P=0.353), respectively. DFS (36 months) was very favorable 100% in ALN-pCR cases that obtained the effect of 1b or higher on evaluation of the main lesion effect and ALN-pCR cases with Her2-positive results on sub-type analysis. Conclusions: ALN-pCR of cytologically proven axillary node metastasis was indicated to be a prognostic factor in cases of breast cancer. Especially, its importance was demonstrated in HER2-positive breast cancer. From these findings, HER2-positive and cytologically axillary lymph node-positive cases were considered the best indications for preoperative chemotherapy.

No significant financial relationships to disclose.
Effects of low molecular weight heparin on angiogenic factors in advanced cancer patients
Authors: Coskun H. , Karakus N. , Vural H. , Akturk O. , Alanoglu G.
Published: March 2020

21158

Background: The relationship between cancer and thrombosis is known for many years. Thrombosis can be associated with the biology and therapy of the disease in cancer patients. Low molecular weight heparin (LMWH) is shown to increase the duration of life in several types of cancer in clinical trials. Low molecular weight heparin is thought to be inhibiting with tumor growth and metastasis in several ways. In our study, we evaluate effects of LMWH therapy on angiogenic factors. Methods: Thirty-two advanced cancer patients who used prophylactic LMWH before chemotherapy. Median age was 64.5 years with range from 18 to 80. Sixteen healthy volunteers that are of same age and gender with study patients comprised the control group. VEGF, HGF, FGF, EGF, PDGF, ICAM, p-selectin, e-selectin levels were evaluated before and 7 days after LMWH therapy. Results: In baseline measurements HGF and ICAM levels were higher in treatment group compared the control group. But, levels of EGF and VEGF were not statistically significant higher in the treatment group compared to control group. After LMWH therapy, VEGF, FGF, PDGF, ICAM, e-selectin levels appeared to decrease and HGF, EGF, p-selectin levels appeared to increase but none of these changes were statistically significant. In a subset of seventeen patients with adenocarcinoma, levels of ICAM declined significantly after LMWH therapy this group. Before LMWH therapy; HGF was correlated with PDGF, VEGF, p-selectin, e-selectin; EGF was correlated with p-selectin; e-selectin was correlated with p- selectin. After therapy HGF was correlated with PDGF and EGF; PDGF was correlated with FGF and p-selectin; EGF was correlated with ICAM levels. Conclusions: In summary, with a short course of LMWH therapy a change in anjiogenic biomarkers was observed only in adenocarcinoma patients and this change was limited only to ICAM levels. The study focused on long term course was warranted.

No significant financial relationships to disclose.
Examining ethnic differences for bevacizumab-induced hypertension and proteinuria
Authors: Choi Y. M. , Shord S. , Cuellar S. , Villano J.
Published: March 2020

21168

Background: Bevacizumab is an increasingly used anti-cancer treatment with common side effects including hypertension (htn) and proteinuria which occur in approximately 10% and 20% of the patients, respectively. Little is known regarding ethnic variations of bevacizumab induced htn and proteinuria, particularly in African-Americans (AA) who have a high prevalence of htn and susceptibility to kidney disease. Methods: We conducted a retrospective chart review of patients who completed bevacizumab alone or as a chemotherapy regimen at the University of Illinois at Chicago for an 18-month study period. We collected blood pressure (BP) measurements and urinanalyses before starting bevacizumab, during bevacizumab and after stopping bevacizumab, in addition to concurrent medications, past medical history and demographics. Htn and proteinuria were graded by CTC v3.0. Patients with less than two successive doses of bevacizumab or unreliable ethnicity were excluded. Results: 27 subjects were eligible. Eighteen AA (67%) and 9 (33%) non-AA were included. Twenty-two (81%) had colorectal cancer. AA received a median of 10 cycles and non-AA received a median of 6 cycles. Six subjects (22%) developed any grade htn toxicity; maximum grade: grade 2=4 (15%), grade 3=2 (7%). Htn toxicity occurred in 28% AA and 11% non-AA (p=NS). Previous history of hypertension was found in 15 subjects (55%): AA=14 vs. non-AA=1 (p=0.002) and was not correlated with hypertensive toxicity. Twelve subjects (44%) developed any grade proteinuria; maximum grade: grade 1=9 (33%), grade 2=3 (11%). Proteinuria toxicity occurred in 50% AA and 33% non-AA (p=NS). Presence of hypertensive toxicity was associated with increased risk of proteinuria. Clinical benefit (PR, SD) was seen in 15 subjects (55%). Rate of clinical benefit was 67% in AA and 33% in non-AA (p=NS). Clinical benefit did not correlate with hypertensive or proteinuria toxicities. Conclusions: AA were more prone than non-AA to bevacizumab induced hypertension and proteinuria toxicity in this retrospective study. Higher clinical benefit was seen in AA.

No significant financial relationships to disclose.
Inmunophenotypic profiles of male breast cancer
Authors: Martin Gomez T. , Torio B. , Ruiz I. , Arranz F. , Arizcun A.
Published: March 2020

21180

Background: Recently, inmunophenotypic characterization methods have allowed identification of female breast carcinomas into separate groups showing different behaviour and response to therapy: luminal A phenotype (RE +, HER2-neu - ), luminal B (RE +, HER2- neu + ), basal like (RE -, HER2-neu - ). In this study, we used immunohistochemistry to investigate the inmunophenotypic profile distribution of male breast cancer. Methods: all the male breast cancers were obtained from the files of the Departments of Pathology of Hospital Río Carrión in Palencia, Spain, since 1996. A total of 9 cases were reviewed to confirm the diagnosis and to characterize each tumour. The following CK immunohistochemistry was performed: 8/18 and 5 (Dako, Carpinteria, CA, USA) in a Dako autostainer. ER was interpreted as positive if > 10% of the cells were staining. Normal skin and tonsils were used as positive controls for the CK and a known breast cancer for the ER immunohistochemistry. Results: five cases expressed RE and were HER2-neu negative, so they have a luminal-A phenotype. The four cases that expressed the luminal-B pehnotype expressed RE and HER2-neu; we demonstrated gene amplification of the HER-neu gene using fluorescent in situ hybridisation (FISH) in those cases. Respect the CKs profile, all cases were positive for CK 8/18 and negative with CK 5, vimentin and p63, characteristic of luminal-like CK expression profile, according wiht the literature. Conclusions: this is the first case series of male breast cancer patients that provides inmunophenotypic profile data on this rare disease in only one center in Spain. We comunicate that the vast majority of these tumours express the phenotype of luminal-like CKs. None of our patients were basal-like tumours. The percentage expression of Her-2 parallels the finding in female breast cancers and this should be analysed for its predictive significance, according to new specific biological treatments.

No significant financial relationships to disclose.
A genotype-directed phase I study of irinotecan in advanced cancer patients
Authors: Innocenti F. , Janisch L. , Das S. , Ramirez J. , House L. , Maitland M. L. , Salgia R. , Ratain M. J.
Published: March 2020

2502

Background: The risk of severe toxicity of irinotecan is in part related to the presence of common polymorphisms in UGT1A1, which codes for the enzyme responsible for the inactivation of SN-38, the active metabolite of irinotecan. The most common polymorphism in non-Asian populations is an extra TA repeat in the promoter (7 vs. 6), which reduces the enzyme expression. The three most common genotypes are: 6/6 (40–50%), 6/7 (40–50%), and 7/7 (10–15%). The 7/7 genotype confers the highest risk of toxicity, and it has been suggested that dose reduction is appropriate for patients with this genotype. As one published study has suggested that doses up to 500 mg/m2 q3w can be tolerated by some patients, we hypothesized that higher doses can be safely administered based on genotype by excluding patients with the 7/7 genotype. This study seeks to find the maximum tolerated dose (MTD) in patients with 6/6 and 6/7 genotypes. Methods: Advanced cancer patients (5 SCLC, 4 esophageal, 5 other tumors) have been genotyped for the UGT1A1 polymorphism. 6/6 and 6/7 patients have been treated with 700–850 mg (flat dose, corresponding to about 400–500 mg/m2) of q3w irinotecan. Dose-limiting toxicity (DLT) at cycle 1 is defined as grade =3 nonhematological toxicity, grade 4 neutropenia lasting more than 4 days, and febrile neutropenia. Results: Three 6/6 patients tolerated 700 mg (no grade >2 toxicity). Three additional 6/6 patients have been treated at 850 mg, and one DLT (grade 4 neutropenia lasting more than 4 days) was observed; 3 additional 6/6 patients will be enrolled at 850. Six 6/7 patients have been treated at 700 mg, with only one DLT (febrile neutropenia). Two additional 6/7 patients have been treated at 850, and one DLT (febrile neutropenia) has been observed. Conclusions: Higher doses of irinotecan can be safely administered in patients with selected UGT1A1 genotypes. On the basis of these interim results, a phase II trial in metastatic colorectal cancer patients with 6/6 and 6/7 genotypes has been designed to evaluate the efficacy of higher doses of irinotecan.

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