Primary Tumor Biopsy Specimens Research Articles

Biomarker Study for Selecting Neoadjuvant Chemotherapy Regimens Based on Prognostic Prediction Using Gastric Cancer Biopsy Specimens from a Phase II Randomized Controlled Trial.

The randomized phase II COMPASS trial revealed that neither the regimen nor the number of courses of preoperative neoadjuvant chemotherapy (NAC) for locally advanced gastric cancer (GC) significantly influence overall survival (OS). However, the impact of NAC regimens on OS may vary from patient to patient. The aim of this study was to identify biomarkers that can predict more appropriate individualized NAC regimens for improved prognosis using biopsy specimens from the COMPASS trial. RNA was extracted from endoscopic biopsy specimens of primary tumors obtained prior to NAC and real-time PCR analysis of 127 genes was conducted to identify those significantly affecting survival in the context of specific NAC regimens. THBS1, MSI1, and IGF2BP3 were identified as significant factors for stratifying survival among different NAC regimens, with statistically significantly interaction p values. Immunohistochemical analysis confirmed that the protein levels of THBS1, MSI1, and IGF2BP3 strongly correlated with their gene expression levels, validating these proteins as reliable biomarkers. This study effectively identified THBS1, MSI1, and IGF2BP3 as promising biomarkers for personalizing NAC regimens in patients with locally advanced GC. By tailoring NAC based on these biomarkers, it is possible to enhance survival outcomes and advance personalized treatment strategies. The findings underscore the potential for incorporating biomarker-guided approaches into clinical trials, aiming to refine and optimize NAC regimens for improved patient-specific treatment efficacy.

Abstract 7623: Biomarker study to personalized neoadjuvant chemotherapy using preoperative gastric cancer specimens of biopsy from the COMPASS trial, a phase II randmazed controlled trial

Abstract Background and Aim: The results of COMPASS, a randomized phase II trial, suggest that preoperative adjuvant chemotherapy (NAC) regimen or course for locally advanced gastric cancer (GC) does not affect overall survival (OS). However, we hypothesized that some NAC regimens may be more effective for OS on a case-by-case basis. Furthermore, we searched for biomarkers, assuming that further improvement in the outcome of locally advanced GC could be expected if appropriate NAC regimens could be selected using biomarkers before treatment initiation. Materials and Methods: RNA was extracted from endoscopic biopsy specimens of primary tumors collected prior to NAC administration in COMPASS patients, and real-time polymerase chain reaction (PCR) of 127 genes was performed to identify genes with significant (P < 0.05) interaction P values for survival with each regimen and stratified OS with each regimen. Genes were identified and biomarkers were searched at the protein level using immunohistochemical analysis. Results: The THBS1, MSI1, and IGF2BP3 genes were identified as genes whose expression levels significantly (P < 0.05) stratified survival for each regimen and had significant (P < 0.05) interaction P values. Furthermore, a strong correlation was observed between each gene and THBS1, MSI1, and IGF2BP protein expression by immunohistochemical analysis, confirming that protein expression levels by immunohistochemical analysis are also useful as biomarkers. Conclusion: Using endoscopic biopsy specimens prior to NAC for locally advanced GC, biomarkers were identified to select NAC regimens over predicted OS. The results of this study may guide the conduct of clinical trials of individualized treatment of NAC using biomarkers. Citation Format: Takashi Oshima. Biomarker study to personalized neoadjuvant chemotherapy using preoperative gastric cancer specimens of biopsy from the COMPASS trial, a phase II randmazed controlled trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7623.

Effect of Lysine Acridone Acetate on the Level of Apoptosis and Expression of Apoptosis-Associated Proteins during Antioncogenic Therapy in Colorectal Cancer in Mice.

We studied the mechanism of action of cytostatics with the addition of lysine acridone acetate to evaluate the possibility of its use for improving the effectiveness of antioncogenic therapy in colorectal cancer. In Nude mouse model, the level of apoptosis (TUNEL) and expression of proteins CD95, p53, Bcl-2, histone H3, and Ki-67 (immunohistochemistry) were assessed in primary tumor biopsy specimens. It has been shown that cytostatic treatment led to stimulation of p53-mediated apoptosis and suppression of proliferation (Ki-67 expression) of tumor cells, and apoptosis level was increased in groups receiving lysine acridone acetate. H3 expression in the experimental groups was changed.

Biomarker analysis to predict the pathological response to neoadjuvant chemotherapy in locally advanced gastric cancer: An exploratory biomarker study of COMPASS, a randomized phase II trial.

Background: The findings of COMPASS, a randomized phase II study, suggested that the regimens and courses of neoadjuvant chemotherapy (NAC) for locally advanced gastric cancer (GC) did not affect the pathological response. However, pathological complete response was achieved in 10% patients who received four courses of either S-1/cisplatin or paclitaxel/cisplatin. We hypothesized that if relevant biomarkers could be used to predict the suitable NAC regimen before treatment initiation, further improvements could be ensured in the outcomes of locally advanced GC.Materials and Methods: mRNA extraction, real-time polymerase chain reaction, and immunohistochemical analyses were performed using endoscopic biopsy specimens of primary tumors, collected prior to NAC, to determine the clinically relevant biomarkers.Results: TIMP1, DSG2, RRM1, MUC2, EGFR, ZDHHC14, and CLDN18.2 were identified as biomarker candidates, since their expression was significantly associated with the pathological responses to each NAC regimen. Furthermore, TIMP1 and DSG2 were identified as predictive biomarkers of the pathological response to each NAC regimen.Conclusions: The effective prediction of the pathological response to NAC regimens in locally advanced GC using biomarkers identified from endoscopic biopsy specimens indicates the possibility of personalizing NAC based on biomarker analysis.

Prediction of lymph node metastasis by tumor-infiltrating lymphocytes in T1 breast cancer

BackgroundLymph node metastasis is more likely in early-stage breast cancer with lower tumor-infiltrating lymphocyte (TIL) density. Therefore, we investigated the correlation between TILs and lymph node metastasis in cT1 breast cancer patients undergoing surgery and the usefulness of TILs in predicting sentinel lymph node metastasis (SLNM) in cT1N0M0 breast cancer.MethodsWe investigated 332 breast cancer patients who underwent surgery as the first-line treatment after preoperative diagnosis of cT1. A positive diagnosis of SLNM as an indication for axillary clearance was defined as macrometastasis in the sentinel lymph node (SLN) (macrometastasis: tumor diameter > 2 mm). Semi-quantitative evaluation of lymphocytes infiltrating the peritumoral stroma as TILs in primary tumor biopsy specimens prior to treatment was conducted.ResultsFor SLN biopsy (SLNB), a median of 2 (range, 1–8) SLNs were pathologically evaluated. Sixty cases (19.4%) of SLNM (macrometastasis: 46, micrometastasis: 16) were observed. Metastasis was significantly greater in breast cancers with tumor diameter > 10 mm than in those with diameter ≤ 10 mm (p = 0.016). Metastasis was significantly associated with lymphatic invasion (p < 0.001). These two clinicopathological factors correlated with SLNM even in patients diagnosed with cN0 (tumor size; p = 0.017, lymphatic invasion; p = 0.002). Multivariate analysis for SLNM predictors revealed lymphatic invasion (p = 0.008, odds ratio [OR] = 2.522) and TILs (p < 0.001, OR = 0.137) as independent factors.ConclusionsOur results suggest a correlation between lymph node metastasis and tumor immune-microenvironment in cT1 breast cancer. TIL density may be a predictor of SLNM in breast cancer without lymph node metastasis on preoperative imaging.

Correlation Between Biomarker Candidate Proteins with the Effect of Neoadjuvant Chemoradiation Therapy on Esophageal Squamous Cell Carcinoma.

While chemoradiation therapy (CRT) is one of the most useful treatments for esophageal squamous cell carcinoma (ESCC), it is important to predict response prior to treatment by using markers because some patients respond well and others do not. Fifty-nine patients with ESCC were treated with neoadjuvant CRT at the Kagoshima University Hospital. The expression of seven types of biomarker candidate proteins in biopsy specimens of untreated primary tumors was evaluated to determine whether it correlated with response and prognosis. The positive expression rates were 47% for p53, 83% for CDC25B, 68% for 14-3-3sigma, 76% for p53R2, 75% for ERCC1, 32% for Gli-1, and 54% for Nrf2. In terms of histological response, tumor grade of the 59 patients was 48.8% for grade 1 as the non-responder, 29.2% for grade 2, and 22.0% for grade 3 as the responder. CRT was significantly effective in p53(-), p53R2(-), ERCC1(-), and Nrf2(-) tumors, while p53(-), p53R2(-), and ERCC1(-) were factors independently correlated with effective histological response. Their combined expression of two or three negative expressions had 100% effective response and was a significant prognostic factor. Our results suggest that two or three negative expressions of p53, p53R2, and ERCC1 in biopsy specimens of primary tumors were associated with a favorable response to CRT for ESCC. Assessment of tumor suppressor and DNA repair protein expressions in biopsy specimens may be useful for the potential utility of CRT therapy for patients with ESCC prior to treatment.

Correlation of Aurora-A expression with the effect of chemoradiation therapy on esophageal squamous cell carcinoma.

BackgroundChemoradiation therapy (CRT) is one of the most useful treatments for esophageal squamous cell carcinoma (ESCC). However, because some patients respond well to CRT and others do not, it is important to be able to predict response to CRT before beginning treatment by using markers. Aurora-A encodes a cell cycle regulated serine/threonine kinase that has essential functions in centrosome maturation and chromosome segregation. In this study, we investigated the relationship between the expression of Aurora-A and the response to CRT in patients with ESCC.MethodsWe immunohistochemically investigated the expression of Aurora-A in biopsy specimens of untreated primary tumors of 78 patients with ESCC and determined the relationship between Aurora-A levels and patient responses to CRT, which consisted of 5-fluorouracil plus cisplatin and 40 Gy of radiation.ResultsTumors were judged as Aurora-A positive when more than 10% of the cancer cells displayed a distinct positive nuclear anti-Aurora-A immunoreaction by immunohistochemical evaluation. The tumors of 46 of 78 patients (58.9%) displayed positive expression of Aurora-A. In terms of clinical response the percentage of patients showing complete response (CR), incomplete response/stable disease of primary lesion (IR/SD), and progressive disease (PD) was 19.2, 69.2, and 11.5%, respectively. In terms of histological response the tumor grade of the 41 patients who underwent surgery was as follows: grade 1, 48.8%; grade 2, 29.2%; grade 3, 22.0%. CRT was effective for patients who had Aurora-A (+) tumors (clinically: P = 0.0003, histologically: P = 0.036).ConclusionsOur results suggest that Aurora-A expression in biopsy specimens of primary tumors is associated with CRT efficacy in patients with ESCC. Assessment of Aurora-A expression in biopsy specimens maybe useful for regarding the potential utility of CRT therapy for patients with ESCC before treatment.

Survivin expression correlates with nodal metastasis in T1–T2 squamous cell carcinoma of the tongue

In this study, the correlation between neck metastasis and recurrence was investigated by studying specimens of tongue squamous cell carcinoma patients immunohistochemical with survivin antibodies in the primary biopsy. A retrospective review was conducted at the Academic University Hospital. 46 patients who had squamous cell carcinoma of the tongue, who underwent various types of glossectomy and neck dissections between 1991 and 2008, were evaluated. The patient's sex, TNM staging, differentiation and recurrence rates were analyzed. There were 20 T1 patients and 26 T2 patients; 27 of the patients were N0 and 19 had metastatic lymph nodes in the neck. Survivin antibodies were applied with streptavidin-biotin method to the sections that were prepared from the primary tumor biopsy specimens of the patients. The correlation between neck metastasis and recurrence and survivins' immunohistochemical staining was analyzed with statistical methods. There were no significant differences between the patient's age, sex, tumor's T stage, tumor differentiation and survivin staining density. Survivin staining was positive in 15 (79 %) of 19 patients with neck metastasis, while it was positive in 16 (59 %) of 27 patients without neck metastasis. Eleven (79 %) of the 14 patients who had recurrence and all 6 patients who had neck recurrence only were stained by survivin. Expression of nuclear and cytoplasmic survivin can be a useful marker for predicting cervical lymph node metastasis in T1-T2 tumors in tongue SCC.

Dysregulation, But Not Mutation Of p53 Signaling Pathway In Breast Implant-Associated Anaplastic Large Cell Lymphoma Cell Lines

A consistent feature of over 100 reported cases of breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is their complex cytogenetic abnormalities, suggesting that genomic instability may drive lymphomagenesis and/or tumor progression. Loss of heterozygosity(LOH) of the TP53 tumor suppressor gene locus on the short arm of chromosome 17 (17p13.1) is a frequent finding. Human p53 plays an important role in cell cycle arrest, DNA repair, and apoptosis and it maintains genome stability by preventing mutations. Recently, three T cell breast lymphoma (TLBR) cell lines were derived from patients' BIA-ALCL primary tumor biopsy specimens. These cell lines are IL-2 dependent, ALK-negative, CD30+activated cytotoxic T cells closely resembling the original tumor cells. Thus, the cell lines may serve as an important tool for studying this newly recognized disease entity. Because of its rarity, the clinical pathologic features, tumor cell biology, and genetics of BIA-ALCL have yet to be fully defined.Here we tested the hypothesis that the p53 signaling pathway is defective in TLBR cells. We initially examined TP53 transcript expression among the cell lines. By qRT-PCR, p53 transcripts were detected in all three lines, with the highest level in TLBR-2. Next we examined p53 protein expression and p53 activation in response to ultraviolet (UV) or gamma irradiation. By Western blotting, all TLBR cell lines expressed much lower levels of p53 protein following UV irradiation (400 J/m2) than Karpas (ALK+ ALCL) cells and failed to show ATM/ATR-induced phosphorylation of p53 on serine 15, an early indicator of p53 activation. Genetic defects (deletion, mutation) of the p53 coding sequence were not found by Sanger sequencing. Interestingly, a polymorphism at p53 codon 72 (Arg72Pro), a normal variant associated with increased susceptibility to breast cancer, was detected in TLBR-1 and -3 (derived from indolent BIA-ALCL), but not in the aggressive BIA-ALCL line TLBR-2. Thus, TLBR cells exhibit defective regulation of the p53 pathway in response to DNA damage, suggesting that their ability to sense DNA damage or the regulation of p53 stability may be impaired.We next examined the DNA damage sensing pathway upstream of p53 in the presence and absence of the DNA demethylating agent 5-aza-2'-deoxycytidine (AZA, 10µM for 48hrs). In all TLBR lines, ATM and ATR transcripts were expressed at much lower levels (qRT-PCR) than normal, and their expression was not significantly affected by AZA. However, compared with human T cells, CHK2 (phosphorylate P53 at Ser20) transcripts were very low in TLBR-1 and -2, but not in TLBR-3 cells. CHK2 and p21 (the main p53 target gene) transcripts after AZA were greatly increased in TLBR-2, mildly elevated in TLBR-3, and unchanged in TLBR-1 cells, suggesting that DNA methylation of the CHK2 and p21 genes may partly explain the defective p53 signaling in TLBR-2 cells. This was confirmed by detecting of CHK2 phorphrylation only in TLBR-3 cells. Mdm2, a major negative regulator of p53 protein stability, was either normal or low (qRT-PCR), and was unaffected by AZA. However, immunobloting with Mdm2 antibodies revealed increased levels of two isoforms following UV of TLBR-1 and -2, but only the small isoform was expressed in TLBR-3 cells and there was little response to UV treatment. Treatment of TLBR cells with 5 µM Nutlin-3 (Mdm2 antagonist, p53 activator, and apoptosis inducer) inhibited cell growth by 40% at day 5 (MTT assay). We conclude that these three BIA-ALCL derived cell lines share dysregulation of the p53 signaling pathway, which may contribute to the genomic instability characteristic of these BIA-ALCL cases.First two authors have equally contributed to this abstract. Disclosures:No relevant conflicts of interest to declare.

Abstract 3744: G-CSF-dependent regulation of STAT3 in neuroblastoma cancer stem cell subpopulations.

Abstract Neuroblastoma is a neural crest derived malignancy arising form peripheral sympathetic nervous system of infants and young children. We have recently defined a highly tumorigenic sub-population of neuroblastoma cells based on surface expression of CD114 (the G-CSF receptor, CSF3R). These cells meet the operational criteria for cancer stem cells based on their self-renewal, differentiation and lineage restricted tumorigenicity, and can be isolated directly from primary tumor biopsy specimens. As STAT3 activation is a canonical downstream event of G-CSF receptor ligation, is critical for neuronal specification, and is implicated in cancer stem cell maintenance, we hypothesized that inhibition of the STAT3 pathway may functionally inhibit the cancer stem cell-like neuroblastoma subpopulation. We used pathway-specific qPCR arrays to evaluate differential gene expression in CD114+ and CD114- tumor subpopulations. As predicted, STAT3 target genes were up-regulated in the CD114+ stem cell-like population. In addition, critical genes controlling early neural crest specification, cell cycle progression, self-renewal, and maintenance of pluripotency were up-regulated in CD114+ population while markers of mature mesenchymal neural crest were increased in CD114- population. Treatment of Neuroblastoma cell lines with G-CSF increased both CD114 expression and STAT3 transcriptional activation in a dose and time dependent manner. Phospho-flow analysis confirmed STAT3 phosphorylation (Y705) in response to exogenous G-CSF. The addition of a STAT3 inhibitor which prevents phosphorylation and dimerization of STAT3 (Stattic), specifically abrogated the effect of exogenous G-CSF treatment. Finally, we identified putative STAT3 binding sites within the 5’UTR of the CSF3R gene and confirmed binding of STAT3 and pSTAT3 using chromatin immunoprecipitation analysis (ChIP-qPCR). We demonstrate significant enrichment of binding in response to exogenous G-CSF within CD114 receptor positive subpopulation in contrast to CD114- population. These data support a model for a positive-feedback loop where G-CSF triggers STAT3 dependent up-regulation of the G-CSF receptor, further sensitizing these cells to exogenous ligand, promoting STAT3 transcriptional activation, and contributing to stem cell maintenance. Our data support further evaluation of the potential clinical role for specific STAT3 inhibitors as anti-cancer stem cell therapy in neuroblastoma. Citation Format: Saurabh Agarwal, Zaowen Chen, Danielle Hsu, Eugene Kim, Jason M. Shohet. G-CSF-dependent regulation of STAT3 in neuroblastoma cancer stem cell subpopulations. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3744. doi:10.1158/1538-7445.AM2013-3744

Histologic Assessment of Tumor-Associated CD45+ Cell Numbers Is an Independent Predictor of Prognosis in Small Cell Lung Cancer

Small cell lung carcinoma (SCLC) continues to have a poor prognosis, with a 2-year survival of < 20%. Studies have suggested that SCLC may affect the immune system to allow it to evade immunologic responses. We hypothesized that any such effect would be characterized by a decrease in the lymphoid cells associated with the tumor in biopsy specimens and that this might relate to patient outcome. Sixty-four SCLC biopsy specimens were immunohistochemically stained with anti-CD45 antibody to identify immune cells associated with the tumor. A mean CD45 count per high-power field for each case was obtained, and the results were correlated with age, sex, stage, performance status (PS), treatment with chemotherapy/radiotherapy, and overall survival. The median CD45 count for all cases was taken as 40 (CD45(40)). Kaplan-Meier plots demonstrated better survival for patients with a CD45(40) > 40 ( P < .009). No relationship between CD45 40 and age, sex, stage, or treatment by chemotherapy or radiotherapy was identified. Although PS was a significant predictor of survival ( P = .014), it did not correlate with CD45 40. In patients with better Eastern Cooperative Oncology Group PS (≤ 2), the CD45(40) demonstrated a highly significant survival advantage for those with CD45(40) > 40 ( P < .0001). The data indicate that (1) simple immunohistochemical assessment of immune cell infiltrates in routinely processed and stained biopsy specimens of primary tumors can provide prognostic information in SCLC and (2) tumor-associated CD45(+) cells in SCLC biopsy specimens may be a good clinical marker to identify patients with poor prognosis despite good PS.

Abstract 3669: Autocrine interleukin/STAT3 signaling in breast implant-associated T cell anaplastic large cell lymphoma cell lines suggests a mechanism for tumor progression and effective therapy

Abstract Recently, over 80 cases of anaplastic large-cell kinase (ALK)-negative, T-cell, anaplastic, non-Hodgkin lymphomas (T-ALCL) have been identified worldwide in patients with textured saline and silicone breast implants. These breast implant-associated T-ALCL cases are striking for their homogeneous clinical presentation and pathology that is distinct from established classes of ALCL. In order to understand the nature of this newly emerged clinical entity, we have established three new cell lines, designated T cell Breast Lymphoma (TLBR) -1, -2, and -3, from patient primary tumor biopsy specimens. Characterization of these pre-clinical models confirmed fidelity to the original tumor biopsy specimens and highlighted unique features that may aid the development of effective treatments for these cancers. Gene expression analysis demonstrated significant up-regulation of survivin and down-regulation of pro-apoptotic genes (BID, BAK, BBC3) by all TLBR cell lines relative to healthy donor T cells. This preliminary finding prompted examination of upstream regulators of cell survival in T cells, namely the interleukin signaling associated with the JAK/STAT pathway. TLBR cell lines were found to secrete high levels of IL-6 and IL-10, and were strongly positive for the cognate receptors. The TLBR cell lines also showed elevated levels of STAT3 and pSTAT3 by immunoblotting techniques, along with a simultaneous reduction in expression of the pSTAT3 phosphatase, SHP-1. These results implicate an unregulated autocrine STAT3 pathway present in the TLBR cell lines that strongly contributes to their tumorigenesis. Assays of various inhibitors of this pathway support this hypothesis since targeting of STAT3 with the tyrosine kinase inhibitor, Sunitinib, led to dramatic in vitro cell death in all TLBR cell lines. More specific inhibition of STAT3 with S3I-201 had comparable results with Sunitinib. Furthermore, IL-6 neutralization by a monoclonal antibody or SHP-1 restoration by 5-azacytidine treatment also demonstrated moderate efficacy in inhibiting TLBR cell line growth in vitro. These discoveries strongly suggest the use of these reagents in the clinical treatment of Breast Implant Associated T-ALCL. The TLBR cell lines closely resemble the primary breast implant-associated lymphomas from which they were derived, and as such provide valuable preclinical models to study the unique biology and provide clinicians with improved knowledge for treatment and diagnosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3669. doi:1538-7445.AM2012-3669

Small Biopsy Specimens Reliably Indicate p16 Expression Status of Oropharyngeal Squamous Cell Carcinoma

Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (SCC) is associated with favorable patient survival. Tumor HPV status at primary diagnosis is critical for proper management, and p16 immunohistochemistry (IHC) has emerged as a reliable, single, surrogate marker. It is not known, however, if small biopsy specimens are completely adequate for p16 evaluation. From a database of oropharyngeal SCC for which p16 IHC and histologic typing were already performed, all patients (32) who had available in-house primary tumor biopsy specimens and also subsequent surgical resections were analyzed. p16 IHC was performed along with histologic typing into: Type 1 keratinizing SCC, Type 2 nonkeratinizing SCC with maturation, and Type 3 nonkeratinizing SCC. Staining was graded on both biopsies and resections as follows: 0=negative; 1+ =1-25% of tumor cells positive; 2+ =26-50%; 3+ =51-75%; 4+ =76-100%. Strictly considering p16 score, perfect biopsy-resection correlation was present in 28 of 32 cases (85%), including 6/9 (67%) Type 1, 6/7 (86%) Type 2, and 16/16 (100%) Type 3 cases. Considering p16 expression binarily as 51% tumor cell staining or more (3+ or 4+) being positive and lesser amounts (0, 1+, or 2+) as being negative, there was perfect biopsy-resection correlation for all 32 cases. With p16 expression in resection specimens considered the gold standard, p16 IHC in biopsies was both 100% sensitive and specific. Our results demonstrate that p16 staining in diagnostic biopsies reliably reflects whole tumor staining results, and suggest that biopsies do not suffer from false negatives or positives.

Key Survival Pathways and Chemotherapeutic Sensitivity of Breast Implant Associated Anaplastic Large Cell Lymphomas,

Abstract 3739Primary lymphomas of the breast are very rare (0.2–1.5% of breast malignancies) and the vast majority (95%) is of B-cell origin. Recently, a series of anaplastic large-cell kinase (ALK)-negative, T-cell, anaplastic, non-Hodgkin lymphomas (T-ALCL) have been reported worldwide in patients with saline and silicone breast implants (Lechner et al. Cancer 2010, Lazzeri D et al. Clin Breast Cancer 2011, Carty et al. Plast Reconstr Surg 2011, Popplewell et al. Leuk Lymphoma 2011, Roden et al. Mod Pathol 2008, de Jong et al. JAMA 2008, and others). These cases are striking for their homogeneous clinical presentation and pathology. Histological and cytogenetic analyses of the primary tumor biopsy specimens consistently demonstrates CD30+ EMA(+) PAX5− lymphoma cells with significant chromosomal atypia that lack the NPM-ALK (2;5) translocation, express pan T-cell markers, and show monoclonal TCR gene rearrangements. Increasing evidence suggests that these seroma-associated ALK− ALCLs are a distinct clinicopathologic entity from either systemic ALK− ALCL or cutaneous ALCL, and while the majority of cases are indolent, several patients have died from this disease.In order to understand the nature of this newly emerging clinical entity, we have established three new cell lines, (TLBR-1, TLBR-2, TLBR-3,) from the primary tumor biopsy specimens of three patients diagnosed with breast implant-associated ALCL. Characterization of these pre-clinical models confirmed fidelity to the original tumor biopsy specimens and highlighted unique features that may aid the development of effective treatments for these cancers. Cytogenetics and FISH performed on TLBR-1, -2, and -3 revealed nuclear atypia with partial or complete trisomy (modal chromosome number 47, 76, and 81, respectively) and absence of the NPM-ALK (2;5) translocation. Phenotypic characterization of TLBR cell lines by flow cytometry and immunocytochemistry showed strong positivity for CD30, CD71, pan T cell CD2/5/7, natural killer (CD56), and antigen presentation (HLA-DR, CD80, CD86) markers, and IL-2 receptors (CD25, CD122). Multiplex or consensus sequence PCR confirmed monoclonal TCR gene rearrangements and showed no onco-virus incorporation (EBV, HTLV1/2), respectively, for TLBR-1, -2, and -3.To identify effective therapies for patients with breast-implant associated ALCL, in vitro studies were used to evaluate the mechanisms by which TLBR evades immune detection and promotes cell cycle dysregulation. Gene expression analysis for proto-oncogenes, tumor suppressor genes, and regulators of apoptosis demonstrated significant up-regulation of survivin and down-regulation of pro-apoptotic genes (BID, BAK, BBC3) by all TLBR cell lines relative to healthy donor T cells. Interestingly, TLBR cell lines shared many features with human regulatory T cells, including phenotype (CD4+/8+, CD25+), high FoxP3 expression, up-regulation of immunosuppressive cytokines IL-10 and TGFβ, and suppression of T cell responses to stimuli. Inhibitor studies were used to identify critical signaling pathways in survival and proliferation of breast implant-associated ALCL models TLBR-1, -2, and -3. Data from these studies revealed that inhibition of the JAK/STAT, PI3K/mTOR, and WNT/beta-catenin signaling pathways resulted in a substantial increase in cell death in the TLBR cell lines. Significantly, TLBR-2, which was derived from a fatal breast implant associated ALCL, showed increased resistance to all inhibitors compared with TLBR-1 and TLBR-3, which were derived from more indolent cases. Analysis of protein lysates showed elevated cleaved Notch1 in TLBR-2, compared with cell lines TLBR-1 and -3, which may drive increased cell proliferation and resistance to apoptotic regulators. Since the treatment of these lymphomas has not been studied, we evaluated the sensitivity of the TLBR cell lines to mainstay chemotherapy regimens (CHOP, methotrexate). These studies found that doxorubicin and vinblastine exert potent cytotoxic effects against all three TLBR cell lines, whereas cyclophosphamide was largely ineffective. In summary, TLBR-1, -2, and -3 closely resemble the primary breast implant-associated lymphomas from which they were derived, and as such provide valuable preclinical models to study the cell of origin and biology of this newly emerging clinical entity. Disclosures:No relevant conflicts of interest to declare.

Overexpression of cyclooxygenase-2 is associated with chemoradiotherapy resistance and prognosis in esophageal squamous cell carcinoma patients

Our objective was to investigate whether cyclooxygenase-2 (COX-2) expression can predict the patient's response to chemoradiotherapy (CRT) and ensuing prognosis in esophageal squamous cell carcinoma (ESCC). The clinicopathological and follow-up data of 112 patients with ESCC who underwent CRT from January 2001 to June 2006 were analyzed retrospectively. The immunohistochemical expression level of COX-2 was examined for all biopsy specimens of primary tumors, and the correlation of COX-2 expression with the patient's response to CRT and prognosis was examined. COX-2 positive immunostaining was detected in 111 (99.1%) of the patients, including overexpression in 54 (48.2%) patients and low expression in 58 (51.8%) of the patients. The response of tumors with a low level expression of COX-2 (70.7%, 41/58) was significantly higher than that of tumors with COX-2 overexpression (42.6%, 23/54; P = 0.003). Patients with a low level of COX-2 expression had a higher downstaged rate than those with a high level of COX-2 expression (9/13 vs 2/8), but the difference was not statistically significant (P = 0.08). In the definitive CRT group (91 cases), COX-2 overexpression was significantly associated with poor 3-year overall survival (P = 0.028). Multivariate analysis showed that only metastatic stage (nonregional node metastasis) was an independent prognosis factor. The assessment of COX-2 status may provide additional information to identify ESCC patients with poor chances of response to CRT and potential candidates for more individualized treatment.

Predictive value of laminin-5 and membrane type 1-matrix metalloproteinase expression for cervical lymph node metastasis in T1 and T2 squamous cell carcinomas of the tongue and floor of the mouth

Laminin-5 (Ln-5) cleaved by membrane type 1-matrix metalloproteinase (MT1-MMP) enhances the migration of tumor cells. The purpose of this study was to determine whether or not enhanced expression of both Ln-5 and MT1-MMP was associated with lymph node metastasis in patients with T1 and T2 squamous cell carcinoma (SCC) of the tongue and floor of the mouth. By use of biopsy specimens of primary tumors from 57 patients, intratumoral expression of Ln-5 and MT1-MMP was evaluated immunohistochemically and its association with node metastasis analyzed. The tumors were categorized into three groups: Ln-5 focal type/MT1-MMP (-) (group I, n = 14), Ln-5 focal type/ MT1-MMP (+) and Ln-5 diffuse type/MT1-MMP (-) (group II, n = 16), and Ln-5 diffuse type/MT1-MMP (+) (group III, n = 27). The incidence of node metastasis (initial and latent metastases) was two of 14 (14.3%), five of 16 (31.3%), and 15 of 27 (55.6%) in groups I, II, and III, respectively. Multivariate analysis identified tumor thickness (odds ratio, 4.751; p = .0152) and Ln-5/ MT1-MMP expression (odds ratio, 3.795, p = .0304) as independent factors of node metastasis. Moreover, in 35 patients with N0 disease, Ln-5/MT1-MMP expression was the only parameter associated with latent node metastasis (odds ratio, 12.800, p = .0247). These results suggest that immunohistochemical evaluation of Ln-5 and MT1-MMP expression is useful for identifying patients with T1 and T2 SCC of the tongue and floor of the mouth who should be treated with elective neck dissection.

Establishment and characterization of four Sinclair Swine cutaneous malignant melanoma cell lines

Cutaneous malignant melanoma of Sinclair Swine (SSCM) is a heritable, congenital neoplasm which either proves fatal to the neonatal animal or undergoes spontaneous regression. Four SSCM cell lines, UISO-SSCM-433, UISO-SSCM-438, UISO-SSCM-5052, and UISO-SSCM-8093, were derived from biopsy specimens of primary tumors removed from swine at 26, 8, and 8 weeks of age, and 15 weeks gestation, respectively. Morphologic features, DOPA oxidase staining, and abnormal karyotype were suggestive of malignant melanoma. Each cell line was morphologically heterogeneous in culture with dendritic, spindle- and cuboidal-shaped cells. Pigmented melanosomes and DOPA oxidase activity were present in all cell lines at passages 20–22. UISO-SSCM-433 and UISO-SSCM-5052 contained hypodiploid and hypotetraploid sublines whereas UISO-SSCM-438 and UISO-SSCM-8093 were hypodiploid and hypotetraploid, respectively. At later passages, all cell lines presented evolutionary, karyotypic changes; the same chromosomes were involved in the alterations, however. Chromosomes 2,6,13, and 14 were the most affected, exhibiting numerical and structural alterations in all four cell lines. Despite the presence of multiple chromosomal anomalies in all cell lines, each with a unique set of chromosomal markers, clonal growth was not detected in soft agar, nor were any of the lines tumorigenic following s.c. inoculation in athymic mice. This suggests that the loss of malignant potential in SSCM may be inherent.