In-depth characterization of a new patient-derived xenograft model for metaplastic breast carcinoma to identify viable biologic targets and patterns of matrix evolution within rare tumor types.

Metaplastic breast carcinoma (MBC) is a rare breast cancer subtype with rapid growth, high rates of metastasis, recurrence and drug resistance, and diverse molecular and histological heterogeneity. Patient-derived xenografts (PDXs) provide a translational tool and physiologically relevant system to evaluate tumor biology of rare subtypes. Here, we provide an in-depth comprehensive characterization of a new PDX model for MBC, TU-BcX-4IC. TU-BcX-4IC is a clinically aggressive tumor exhibiting rapid growth in vivo, spontaneous metastases, and elevated levels of cell-free DNA and circulating tumor cell DNA. Relative chemosensitivity of primary cells derived from TU-BcX-4IC was performed using the National Cancer Institute (NCI) oncology drug set, crystal violet staining, and cytotoxic live/dead immunofluorescence stains in adherent and organoid culture conditions. We employed novel spheroid/organoid incubation methods (Pu·MA system) to demonstrate that TU-BcX-4IC is resistant to paclitaxel. An innovative physiologically relevant system using human adipose tissue was used to evaluate presence of cancer stem cell-like populations ex vivo. Tissue decellularization, cryogenic-scanning electron microscopy imaging and rheometry revealed consistent matrix architecture and stiffness were consistent despite serial transplantation. Matrix-associated gene pathways were essentially unchanged with serial passages, as determined by qPCR and RNA sequencing, suggesting utility of decellularized PDXs for in vitro screens. We determined type V collagen to be present throughout all serial passage of TU-BcX-4IC tumor, suggesting it is required for tumor maintenance and is a potential viable target for MBC. In this study we introduce an innovative and translational model system to study cell-matrix interactions in rare cancer types using higher passage PDX tissue.

Neuropilin-2 gene expression correlates with malignant progression in cutaneous melanoma.

BACKGROUND: It is currently not possible to predict the metastatic potential of early-stage melanoma lesions by histological examination alone; however, a significant number of thin melanomas will progress over time to advanced disease. Molecular biomarkers that could identify patients with melanoma at high risk at the time of original diagnosis would contribute significantly to improved patient outcomes and increased survival. Neuropilin-2 (NRP2), a cell surface receptor involved in tumour-associated angiogenesis and lymphangiogenesis, has recently been shown to be expressed in melanoma. OBJECTIVES: To evaluate the potential value of NRP2 gene transcript levels as biomarkers for malignant melanoma progression. METHODS: We measured NRP2 gene expression in a panel of formalin-fixed paraffin-embedded tissue specimens consisting of naevi, primary melanomas and metastatic melanomas using quantitative reverse transcriptase-polymerase chain reaction technique. RESULTS: NRP2 levels are clearly segregated among the groups of naevi, primary and metastatic melanoma samples with a statistical trend towards increasing NRP2 gene expression correlating with disease progression. Logistic regression analysis reveals that the probability of malignant progression increases with elevated levels of NRP2 (odds ratio of 2·60 with confidence interval 1·29-5·21). Within the group of primary melanomas, there is a positive correlation (r = 0·823) between NRP2 expression and Breslow depth. This correlation was validated in an independent sample set of patients with melanoma. CONCLUSIONS: This preliminary study strongly supports the significance of NRP2 as a useful biomarker for malignant progression of melanoma, which may be useful for early identification of patients with melanoma at high risk.

Melanoma antigen expression in serial fine-needle aspiration samples in patients with metastatic malignant melanoma participating in immunotherapy clinical trials: a preliminary look.

BACKGROUND: MART-1 and gp100 currently are utilized as targets in immunotherapy protocols for metastatic malignant melanoma (MMM). Enrollment of patients into ongoing peptide vaccination trials at the National Cancer Institute includes immunophenotyping of samples of metastatic lesions obtained by fine-needle aspiration (FNA). As therapy progresses, immunocytochemistry is performed on serial FNAs of metastatic lesions to monitor changes in antigen expression during treatment. It is theorized that antigen expression of melanoma cells may be diminished because of selective immunodestruction of tumor cells, or perhaps intentionally, to escape immunosurveillance. METHODS: Thirty-eight lesions from 33 patients were serially monitored for the expression of gp100 (clone HMB-45) and MART-1 (clone M2-7C10), using an avidin-biotin peroxidase technique. The staining intensity of tumor cells was scored on a scale of 0 to 3+, with the proportion of positive cells categorized as less than 25%, 25-50%, 50-75%, and greater than 75%. All lesions were examined within approximately 2 months after the start of peptide vaccination, providing a consistent timepoint for analysis. RESULTS: Using the Wilcoxon signed rank test, the authors found that there were no significant changes from baseline compared with 2 months later for quantitative antigen expression of HMB-45 or MART-1. However, there was a trend toward a decline in staining intensity of tumor cells for HMB-45. CONCLUSIONS: Preliminary results evaluating antigen expression during selective immunotherapy indicate a trend in the decline of staining intensity of tumor cells to HMB-45. Thus, although other studies have shown that peptide-based immunotherapy results in immune selection, this does not hinder the diagnostic utility of antibodies to HMB-45 and MART-1 in FNA samples of MMM.

Short-term kinetics of tumor antigen expression in response to vaccination.

The melanoma patient's immune response to tumor has been extensively studied. Yet, the frequently observed coexistence of tumor-associated Ag (TAA)-specific T cells with their target cells in vivo remains unexplained. Loss of TAA expression might contribute to this paradox. We studied TAA expression in metastases by obtaining fine-needle aspirations from 52 tumor lesions in 30 patients with melanoma before and soon after immunotherapy. Limitations due to low amounts of starting material were overcome with a high fidelity antisense RNA amplification method. TAA expression was measured by quantitative real-time PCR of anti-sense RNA. Decrease in gp100/Pmel-17 TAA preceded tumor disappearance in several instances and could be best explained by immune selection because most patients had received gp100/Pmel-17-specific vaccination. Conversely, immune selection was absent in nonregressing lesions. These observations suggest that vaccination, when successful, triggers a broad inflammatory reaction that can lead to tumor destruction despite immune selection. Additionally, lack of clinical response might be attributed to lack of this initiating event rather than immune escape. This study provides an insight into the natural history of tumors and defines a strategy for the characterization of gene expression in tumors during therapy.

Utility of fine-needle aspiration biopsy for prospective analysis of patients undergoing therapy for metastatic melanoma.

Many of the recent advancements in the area of tumor immunobiology have allowed us to focus our efforts on the experimental treatment of patients with metastatic melanoma. It has been both a frustrating and often a seemingly futile effort on the parts of physicians and researchers alike in treating such patients. The last decade of research has resulted in a paradigm shift in our understanding of the immunologic interactions between a tumor cell and the host immune response. The discovery and clinical application of tumor-associated antigens has allowed for a selective and highly specific approach to the treatment of patients with stage IV metastatic melanoma (1).

Tyrosinase immunoreactivity in fine-needle aspiration samples of metastatic malignant melanoma.

BACKGROUND: Tyrosinase, the rate-limiting enzyme in melanin synthesis, is a melanoma associated antigen that is recognized by both CD4+ and CD8+ T-cells in an HLA-restricted fashion. Peptides derived from the tyrosinase antigen currently are being utilized as a target for T-cells in several immunotherapy protocols for metastatic malignant melanoma (MMM) at the National Institutes of Health/National Cancer Institute. Serial fine-needle aspirations of metastatic lesions are performed to monitor the antigen expression of tyrosinase during treatment by immunostaining cytologic preparations with the monoclonal antibody T311. METHODS: In the current study, 62 samples of MMM were evaluated for tyrosinase immunoreactivity on air-dried, acetone fixed cytospins and the corresponding formalin fixed, paraffin embedded cell block using an avidin-biotin immunoperoxidase method. RESULTS: Positive immunoreactivity revealed a granular cytoplasmic staining in melanocytic cells. The current study results showed that 92% of samples (57 of 62) were T311 immunoreactive on cell block preparations, whereas only 61% (38 of 62) were immunoreactive on cytospin preparations. In 66% of samples (41 of 62) immunoreactivity for T311 was greater in the cell block sample than in the corresponding cytospin, whereas in only 3% of samples (2 of 62) was it greater in the cytospins. In 31% of samples (19 of 62) there was no significant difference in immunoreactivity between the 2 sample types. CONCLUSIONS: The results of the current study show that tyrosinase is a sensitive marker for the detection of MMM; however, the optimal method of sample preparation for immunoperoxidase staining appears to be formalin fixation and paraffin embedding as tyrosinase immunoreactivity is diminished significantly in air-dried cytospin samples despite subsequent acetone fixation. Cancer (Cancer Cytopathol)

Threshold levels of gene expression of the melanoma antigen gp100 correlate with tumor cell recognition by cytotoxic T lymphocytes.

The level of expression of melanoma antigens (MA) may modulate the host immunologic response. Thus, the accurate measurement of MA expression may allow proper patient selection for antigen-specific therapies and yield important information for the evaluation of clinical results. In this study, we measured the absolute levels of MA messenger ribonucleic acid (mRNA) in tumor cell lines utilizing real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). mRNA levels of MART-1, gp100, tyrosinase, TRP-1 and TRP-2 melanoma differentiation antigens and MAGE-1, MAGE-3 and ESO-1 cancer testis (CT) antigens were compared in 24 early-passage (<5 passages in culture) and 12 archival melanoma cell lines. MA mRNA expression was extremely variable among cell lines, occasionally reaching levels comparable to ribosomal RNA (rRNA). gp100 and MART-1 mRNA levels correlated with protein expression measurement obtained by FACS analysis. More significantly, a threshold of gp100 mRNA expression required for T-cell stimulation and target-cell killing was identified. This threshold level corresponded to approximately 500 mRNA copies per 10(8) copies of rRNA. Our results suggest that the measurements of MA mRNA levels may yield useful information relevant to the interpretation of clinical outcome during antigen-specific treatments.

Functional analysis of antigen-specific T lymphocytes by serial measurement of gene expression in peripheral blood mononuclear cells and tumor specimens.

The cloning of cancer Ags recognized by T cells has provided potentially new tools to enhance immunity against metastatic cancer. The biological monitoring of effective immunization has, however, remained a dilemma. We describe here a sensitive molecular quantitation methodology that allows analysis of in vivo immune response to vaccination. Metastatic melanoma patients were immunized with a synthetically modified peptide epitope (209-2M) from the melanoma self-Ag gp100. Using serial gene expression analysis, we report functional evidence of vaccine-induced CTL reactivity in fresh cells obtained directly from the peripheral blood of postimmunized patients. Further, we demonstrate in vivo localization of vaccine-induced immune response within the tumor microenvironment. The results of these molecular assays provide direct evidence that peptide immunization in humans can result in tumor-specific CTL that localize to metastatic sites.

Development and characterization of melanoma cell lines established by fine-needle aspiration biopsy: advances in the monitoring of patients with metastatic melanoma.

The establishment of melanoma cell lines from fine-needle aspiration biopsies (FNAB) has allowed for an enhanced understanding of the complex interactions that occur between T cells and tumor cells. The technique of FNAB offers the advantage of providing a sequential analysis of the same tumor nodules throughout treatment. The expression of melanoma antigens (MAs) was assessed in fresh melanoma FNAB samples and from tumor cell lines derived from these samples using several different approaches. Cytospin preparations of freshly isolated tumor cell explants were analyzed by immunocytochemistry (ICC), while the daughter cell line was analyzed by fluorescent activated cell sorting (FACS) analysis, and semiquantitative and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR, qRT-PCR). As assessed by these methods, the level of MA expression by the original tumor cell explants correlated with the expression in established in vitro cell lines. Molecular analysis of the established cell lines utilizing PCR technology improved the sensitivity of detection of MA expression. Thus FNAB of melanoma is an efficient and effective method of tissue procurement, capable of generating, sequentially and from the same lesion, fresh tumor cells, tumor infiltrating lymphocytes (TIL), and long-term melanoma cell lines.

Functional dissociation between local and systemic immune response during anti-melanoma peptide vaccination.

Peptide vaccination against tumor Ags can induce powerful systemic CTL responses. However, in the majority of patients, no tumor regression is noted. To study this discrepancy, we analyzed CTL reactivity in a melanoma patient (F001) vaccinated with g209-2M peptide, a single residue variant of gp100(209-217). G209/g209-2M-reactive CTL were identified in post- but not prevaccination PBL. Limiting dilution analysis identified one predominant CTL clone (C1-35), with TCR Vbeta6s2, recognizing g209/HLA-A*0201-expressing targets. Additionally, two autologous melanoma lines (F001TU-3 and -4) and 20 separate tumor-infiltrating lymphocyte cultures were generated from a fine needle aspirate of a metastatic lesion progressing after initial response to vaccination. Both F001TU did not express gp100 and were not recognized by C1-35. Loss of gp100 by F001TU correlated with a marked reduction of gp100 expression in the same metastatic lesion compared with prevaccination. Thus, ineffectiveness of C1-35 and tumor progression could be best explained by loss of target Ag expression. Interestingly, 12 of 20 tumor-infiltrating lymphocyte cultures recognized F001TU, but none demonstrated g209/g209-2M reactivity, suggesting a functional dissociation between systemic and local immune response. This study suggests that vaccination effects must be analyzed in the target tissue, rather than in the systemic circulation alone.

Evidence of gender bias in patients undergoing flexible sigmoidoscopy.

Some studies have revealed gender bias against women in various aspects of medical care. There is no substantial evidence of gender bias in patients undergoing cancer evaluations, specifically colorectal cancer screening and diagnosis of colorectal complaints. This study was designed to examine the role of gender bias related to patients undergoing flexible sigmoidoscopy. At the University of South Florida, we conducted a retrospective study of 1910 patients at three distinct flexible sigmoidoscopy clinics over several years, through 1992. The proportions of male and female patients who underwent the procedure for indications of either screening for colorectal cancer or the diagnosis of colorectal complaints were determined. These proportions were compared with the respective male and female patient proportion from the total number of currently active patients at each site who were eligible to have the procedure for an appropriate indication. At all three sites, a significantly smaller proportion of women (p < 0.01) underwent the procedure than expected. This was true for both screening and diagnostic indications. Conversely, at all sites significantly more men (p < 0.01) underwent the procedure for both indications. The results of this study suggest gender bias against women for patients undergoing flexible sigmoidoscopy for both screening and diagnosis. This bias may adversely affect the lethality of colorectal cancer in women. It is important to determine if such biases are influenced by the physician's recommendation or mainly due to patient attitudes.