The Predictive Utility of MammaPrint and BluePrint in Identifying Patients with Locally Advanced Breast Cancer Who are Most Likely to Have Nodal Downstaging and a Pathologic Complete Response After Neoadjuvant Chemotherapy.

BACKGROUND: Neoadjuvant chemotherapy (NCT) increases the feasibility of surgical resection by downstaging large primary breast tumors and nodal involvement, which may result in surgical de-escalation and improved outcomes. This subanalysis from the Multi-Institutional Neo-adjuvant Therapy MammaPrint Project I (MINT) trial evaluated the association between MammaPrint and BluePrint with nodal downstaging. PATIENTS AND METHODS: The prospective MINT trial (NCT01501487) enrolled 387 patients between 2011 and 2016 aged ≥ 18 years with invasive breast cancer (T2-T4). This subanalysis includes 146 patients with stage II-III, lymph node positive, who received NCT. MammaPrint stratifies tumors as having a Low Risk or High Risk of distant metastasis. Together with MammaPrint, BluePrint genomically (g) categorizes tumors as gLuminal A, gLuminal B, gHER2, or gBasal. RESULTS: Overall, 45.2% (n = 66/146) of patients had complete nodal downstaging, of whom 60.6% (n = 40/66) achieved a pathologic complete response. MammaPrint and combined MammaPrint and BluePrint were significantly associated with nodal downstaging (p = 0.007 and p < 0.001, respectively). A greater proportion of patients with MammaPrint High Risk tumors had nodal downstaging compared with Low Risk (p = 0.007). When classified with MammaPrint and BluePrint, more patients with gLuminal B, gHER2, and gBasal tumors had nodal downstaging compared with HR+HER2-, gLuminal A tumors (p = 0.538, p < 0.001, and p = 0.013, respectively). CONCLUSIONS: Patients with genomically High Risk tumors, defined by MammaPrint with or without BluePrint, respond better to NCT and have a higher likelihood of nodal downstaging compared with patients with gLuminal A tumors. These genomic signatures can be used to select node-positive patients who are more likely to have nodal downstaging and avoid invasive surgical procedures.

Phyllodes tumours of the breast: a consensus review.

Phyllodes tumours constitute an uncommon but complex group of mammary fibroepithelial lesions. Accurate and reproducible grading of these tumours has long been challenging, owing to the need to assess multiple stratified histological parameters, which may be weighted differently by individual pathologists. Distinction of benign phyllodes tumours from cellular fibroadenomas is fraught with difficulty, due to overlapping microscopic features. Similarly, separation of the malignant phyllodes tumour from spindle cell metaplastic carcinoma and primary breast sarcoma can be problematic. Phyllodes tumours are treated by surgical excision. However, there is no consensus on the definition of an appropriate surgical margin to ensure completeness of excision and reduction of recurrence risk. Interpretive subjectivity, overlapping histological diagnostic criteria, suboptimal correlation between histological classification and clinical behaviour and the lack of robust molecular predictors of outcome make further investigation of the pathogenesis of these fascinating tumours a matter of active research. This review consolidates the current understanding of their pathobiology and clinical behaviour, and includes proposals for a rational approach to the classification and management of phyllodes tumours.

Menopausal status does not predict Oncotype DX recurrence score.

BACKGROUND: Adjuvant treatment for early stage, estrogen receptor (ER) positive invasive breast cancer has been based on prognosticators such as menopausal status. The recurrence score (RS) from the 21-gene assay Oncotype DX (ODX) is predictive of a 10-y distant recurrence in this population but is rarely applied to premenopausal patients. The relationship between menopausal status and RS was evaluated. MATERIALS AND METHODS: An institutional review board-approved retrospective review was conducted of invasive breast cancer patients with known RS. ODX eligibility was based on National Comprehensive Cancer Network guidelines or physician discretion. Perimenopausal women were classified as premenopausal for statistical analyses. Comparisons of menopausal status and RS were made using general linear regression model and the exact Wilcoxon rank-sum test. RESULTS: Menopausal status was available for 575 patients (142 premenopausal, 433 postmenopausal). Median age was 46 y for premenopausal and 62 y for postmenopausal. Median invasive tumor size was 1.5 cm for both cohorts. Mastectomy rate was higher in the premenopausal group (54.8%) than postmenopausal (42%; P = 0.0001). Premenopausal women had a higher local-regional recurrence rate (2.8% versus 0%; P = 0.0384) but distant recurrence and overall survival were not statistically different (P = 0.6808). Median ER H-score was lower in premenopausal (H-score = 270) than postmenopausal women (H-score = 280; P < 0.0001). Median RS was 16 for both premenopausal (range, 0-54) and postmenopausal (range, 0-63) women. Menopausal status as a categorical variable was not predictive of RS (P-value = 0.6780). CONCLUSIONS: Menopausal status has limited predictive power for distant recurrence. Therefore, menopausal status alone should not preclude performance of ODX in ER-positive, early stage breast cancer.

Comparison of Oncotype DX and Mammostrat risk estimations and correlations with histologic tumor features in low-grade, estrogen receptor-positive invasive breast carcinomas.

Several molecular tests have been developed to estimate risk of distant recurrence and help clinical decision-making regarding adjuvant chemotherapy in patients with early stage breast carcinoma. Both Oncotype DX, a 21-gene expression profile, and Mammostrat, an immunohistochemistry-based assay, are validated to stratify patients into groups with low, intermediate and high risk of distant recurrence. However, they have not been compared head-to-head and little data are available regarding their correlation with clinicopathologic tumor features. In this study, we compared the clinicopathologic tumor features with risk estimations by Oncotype DX and Mammostrat in 106 low-grade estrogen receptor (ER)-positive breast carcinomas. Double immunohistochemical stain for pancytokeratin and Ki-67 was performed to assess cell proliferation in cancer vs stromal/inflammatory cells. Tumors showing intermediate/high risk by Oncotype DX, but not by Mammostrat, showed increased stromal cellularity, presence of inflammatory cells and increased proliferation in stromal/inflammatory cells. Discrepant cases showing intermediate/high risk by Oncotype DX but low risk by Mammostrat were associated with increased stromal cellularity, presence of inflammatory cells and increased proliferation in stromal/inflammatory cells, compared with concordant cases showing low risk by both assays. Our results suggest that low-grade ER-positive breast carcinomas with increased stromal/inflammatory cell proliferation may show an apparent increased risk of distant recurrence as assessed by Oncotype DX, which uses RNA extracted from a mixture of tumor and stromal/inflammatory cells in the assay. Mammostrat, which examines cancer cells only, may provide a better estimation of likely tumor behavior in a subgroup of low-grade breast carcinomas.

Melanocytic tumors express connexin 43 but not 26: immunohistochemical analysis with potential significance in melanocytic oncogenesis.

Connexins (Cx) are structural proteins that form gap junctions, which are vital to cell-cell communication and help to regulate cell division. The purpose of this study was to evaluate if there are diagnostically important differences in immunostaining for connexins 43 (Cx43) and 26 (Cx26) in melanoma compared with nevi. Formalin-fixed paraffin-embedded sections of 34 histologically well-characterized melanocytic lesions, 17 primary malignant melanomas (MM), and 17 nevi were stained with a polyclonal antibody to Cx43 and a polyclonal antibody to Cx26. Immunoreactivity in tumor cells was evaluated semiquantitatively based on extent (1%-100%) and intensity (0-3) of reactivity. A score of 0-300 was generated by the product of the extent and intensity readings in each case. Significantly higher Cx43 immunoreactivity was detected in MM (mean intensity score = 253.5; 95% confidence interval, 227.9-279.2; P = 0.002) compared with nevi (mean intensity score = 152.4; 95% confidence interval, 104.9-199.8). In contrast, Cx26 immunoreactivity was less than 5% or entirely absent in all melanocytic tumors (n = 34). The significantly higher Cx43 staining in MM when compared with nevi suggests an oncogenic role for this protein in melanocytic tumor progression. Consequently, the evaluation of immunohistochemical staining for Cx43 in conjunction with other ancillary stains and tumor histology may be helpful in distinguishing MM from nevi, although positive Cx26 reactivity suggests that a cutaneous neoplasm is of nonmelanocytic origin.

A mitotically active, cellular tumor stroma and/or inflammatory cells associated with tumor cells may contribute to intermediate or high Oncotype DX Recurrence Scores in low-grade invasive breast carcinomas.

Oncotype DX is an RT-PCR-based 21-gene assay validated to provide prognostic and predictive information in the form of a Recurrence Score in patients with estrogen receptor-positive, lymph node-negative breast cancer. Although the Recurrence Score was shown to correlate with several histopathological tumor features, there is a significant proportion of cases showing an apparent discrepancy between Recurrence Score and risk estimates based on the traditional clinicopathological tumor features. In this study, we tested whether a proliferating, cellular stroma and/or admixed inflammatory cells may result in an artificially increased Recurrence Score in low-grade invasive breast cancers. We analyzed the histopathological features in 141 low-grade invasive breast carcinomas, including 41 special type (tubular, cribriform and mucinous) carcinomas, with available Recurrence Score. The tumor stroma was evaluated for increased cellularity and presence of inflammatory cells. Double immunohistochemical stains for pancytokeratin and Ki-67 was performed to assess the cell proliferation in tumor vs stromal/inflammatory cells. The clinicopathological features of tumors with Recurrence Score <18 (low risk) were compared with those with Recurrence Score ≥18 (intermediate/high risk). Carcinomas associated with Recurrence Score ≥18 showed lower progesterone receptor immunoreactivity, increased stromal cellularity and presence of inflammatory cells associated with the tumor. Double immunohistochemical stains showed significantly increased proliferation in stromal/inflammatory cells compared with carcinoma cells in cases associated with Recurrence Score ≥18. A Ki-67-positive stromal/tumor cells ratio of >1 predicted Recurrence Score ≥18 with an area under the curve of 0.8967 on receiver operator curve analysis (P<0.0001). Our results suggest that the presence of increased stromal cellularity and/or associated inflammatory cells in low-grade invasive breast carcinomas may contribute to an apparently increased risk of recurrence according to Oncotype DX Recurrence Score. Careful assessment and correlation with histopathological features in such cases may help in determining the appropriate patient management.

The extent of retraction clefts correlates with lymphatic vessel density and VEGF-C expression and predicts nodal metastasis and poor prognosis in early-stage breast carcinoma.

Although the earliest feature of disseminated disease in breast cancer is regional lymph node involvement, little is known about the mechanisms whereby cancer cells interact with lymphatic endothelial cells and enter the lymphatic system. We have previously reported that the extensive presence of retraction clefts in breast carcinomas highly significantly correlates with lymphatic tumor spread and predicts poor outcome, suggesting that retraction clefts are not just fixation artifacts, but real potential spaces that are exaggerated by tissue processing and may reflect an early stage of lymphatic invasion. In this study, we examined the correlation between the extent of retraction clefts and lymphangiogenesis, as assessed by lymphatic vessel density and vascular endothelial growth factor-C (VEGF-C) expression in a series of 256 early-stage breast carcinomas. The presence and extent of retraction clefts around tumor cell nests was determined by review of all hematoxylin- and eosin-stained tumor sections. Lymphatic vessels were detected by podoplanin immunohistochemistry and lymphatic vessel density was measured using the hot-spot method. The expression of VEGF-C in the tumor cells was determined by immunohistochemistry and analyzed semiquantitatively on a four-tiered scale. High levels of retraction clefts, peritumor lymphatic vessel density and VEGF-C expression at the invasive edge in breast carcinomas significantly correlated with tumor size, histological grade, lymphatic invasion and nodal metastasis. Breast carcinomas showing extensive retraction clefts (>20% of tumor volume) were found to have significantly higher lymphatic vessel density and VEGF-C expression levels compared to tumors without this feature. High retraction clefts, peritumor lymphatic vessel density and VEGF-C expression predicted poor outcome in breast carcinomas. Our results support the hypothesis that retraction clefts are real potential spaces that may represent 'pre-lymphatic spaces' facilitating initial lymphatic invasion and that growth factors secreted by the tumor cells may stimulate tumor-associated lymphangiogenesis by promoting the endothelialization of these 'pre-lymphatic channels'.

The effect of Oncotype DX recurrence score on treatment recommendations for patients with estrogen receptor-positive early stage breast cancer and correlation with estimation of recurrence risk by breast cancer specialists.

PURPOSE: The Oncotype DX assay predicts likelihood of distant recurrence and improves patient selection for adjuvant chemotherapy in estrogen receptor-positive (ER-positive) early stage breast cancer. This study has two primary endpoints: to evaluate the impact of Oncotype DX recurrence scores (RS) on chemotherapy recommendations and to compare the estimated recurrence risk predicted by breast oncology specialists to RS. METHODS: One hundred fifty-four patients with ER-positive early stage breast cancer and available RS results were selected. Clinicopathologic data were provided to four surgeons, four medical oncologists, and four pathologists. Participants were asked to estimate recurrence risk category and offer their chemotherapy recommendations initially without and later with knowledge of RS results. The three most important clinicopathologic features guiding their recommendations were requested. RESULTS: Ninety-five (61.7%), 45 (29.2%), and 14 (9.1%) tumors were low, intermediate, and high risk by RS, respectively. RS significantly correlated with tumor grade, mitotic activity, lymphovascular invasion, hormone receptor, and HER2/neu status. Estimated recurrence risk by participants agreed with RS in 54.2% ± 2.3% of cases. Without and with knowledge of RS, 82.3% ± 1.3% and 69.0% ± 6.9% of patients may be overtreated, respectively (p = 0.0322). Inclusion of RS data resulted in a 24.9% change in treatment recommendations. There was no significant difference in recommendations between groups of participants. CONCLUSIONS: Breast oncology specialists tended to overestimate the risk of tumor recurrence compared with RS. RS provides useful information that improves patient selection for chemotherapy and changes treatment recommendations in approximately 25% of cases.

Connexin 43 is a potential prognostic biomarker for ewing sarcoma/primitive neuroectodermal tumor.

Connexins (Cxs) are building unit proteins of gap junctions (GJs) that are prognostic markers in carcinomas. To investigate the role of Cx in Ewing sarcoma (EWS)/primitive neuroectodermal tumor (PNET), we examined the expression of Cx43 and Cx26 in 36 EWS/PNETs and found (1) cytoplasmic Cx43 reactivity in 28/36 (78%) cases. (2) Cx43 score was significantly correlated with overall survival (P = .025). The average scores for patients alive and dead at 3 years are 46.08 and 96.98 (P = .004) at 5 years are 46.06 and 96.42 (P = .002). (3) Metastasis had a significant effect on the overall survival (P = .003). (4) Cytoplasmic Cx26 reactivity was detected in 2 of 36 (6%) patients who died with metastasis. Our results suggest a possible oncogenic and prognostic role for Cx43 and Cx26 in EWS/PNET. The lack of membranous immunoreactivity suggests that the effect of Cx in EWS/PNET is via a GJ function-independent mechanism.

RNA interference-mediated inhibition of erythropoietin receptor expression suppresses tumor growth and invasiveness in A2780 human ovarian carcinoma cells.

Although recombinant human erythropoietin (rHuEpo) has revolutionized the treatment of anemia, recent clinical trials suggested that rHuEpo use may be associated with decreased survival in cancer patients. Although the expression of erythropoietin (Epo) receptor (EpoR) has been demonstrated in various human cancers, the effect of exogenous Epo on the growth and therapy resistance of EpoR-bearing tumor cells is unclear at present. In the current study, we examined the hypothesis that EpoR may contribute to tumor growth independent of Epo in A2780 human ovarian carcinoma cells. A2780 human ovarian carcinoma cells showed high levels of EpoR expression, but lacked expression of Epo mRNA and biologically active Epo protein under both normoxic and hypoxic conditions. Exogenous Epo did not stimulate EpoR-mediated signaling, proliferation, invasiveness, or resistance to cytotoxic drugs in A2780 cells. In contrast, specific inhibition of EpoR expression using a short hairpin RNA (shRNA) expression plasmid resulted in markedly reduced proliferation and invasiveness in vitro. In addition, inhibition of EpoR expression led to abrogated in vivo ovarian cancer cell growth in a tumor xenograft system and resulted in decreased EpoR signaling. Our findings suggest that EpoR may be constitutively active in some cancer cells in the absence of Epo and provide the first evidence for a potential role of an Epo-independent, EpoR-mediated pathway in the growth of some human cancers.

Gene expression profiling in breast cancer.

BACKGROUND: Breast cancer is a heterogeneous group of different tumor subtypes that vary in prognosis and response to therapy. This heterogeneity has spawned an era of molecular assays striving to classify and thus predict outcome, thereby guiding the future in targeted personalized treatment strategies. METHODS: This article provides an overview of the development and application of molecular assays as applied to breast cancer. Differences in the technology used for these tests as well as scientific evidence supporting the validity of the gene expression profile are discussed. Examples of the clinical applicability of these assays are provided, but these represent only a fraction of the potential uses yet to be discovered. A comparison of the three most commonly used assays is included. RESULTS: Molecular assays have provided new genetic approaches to unravel the complexities of clinical specimens relevant to breast cancer treatment planning and assessment of outcome. In particular, on a molecular level specific to the woman's tumor, these assays allow a prediction of outcome (prognosis) in terms of low and high risk for the future development of distant metastatic disease. Additionally, one assay, Oncotype DX (Genomic Health Inc, Redwood City, CA), allows for the prediction of benefit of the addition of chemotherapy to hormone therapy alone. CONCLUSIONS: While incorporation of molecular assays into the treatment planning strategy of breast cancer continues to be a work in progress, this approach is evolving quickly due to strong scientific evidence to become standard of practice in the near future. The possibilities of these assays in terms of clinical investigation are limitless, but currently their general applicability is limited to less than half of the population of women presenting with breast cancer.

Mutant V600E BRAF increases hypoxia inducible factor-1alpha expression in melanoma.

Mutations in the BRAF serine/threonine kinase gene are frequently found in cutaneous melanomas. Activation of hypoxia inducible factor-1alpha (HIF-1alpha) in response to both hypoxic stress and oncogenic signals has important implications in cancer development and progression. Here, we report that mutant BRAF(V600E) increases HIF-1alpha expression in melanoma cells. Our microarray profiling data in 35 melanoma and melanocyte cell lines showed that HIF-1alpha gene expression was significantly increased in melanomas harboring BRAF(V600E) mutation. Stable suppression of mutant BRAF(V600E) or both wild-type and mutant BRAF(V600E) by RNA interference in melanoma cells resulted in significantly decreased HIF-1alpha expression. Knockdown of mutant BRAF(V600E) induced significant reduction of cell survival and proliferation under hypoxic conditions, whereas knockdown of both wild-type and mutant BRAF(V600E) resulted in further reduction. The effects of BRAF knockdown can be rescued by reintroducing BRAF(V600E) into tumor cells. Transfection of BRAF(V600E) into melanoma cells with wild-type BRAF induced significantly more hypoxic tolerance. Knockdown of HIF-1alpha in melanoma cells resulted in decreased cell survival under hypoxic conditions. Pharmacologic inhibition of BRAF by BAY 43-9006 also resulted in decreased HIF-1alpha expression. Although HIF-1alpha translational rate was not changed, the protein was less stable in BRAF knockdown cells. In additional, von Hippel-Lindau protein expression was significantly increased in BRAF knockdown cells. Our data show for the first time that BRAF(V600E) mutation increases HIF-1alpha expression and melanoma cell survival under hypoxic conditions and suggest that effects of the oncogenic V600E BRAF mutation may be partially mediated through the HIF-1alpha pathway.

Nuclear orphan receptor TR3/Nur77 mediates melanoma cell apoptosis.

TR3 was originally recognized for its role in the regulation of cell survival and differentiation, however, it was recently found to be a potent pro-apoptotic protein. In order to characterize the role of TR3 in melanoma cell apoptosis, we studied expression of TR3 in melanoma cell lines and tissues, its subcellular distribution and function during apoptosis using various expression and RNA interference vectors. We found that TR3 was constitutively expressed in both cultured melanoma cells and melanoma tissues. TR3 expression was significantly decreased in advanced melanomas comparing to benign nevi. Over-expression of wild type TR3 or mutant TR3 lacking the DNA binding domain resulted in massive apoptosis in melanoma cells, whereas stable knockdown of TR3 using RNA interference resulted in melanoma cell resistance to apoptosis induced by chemotherapeutic agents ATRA and cisplatin. We further demonstrated that apoptosis in melanoma cells was mediated, at least partially, through TR3 mitochondrial translocation but not alteration in TR3 expression levels. Our results suggest that TR3 is an important apoptosis inducing factor in melanoma cells. Decreased expression of TR3 in metastatic melanoma cells may contribute to their reduced apoptotic potential and increased resistance to chemotherapy.

Extensive retraction artifact correlates with lymphatic invasion and nodal metastasis and predicts poor outcome in early stage breast carcinoma.

Retraction artifact resulting in clear spaces around tumor cell nests is frequently seen in histologic material and may present difficulty in their differentiation from lymphovascular invasion. We noticed that retraction artifact seemed to be more common around groups of breast cancer cells compared with benign acini, and when extensively present, metastasis to axillary lymph nodes was often seen. Thus, we performed a study of 304 cases of stage pT1 and pT2 breast carcinomas to test our hypothesis that extensive retraction artifact in tumors correlates with lymphatic spread and outcome. Tumors were evaluated to determine the presence and extent of retraction artifact around tumor cell nests and the presence of lymphatic invasion. Lymphatic invasion was confirmed by D2-40 immunostaining. The extent of retraction artifact in tumors was correlated with clinicopathologic tumor features and patient outcome. Variable degree of retraction artifact was present in 183 of 304 (60%) invasive carcinomas, with its extent ranging from 0% to 90% (median 5%). The extent of retraction artifact showed a significant correlation with tumor size, histologic type, histologic grade, presence of lymphovascular invasion, and nodal metastasis. Further, extensive retraction artifact was significantly associated with poor overall and disease-free survival in both univariate and multivariate analyses. We propose that the apparent retraction of the stroma from cells of invasive breast carcinoma on routine histologic sections is not a phenomenon merely due to inadequate fixation as currently believed. Rather, it likely signifies important biologic changes that alter tumor-stromal interactions and contribute to lymphatic spread and tumor progression.

Podoplanin is a highly sensitive and specific marker to distinguish primary skin adnexal carcinomas from adenocarcinomas metastatic to skin.

Distinction of primary skin adnexal carcinomas from cutaneous metastasis of adenocarcinomas is challenging. In this study, we evaluated podoplanin immunoreactivity in a series of primary skin adnexal tumors and adenocarcinomas metastatic to skin using a D2-40 antibody. The initial test series were composed of a total of 93 cases including 32 primary skin adnexal carcinomas, 46 benign primary adnexal tumors, and 15 cutaneous metastatic adenocarcinomas. We found that variable D2-40 reactivity was seen in all of the primary cutaneous carcinomas including sebaceous carcinomas (10/10), squamous cell carcinomas (10/10), porocarcinomas (4/4), trichilemmal carcinomas (4/4), skin adnexal carcinomas not otherwise specified (4/4), and in the majority of benign skin adnexal tumors. In contrast, no podoplanin immunoreactivity was seen in any of the 15 (0/15) cutaneous metastases. To confirm the initial findings and to further explore the utility of podoplanin reactivity in the distinction of these tumors, we also examined a test set of 35 unknown cases, including 21 adenocarcinomas metastatic to skin and 14 primary adnexal tumors, in a blinded fashion. In this test set of cases, podoplanin was negative in 22 cases and positive in 13 cases. Of the 22 podoplanin negative cases, 20 were proven to be metastatic adenocarcinoma. Of the 13 D2-40 positive cases, 12 were proven to be primary adnexal tumors. Our results suggest that podoplanin can be a useful tool to distinguish primary skin adnexal carcinomas form adenocarcinomas metastatic to skin with high sensitivity (94.5%) and specificity (97.2%).

High-risk benign breast lesions: current strategies in management.

BACKGROUND: High-risk benign breast lesions can create confusion for both the patient and the clinician. This paper reviews the characteristics of these lesions to help direct appropriate management. METHODS: The authors reviewed the literature regarding high-risk breast lesions and include management guidelines that we employ at our institute. RESULTS: High-risk breast lesions offer varying degrees of increased risk for the future development of breast cancer. Chemoprevention may be used to help decrease the risks from some lesions. CONCLUSIONS: The management of high-risk benign breast lesions can be confusing. Clinicians should assess the risk of future breast cancer and develop a proper screening and prevention strategy for each individual patient.

Survival and invasiveness of astrocytomas promoted by erythropoietin.

OBJECT: The hypoxia-inducible pleiotropic hormone, erythropoietin (EPO), has recently been found to promote the development and survival of neurons and astrocytes. Since hypoxia has been implicated in the malignant progression of some human cancers, the authors investigated whether EPO signaling influenced the malignant properties of human astrocytoma cells. METHODS: Reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunohistochemical studies were used to measure EPO and its receptor (EPOR). Cell viability, Matrigel invasion assays, metalloprotease assays, EPO neutralizing antibodies, and EPOR overexpression were used to study the biological actions of EPO. Expression of both EPO and EPOR was observed in the hypoxic regions and invasive margins of glioma specimens obtained at biopsy, and expression of EPOR correlated with the stage of the tumor. The EPOR was also functionally upregulated by hypoxia in cultured glioblastoma multiforme (GBM) cells. Both hypoxia and EPO protected cultured GBM cells from cisplatin cytotoxicity and promoted the invasiveness of GBM cells through Matrigel by potentiating metalloprotease activity. Hypoxia-enhanced cell invasion was attenuated in cells that overexpressed a nonfunctional EPOR. CONCLUSIONS: Hypoxia-inducible autocrine and paracrine EPO signaling participates in the malignant progression of GBMs.

Repeat operative sentinel lymph node biopsy.

Because sentinel lymph node (SLN) biopsy continues to be used for staging in patients with breast cancer, physicians treating these patients will be faced with in-breast recurrences and new primary breast cancers in the treated breast. Repeat operative SLN biopsy might be feasible in this clinical scenario. This report describes the case of a patient with an ipsilateral different-site, recurrent, infiltrating ductal carcinoma 14 months after lumpectomy; negative SLN biopsy result; and radiation therapy, now with a positive SLN biopsy result.

Can Melan-A replace S-100 and HMB-45 in the evaluation of sentinel lymph nodes from patients with malignant melanoma?

The sentinel lymph node (SLN) biopsy has become an increasingly important procedure used in the primary staging of malignant melanoma. However, micrometastases in a lymph node can be easily missed on routine H&E-stained sections. Therefore, S-100 and HMB-45 IHC stains are standardly performed on grossly negative SLNs for detection of metastatic melanoma. Each of these IHC markers, however, is not ideal. The authors investigated whether the newer IHC marker Melan-A would improve the detection of metastatic melanoma in SLN biopsies. Forty lymph nodes previously diagnosed with metastatic melanoma were retrospectively evaluated for S-100, HMB-45, and Melan-A expression. In addition, 42 SLN biopsies for metastatic melanoma detection were prospectively collected and evaluated for S-100, HMB-45, and Melan-A expression. All lymph nodes with metastatic melanoma from the retrospective study demonstrated S-100 reactivity. Five of the lymph nodes with metastatic melanoma from the retrospective study failed to express either HMB-45 or Melan-A, all of which displayed a desmoplastic morphology. One of the metastases positive for S-100 and HMB-45 failed to show reactivity with Melan-A (3%). The prospective study found 10 lymph nodes from 42 cases to be positive for metastatic melanoma, which were positive for S-100 (100%). Nine of the involved lymph nodes were positive for HMB-45(90%), and nine were positive for Melan-A (90%). Melan-A, although very specific, cannot replace the use of S-100 and HMB-45 for the detection of metastatic melanoma in SLNs. It can, however, substitute for HMB-45 with equally good results.

Erythropoietin activates the phosphoinositide 3-kinase/Akt pathway in human melanoma cells.

Erythropoietin (Epo) is used commonly to treat cancer and/or therapy-related anemia. Until recently, Epo was considered to be a specific stimulator of erythropoiesis, acting via its receptor, EpoR. It becomes clear, however, that EpoR is expressed in a variety of cell types other than hematopoietic cells, and that Epo is a potent cytoprotective cytokine increasing cell survival under hypoxic conditions. Epo and EpoR are also expressed in various malignant tumors, and EpoR expression shows association with tumor invasion and progression. Recently, a functional Epo autocrine signaling mechanism was also detected in human melanoma cells. In this study, we examined the hypothesis that Epo activates the Akt signaling pathway in human melanoma cells and thus promotes the survival of tumor cells. The Akt signaling pathway in response to Epo was examined in melanoma. Similar to Epo, the expression of EpoR was up-regulated in response to hypoxia and Epo stimulation in melanoma cells. Melanoma cells constitutively expressed Akt with variable expression of mammalian target of rapamycin, and Epo dose-dependently induced their activity. Epo increased Akt kinase activity, which was abrogated by co-treatment with LY294002, a specific blocker of phosphoinositide 3-kinase. LY294002 also inhibited the cytoprotective effects of Epo in melanoma cells under both normoxic and hypoxic conditions. Our results suggest that Epo promotes melanoma cell survival by activating an Akt-dependent signaling pathway.