A cut-off of 2150 cytokeratin 19 mRNA copy number in sentinel lymph node may be a powerful predictor of non-sentinel lymph node status in breast cancer patients.

Since 2007, one-step nucleic acid amplification (OSNA) has been used as a diagnostic system for sentinel lymph node (SLN) examination in patients with breast cancer. This study aimed to define a new clinical cut-off of CK19 mRNA copy number based on the calculation of the risk that an axillary lymph node dissection (ALND) will be positive. We analyzed 1529 SLNs from 1140 patients with the OSNA assay and 318 patients with positive SLNs for micrometastasis (250 copies) and macrometastasis (5000 copies) underwent ALND. Axillary non-SLNs were routinely examined. ROC curves and Youden's index were performed in order to identify a new cut-off value. Logistic regression models were performed in order to compare OSNA categorical variables created on the basis of our and traditional cut-off to better identify patients who really need an axillary dissection. 69% and 31% of OSNA positive patients had a negative and positive ALND, respectively. ROC analysis identified a cut-off of 2150 CK19 mRNA copies with 95% sensitivity and 51% specificity. Positive and negative predictive values of this new cut-off were 47% and 96%, respectively. Logistic regression models indicated that the cut-off of 2150 copies better discriminates patients with node negative or positive in comparison with the conventional OSNA cut-off (p<0.0001). This cut-off identifies false positive and false negative cases and true-positive and true negative cases very efficiently, and therefore better identifies which patients really need an ALND and which patients can avoid one. This is why we suggest that the negative cut-off should be raised from 250 to 2150. Furthermore, we propose that for patients with a copy number that ranges between 2150 and 5000, there should be a multidisciplinary discussion concerning the clinical and bio-morphological features of primary breast cancer before any decision is taken on whether to perform an ALND or not.

Elaboration of a nomogram to predict nonsentinel node status in breast cancer patients with positive sentinel node, intraoperatively assessed with one step nucleic amplification: Retrospective and validation phase.

BACKGROUND: Tumor-positive sentinel lymph node (SLN) biopsy results in a risk of non sentinel node metastases in micro- and macro-metastases ranging from 20 to 50%, respectively. Therefore, most patients underwent unnecessary axillary lymph node dissections. We have previously developed a mathematical model for predicting patient-specific risk of non sentinel node (NSN) metastases based on 2460 patients. The study reports the results of the validation phase where a total of 1945 patients were enrolled, aimed at identifying a tool that gives the possibility to the surgeon to choose intraoperatively whether to perform or not axillary lymph node dissection (ALND). METHODS: The following parameters were recorded: Clinical: hospital, age, medical record number; Bio pathological: Tumor (T) size stratified in quartiles, grading (G), histologic type, lymphatic/vascular invasion (LVI), ER-PR status, Ki 67, molecular classification (Luminal A, Luminal B, HER-2 Like, Triple negative); Sentinel and non-sentinel node related: Number of NSNs removed, number of positive NSNs, cytokeratin 19 (CK19) mRNA copy number of positive sentinel nodes stratified in quartiles. A total of 1945 patients were included in the database. All patient data were provided by the authors of this paper. RESULTS: The discrimination of the model quantified with the area under the receiver operating characteristics (ROC) curve (AUC), was 0.65 and 0.71 in the validation and retrospective phase, respectively. The calibration determines the distance between predicted outcome and actual outcome. The mean difference between predicted/observed was 2.3 and 6.3% in the retrospective and in the validation phase, respectively. The two values are quite similar and as a result we can conclude that the nomogram effectiveness was validated. Moreover, the ROC curve identified in the risk category of 31% of positive NSNs, the best compromise between false negative and positive rates i.e. when ALND is unnecessary (<31%) or recommended (>31%). CONCLUSIONS: The results of the study confirm that OSNA nomogram may help surgeons make an intraoperative decision on whether to perform ALND or not in case of positive sentinel nodes, and the patient to accept this decision based on a reliable estimation on the true percentage of NSN involvement. The use of this nomogram achieves two main gools: 1) the choice of the right treatment during the operation, 2) to avoid for the patient a second surgery procedure.

Topographic expression of the Hippo transducers TAZ and YAP in triple-negative breast cancer treated with neoadjuvant chemotherapy.

BACKGROUND: The Hippo signaling acts as a tumor-suppressor pathway that negatively regulates TAZ and YAP. Increasing evidence supports the activation of TAZ and YAP in breast cancer. Moreover, the Hippo pathway is involved in the biology of non-neoplastic cells residing in the tumor microenvironment. On this basis, we herein assessed TAZ and YAP in triple-negative breast cancer and its surrounding microenvironemnt in order to investigate their impact on pathological complete response (pCR) and tumor recurrence. METHODS: Sixty-one triple-negative breast cancer patients treated with neoadjuvant chemotherapy were retrospectively evaluated. TAZ and YAP were assessed by immunohistochemistry and classified as positive or negative according to the percentage of tumor-expressing cells, cellular localization, and staining intensity. TAZ and YAP expression was also evaluated in non-lymphocytic stromal cells, tumor-infiltrating lymphocytes (TILs) and endothelial cells. The Pearson's Chi-squared test of independence was used to test the association between TAZ/YAP and clinical-molecular factors. A multivariate logistic regression model was generated to identify variables impacting pCR. The Kaplan-Meier method and the log-rank test were used for estimating and comparing survival curves. Cox proportional regression models were built to evaluate the risk of recurrence for the variables considered. Internal validation was carried out with a re-sampling without replacement method. RESULTS: We did not observe any impact on pCR rate when TAZ and YAP were addressed singularly. Conversely, the combined expression of YAP in tumor cells and non-lymphocytic stromal cells was an independent predictor of reduced pCR rate in the multivariate model (OR 7.13, 95% CI: 1.23-41.41, p = 0.029). Next, the combined expression of TAZ and YAP was associated with shorter disease-free survival (DFS) in multivariate analysis (HR 3.07, 95% CI: 1.24-7.61, p = 0.016). The robustness of these findings were internally validated. CONCLUSIONS: The combined expression of YAP in TNBC cells and in the surrounding stroma seems to be associated with a decreased likelihood to achieve pCR. Conversely, the combined expression of TAZ and YAP in tumor cells conferred poor survival outcomes.

Body Mass Index and Treatment Outcomes in Metastatic Breast Cancer Patients Treated With Eribulin.

Eribulin has shown survival advantage and manageable toxicity in heavily pre-treated metastatic breast cancer (mBC). We assessed whether body mass index (BMI) impacts treatment outcomes in 101 patients treated with eribulin at six Italian Oncologic Centers. BMI was addressed as a categorical variable (18.5-24.9 vs. at least 25). Clinical benefit rate (CBR) was assessed overall and in subgroups defined by BMI, line of therapy (LOT), and hormone receptor (HR) status. Analysis of CBR by LOT and HR status were further stratified by BMI. Survival curves were compared using the Kaplan-Meier method and log-rank test. Predictors of survival were tested in Cox models. Patients treated with eribulin as third line showed greater CBR when their BMI was in the lowest category (77.8 vs. 58.1%, P = 0.03). Median progression free survival (PFS) and overall survival (OS) in normal and overweight patients were 4 (95% CI, 3-5) versus 3 (2.1-4) months, P = 0.02 and 13 (11-15) versus 12 (6-18) months, P = 0.96, respectively. Median PFS and OS in estrogen receptor (ER) positive and negative tumours were 4 (3-5) versus 3 (2-4) months, P = 0.005 and 14 (10-18) versus 7 (4-10), P = 0.02, respectively. In multivariate analyses, BMI impacted PFS at a nearly significant extent (P = 0.05), while ER expression significantly affected PFS and OS (P = 0.01 and 0.02, respectively). No relevant findings emerged concerning toxicity. We found evidence of greater efficacy of eribulin in leaner mBC patients, particularly if given as third line and in ER positive tumors. Further studies are warranted to confirm our findings.

Modeling the positioning of single needle electrodes for the treatment of breast cancer in a clinical case.

BACKGROUND: Breast cancer is the most common cancer in women worldwide and is the second most common cause of cancer death in women. Electrochemotherapy (ECT) used in early-phase clinical trials for the treatment of primary breast cancer resulted in a not complete tumor necrosis in most cases. The present study was undertaken to analyze the feasibility to use ECT to treat patients with histologically proven unifocal ductal breast cancer. In particular, results of ECT treatment in a clinical case are compared with the ones of a simplified 3D dosimetric model. METHODS: This clinical study was conducted with the pulse generator Cliniporator Vitae (IGEA, Carpi, Italy). ECT procedures were performed according to ESOPE standard operating procedures. Five single needle electrodes were used with one positioned in the center of the tumor, and the other four distributed around the nodule. Histological images of the resected tumor are compared with the maps of the electric field obtained with a simplified 3D model in Comsol Multiphysics v 4.3. RESULTS: The results of the clinical case demonstrated a reduced efficacy of the ECT treatment described. The proposed simple numerical model of the breast tumor located in a low conductive tissue suggests that this is due to the reduced electric field induced inside the tumor with such 5 electrodes placement. However, where the electric field is predicted higher than the reversible electroporation threshold (E>400 V/cm), also the histological images confirm the necrosis of the target with a good agreement between the modeled and clinical results. CONCLUSIONS: The results suggest the dependence of the effectiveness of the treatment on the careful placement of the electrodes. A detailed planned procedure for the tumor analysis after the treatment is also needed in order to better correlate the single electrode positions and the histological images. Simulation models could be used to identify better electrodes configuration in planning the experimental protocol for ECT treatment of breast tumors.

Hot flushes in women with breast cancer: state of the art and future perspectives.

Although not life-threatening, vasomotor symptoms might have a detrimental effect on quality of life and represent a major determinant of poor therapeutic compliance in breast cancer patients. Limitations of hormonal therapies have fostered the use of non-estrogenic pharmacological agents, which mainly include centrally acting compounds, antidepressant drugs, serotonin-norepinephrine reuptake inhibitors and serotonin reuptake inhibitors. Integrating therapeutic tools have recently come from a wide range of heterogeneous approaches varying from phytoestrogens use to ganglion block. We herein critically review the most updated evidence on the available treatment options for management of vasomotor symptoms. The need for a patient-oriented approach following systematic evaluation of the presence and degree of vasomotor disturbances is also discussed and future perspectives in therapeutics are summarized.

A melanoma immune response signature including Human Leukocyte Antigen-E.

Paired cultures of early-passage melanoma cells and melanocytes were established from metastatic lesions and the uninvolved skin of five patients. In this stringent autologous setting, cDNA profiling was used to analyze a subset of 1477 genes selected by the Gene Ontology term 'immune response'. Human Leukocyte Antigen E (HLA-E) was ranked 19th among melanoma-overexpressed genes and was embedded in a transformation signature including its preferred peptide ligand donors HLA-A, HLA-B, HLA-C, and HLA-G. Mostly undetectable in normal skin and 39 nevi (including rare and atypical lesions), HLA-E was detected by immunohistochemistry in 17/30 (57%) and 32/48 (67%) primary and metastatic lesions, respectively. Accordingly, surface HLA-E was higher on melanoma cells than on melanocytes and protected the former (6/6 cell lines) from lysis by natural killer (NK) cells, functionally counteracting co-expressed triggering ligands. Although lacking HLA-E, melanocytes (4/4 cultures) were nevertheless (and surprisingly) fully protected from NK cell lysis.

Surgical treatment of melanoma: a survey of Italian hospitals.

Surgery is the first option for treating melanoma regardless of stage at presentation. We surveyed a representative sample of hospitals to evaluate management and quality of surgical indications for melanoma in Italy. At analysis, hospitals were grouped into high- or low-volume centers, with the population median of 25 diagnoses serving as the cut-off. Surgery for primary melanoma was similar between hospital groups. More high-volume centers were organized to perform sentinel node biopsy (91 vs. 56%). There were no major differences between high- and low-volume centers concerning the surgical approach to stage III and IV disease.

Follow-up of melanoma: a survey of Italian hospitals.

Follow-up is managed internally in 94% of centers and is programmed according to international guidelines in 52% of high-volume hospitals (>25 melanoma diagnoses per year); the remainder use internal guidelines; fewer low-volume centers (≤ 25 diagnoses per year) have internal guidelines (25%, p = 0.001). Instrumental examinations for stage III and IV disease are similar, while the examination interval changes from 3/4 months for stage III to 2/3 months for stage IV, and use of PET/CT increases from 44 to 54%. Overall, thoracic and abdominal CT is used most for follow-up in stage III (83%), while bone scintigraphy is used more commonly in low-volume centers (41 vs. 19%, p = 0.003), despite similar use of PET/CT (48 vs. 41%). Brain CT or MRI is more common in high-volume centers (63 vs. 39%, p > 0.0001), as is echography of draining lymph nodes (71 vs. 52%, p = 0.01). Hepatic/abdominal echography and thoracic radiography are used in about 50% of centers, regardless of type. In stage IV, use of bone scintigraphy is similar among groups (ca. 40%); brain CT/NMR use increases from 51 to 64% and is more common in high-volume centers (p = 0.03). Lymph node echography is more common in high-volume centers (56 vs. 39%, p = 0.03).

Quantitative molecular analysis of sentinel lymph node may be predictive of axillary node status in breast cancer classified by molecular subtypes.

To determine the performance of intraoperative one-step nucleic acid amplification (OSNA) assay in detecting sentinel lymph node metastases compared to postoperative histology taking into account breast cancer molecular classification and to evaluate whether the level of cytokeratin 19 mRNA copy number may be useful in predicting the likelihood of a positive axillary lymph node dissection. OSNA assay was performed in a prospective series of 903 consecutive sentinel lymph nodes from 709 breast cancer patients using 2 alternate slices of each sentinel lymph node. The remaining 2 slices were investigated by histology. Cytokeratin 19 mRNA copy number, which distinguishes negative cases (<250 copies), micrometastases (+, ≥250≤5000 copies) and macrometastases (++, >5000 copies), was compared to axillary lymph node dissection status and to the biological tumor profile. Concordance between OSNA and histopathology was 95%, specificity 95% and sensitivity 93%. Multiple Corresponce Analysis and logistic regression evidenced that positive axillary lymph node dissection was significantly associated with a higher cytokeratin 19 mRNA copy number (>5000; p<0.0001), HER2 subtype (p = 0.007) and lymphovascular invasion (p<0.0001). Conversely, breast cancer patients with cytokeratin 19 mRNA copy number <2000 mostly presented a luminal subtype and a negative axillary lymph node dissection. We confirmed that OSNA assay can provide standardized and reproducible results and that it represents a fast and quantitative tool for intraoperative evaluation of sentinel lymph node. Omission of axillary lymph node dissection could be proposed in patients presenting a sentinel lymph node with a cytokeratin 19 mRNA copy number <2000 and a Luminal tumor phenotype.

Melanoma molecular classes and prognosis in the postgenomic era.

Gene expression profiling is a powerful method to classify human tumours on the basis of biological aggressiveness, response to therapy, and outcome for the patient, but its application in melanoma lags behind that of other cancers. From more than 100 articles available on the topic, we selected 14 focusing on patients' outcome. We review and briefly discuss salient findings, and list ten reasons why melanoma molecular classes are not yet used in clinical diagnosis and prognosis. The available evidence suggests that we are on the verge of creating a framework for the use of melanoma molecular classes in prognosis, but so far there is little consensus to put together informative diagnostic and prognostic gene sets.

HER2 protein and gene variation between primary and metastatic breast cancer: significance and impact on patient care.

PURPOSE: To analyze HER2 status in primary breast cancer (PBC) compared with correspondent metachronous metastases and to investigate whether BC phenotype may be predictive of change in HER2 expression. EXPERIMENTAL DESIGN: HER2 was investigated by immunohistochemistry, silver in situ hybridization (SISH), and FISH, in a series of 137 tumors, building up a tissue microarray to concurrently analyze each single PBC and metastatic (MBC) on the same slide. RESULTS: HER2 status was discordant in 14 cases (10%): 12 negative in PBC and positive in metastases and two positive in PBC and negative in metastases (P = 0.04). These findings were confirmed by a PCR based test termed Multiplex Ligation-dependent Probe Amplification (MLPA). HER2 status changed in hormone receptor-positive BC more frequently than in negative ones (P = 0.002). In addition, we evaluated HER2 gene and chromosome 17 copy number by SISH in the 123 cases with unchanged HER2 status during progression. We found consistent HER2 gene copy number stability in the 100 nonamplified cases. Conversely, of the 23 amplified PBC, 13 (57%) demonstrated a significant increase in the HER2 gene and chromosome 17 copy number in their paired metastases (P = 0.01), as defined by SISH (k = 0.54, P < 0.0001) and MLPA. Patients who changed HER2 status from negative to positive, presented significant longer time to progression when treated with trastuzumab compared to those who were untreated (P = 0.04). CONCLUSIONS: When feasible, HER2 reassessment in metastatic lesions should be carefully taken into account, especially for metastases coming from primary hormone receptor-positive BC.

The Gore-Tex biomaterial mesh as temporary divisor in two times surgery for spinal and pelvic tumors.

In muscular skeletal spine and pelvic tumor, surgery can be performed by a double anterior and posterior approach to decrease the risk of bleeding and opening the tumor; in first one, neurovascular bundles are divided by the mass; in second one, the tumor is resected by posterior or postero-lateral approach. A Gore-Tex mesh could be used as divisor between neurovascular bundles and the tumor to decrease the adhesion formation risk and facilitate tumor removal during second operation if performed after more days. The cohort was composed by a consecutive series of 11 patients underwent to surgery for spine and pelvic tumor where Gore-Tex mesh spacer was placed. In this study, efficiency and tolerability of Gore-Tex mesh were evaluated. No case of adhesion between Gore-Tex mesh and surrounding structures is reported, no case of migration or complications occurred. Gore-Tex mesh use can be considered a useful and safe procedure to decrease the risk of adhesion formation between tumor and surrounding tissue so that to allow the tumor removal easier.

Peritoneal carcinomatosis from ovarian epithelial primary: combined aggressive treatment.

UNLABELLED: Cytoreductive surgery followed by platinum based systemic chemotherapy is an effective treatment for advanced ovarian epithelial carcinoma, resulting in up to 80% complete response (CR) rate; however only 30% of patients reaches 5-year survival. The low extra-abdominal relapse attitude leads to consider the opportunity of treatment intensification combining aggressive cytoreductive surgery with locoregional chemotherapy for FIGO stage III/IV ovarian carcinoma recurrent after the first-line chemotherapy, having still a curative intent. PATIENTS AND METHODS: An "open" intra-abdominal hyperthermic perfusion with 25 mg/m(2)/lt cisplatin of perfusate or 50 mg/m(2)cisplatin plus 15 mg/m(2)doxorubicin was carried out throughout the abdomino-pelvic cavity on 42 patients affected by peritoneal carcinomatosis from ovarian primary, soon after tumor removal en bloc with regional involved peritoneum. Clinical and oncologic data have been prospectively recorded on a dedicated database. RESULTS: Forty-two patients, submitted to peritonectomy, achieved no residual macroscopic disease in 83% of the cases. Hyperthermic chemoperfusion was performed in 95% of the patients. Major complications were observed in 21.4%, being directly correlated to the duration of the surgical procedure (p=0.03). The operative mortality was 4.7%. At a mean follow up of 22 months, the overall 3-year survival was 61.4%, with a median survival of 41 months. CONCLUSION: Complete cytoreduction is possible for the majority of patients, allowing encouraging survival to be reached. Careful selection of patients could reduce surgical risk and further improve survival.

Clinical considerations on sentinel node biopsy in melanoma from an Italian multicentric study on 1,313 patients (SOLISM-IMI).

BACKGROUND: Although widely used for the management of patients with cutaneous melanoma, the sentinel lymph node (SLN) biopsy (SNB) procedure raises several issues. This study was designed to investigate: the predictive factors of SLN status, the false-negative (FN) rate, and patients' prognosis after SNB. PATIENTS AND METHODS: This is an observational, prospective study conducted on a large series of consecutive patients (n = 1,313) enrolled by 23 Italian centers from 2000 through 2002. A commonly shared protocol was adopted for the SNB surgical procedure and the SLN pathological examination. RESULTS: The SLN positive and false-negative (FN) rates were 16.9% and 14.4%, respectively (median follow-up, 4.5 years). At multivariable logistic regression analysis, the frequency of positive SLN increased with increasing Breslow thickness (p < 0.0001) and decreased in patients with melanoma regression (p = 0.024). At the multivariable Cox regression analysis, SLN status was the most important prognostic factor (hazards ratio (HR) = 3.08) for overall survival; the other statistically significant factors were sex, age, Breslow thickness, and Clark's level. Considering SLN and NSLN status, including FN cases, we identified four groups of patients with different prognoses. The 5-year overall survival of patients with positive SLNs was 71.3% in those with negative nonsentinel lymph nodes (NSLNs) and 50.4% if NSLNs were positive. CONCLUSIONS: Regression in the primary melanoma seems to be a protective factor from metastasis in the SLN. When correctly calculated, the SNB FN rate is 15-20%. Furthermore, the SNB is important to more precisely assess the prognosis of patients with melanoma.

Gemcitabine at fixed dose-rate in patients with advanced soft-tissue sarcomas: a mono-institutional phase II study.

PURPOSE: To explore the clinical activity and toxicity of gemcitabine infused at the fixed dose of 10 mg/m(2)/min over 100 min in patients with soft tissue sarcomas (STSs). PATIENTS AND METHODS: Fourteen patients with advanced locally unresectable and/or metastatic, pretreated STSs (seven leiomyosarcoma, three malignant schwannoma, one synovialsarcoma, one malignant fibrous histiocytoma, one endometrial stromal cell sarcoma, one undifferentiated) were treated with gemcitabine 10 mg/m(2)/min/week over 100 min given for 3 weeks out of 4. The median age was 52 years (range 27-77), male/female ratio was 3/11, and the median WHO performance status was 0 (range 0-1). The median number of previous medical treatments for advanced disease was 1 (range 1-2). RESULTS: A median number of three cycles (range 1-10 cycles) and a total of 151 weekly administrations (median 9, range 3-27) of gemcitabine were administered. Treatment was well tolerated and the main causes of dose-reduction or omission/delay were hematological and liver toxicities. One patient (7%; 95% confidence interval: 0.2-33.9%) with a metastatic uterine leiomyosarcoma obtained a partial response that lasted for 6.5 months. Three patients (two leiomyosarcoma and one schwannoma) (21%) obtained a stabilization of disease. The median time to progression was 3.1 months (range 1.0-9.5). The median overall survival was 11.8 months (range 1.0-54.5+). CONCLUSIONS: Gemcitabine infused at the fixed dose of 10 mg/m(2)/min over 100 min shows a good tolerability but an overall modest activity in unselected STSs histotypes. Nevertheless, an interesting tumor growth control rate was observed in specific histological variants (i.e., leiomyosarcoma), thus confirming data from recent controlled clinical trials.

Does granulocyte colony-stimulating factor worsen anemia in early breast cancer patients treated with epirubicin and cyclophosphamide?

PURPOSE: We report on the effects of granulocyte colony-stimulating factor (G-CSF) on hemoglobin (Hb) value in early breast cancer patients receiving high-dose epirubicin and cyclophosphamide (EC) adjuvant treatment. METHODS: Five hundred and six stage I or stage II female breast cancer patients were treated with E 120 mg/m2 and C 600 mg/m2 with or without G-CSF and randomly assigned to receive in a factorial 2 x 2 design: EC; EC + lonidamine; EC + G-CSF; EC + lonidamine + G-CSF. Five consecutive G-CSF schedules tested 100 randomly assigned patients each: (1) 480 microg subcutaneously on days 8 to 14; (2) 480 microg on days 8, 10, 12, 14; (3) 300 microg on days 8 to 14; (4) 300 microg on days 8, 10, 12, and 14; and (5) 300 microg on days 8 and 12. The mean Hb level of 246 patients receiving EC plus G-CSF was compared with that of 240 patients receiving EC alone. The data presented are derived from an exploratory hypothesis-generating analysis. RESULTS: The EC dose intensity did not statistically differ between the G-CSF and the control arm. From the third cycle onward, the mean Hb value resulted significantly lower in G-CSF arm compared with control at each time point of each cycle (P < .0001). No statistically significant difference in the mean Hb level was observed between schedule 5 and control. Of interest, from the second course onward, the mean Hb level tended to be lower in patients receiving seven or four G-CSF injections compared with those patients who received only two injections. CONCLUSION: Our data suggest that a G-CSF dose-related effect may play a role in worsening anemia in patients receiving adjuvant EC.

cDNA-array profiling of melanomas and paired melanocyte cultures.

Three paired (from the same donor) sets of melanoma cells and normal melanocytes, established as early-passage cultures from metastatic lesions and the uninvolved skin of three patients, were comparatively cDNA profiled by macroarrays (approximately 1,200 genes) and reverse transcription (RT)-PCR. While 145 gene products were significantly (at least twofold) upregulated or downregulated in at least 1 pair, and 23 were in at least 2 pairs, only 3 (the signal transducer and activator of transcription STAT2, collagen type VI, and CD9) were concordantly modulated (downregulation) in all 3 pairs. Array results were validated by RT-PCR on a small panel of surgically removed nevocellular nevi and metastatic melanoma lesions, and by immunohistochemistry on a large panel of benign and malignant lesions of the nevomelanocytic lineage. The three gene products were downregulated at different stages of melanoma progression. STAT2 was detectable in nevi (5/5) and most primary melanomas (11/12), but was lost in 10/15 metastatic lesions. Collagen type VI was expressed in nevi (5/5) and primary melanomas below a Breslow thickness of 1 mm (3/3), but was lost in 24/24 primary melanomas above this threshold, and in metastatic melanomas (10/10). The tetraspanin CD9 molecule was expressed in 18/18 nevi, but was lost in 20/28 primary melanomas (including thin lesions), and in 24/52 metastatic lesions. These data provide the proof of principle that cDNA profiling of paired melanocyte/melanoma cultures sorts out novel, early signatures of melanocyte transformation that could contribute to the clinical management of patients at high risk of metastatic disease.