Molecular analysis of melanoma-induced sentinel lymph node immune dysfunction

Introduction  

Sentinel lymph nodes (SLNs) of melanoma patients show evidence of tumor-induced immune dysfunction. Our previous works have shown that IL-10 and IFNγ co-regulate indoleamine-2,3-dioxygenase (IDO)-expressing immunosuppressive dendritic cells (DCs) in melanoma SLNs. The goal of this study is to examine the relationship between melanoma SLN tumor burden and the degree of SLN immune dysfunction as a model to study tumor-induced immune dysfunction. We hypothesize that SLN tumor burden correlates with the degree of SLN immune dysfunction.     

Evaluation of Tumor Cell Proliferation by Ki-67 Expression and Mitotic Count in Lymph Node Metastases from Breast Cancer

Few studies have addressed the risk of recurrence by assessing proliferation markers in lymph node metastasis from breast cancer. Here, we aimed to examine Ki-67 expression and mitotic count in lymph nodes in comparison with primary tumors. A cohort of node positive breast cancer (n = 168) was studied as a part of the prospective Norwegian Breast Cancer Screening Program (1996–2009). The percentage of Ki-67 positivity was counted per 500 tumor cells in hot-spot areas (x630). Mitotic count was conducted in the most cellular and mitotic active areas in 10 high power fields (x400). Our results showed that Ki-67 and mitotic count were significantly correlated between primary tumor and lymph nodes (Spearman`s correlation 0. 56 and 0.46, respectively) and were associated with most of the histologic features of the primary tumor. Univariate survival analysis (log-rank test) showed that high Ki-67 and mitotic count in the primary tumor and lymph node metastasis significantly predicted risk of recurrence. In multivariate analysis, mitotic count in the lymph node metastasis was an independent predictor of tumor recurrence. In conclusion, proliferation markers in lymph node metastases significantly predicted disease free survival in node positive breast cancer.     

Histopathologic variables differentially affect melanoma survival by age at diagnosis

We evaluated clinical, phenotypic, behavioral, and histopathologic variables in relationship to melanoma‐specific survival by age at diagnosis among 650 population‐based melanoma patients in Connecticut, with 20 years of follow‐up. Only one variable, skin awareness, was significantly associated with melanoma mortality in both groups. The variables that differed between the age‐groups were anatomic site, Breslow thickness, histologic subtype, mitoses, tumor‐infiltrating lymphocytes (TILs), and solar elastosis. Head and neck melanoma, Breslow thickness, nodular melanoma, and solar elastosis were all significantly more likely to be associated with mortality among the older subjects; among the younger subjects, the presence of mitoses was associated with an increased probability of dying and TILs were associated with a reduced risk of mortality.     

Survival According to BRAF-V600 Tumor Mutations – An Analysis of 437 Patients with Primary Melanoma

The prognostic impact of BRAF-V600 tumor mutations in stage I/II melanoma patients has not yet been analyzed in detail. We investigated primary tumors of 437 patients diagnosed between 1989 and 2006 by Sanger sequencing. Mutations were detected in 38.7% of patients and were associated with age, histological subtype as well as mitotic rate. The mutational rate was 36.7% in patients with disease-free course and 51.7% in those with subsequent distant metastasis (p = 0.031). No difference in overall survival (p = 0.119) but a trend for worse distant-metastasis-free survival (p = 0.061) was observed in BRAF mutant compared to BRAF wild-type patients. Independent prognostic factors for overall survival were tumor thickness, mitotic rate and ulceration. An interesting significant prognostic impact was observed in patients with tumor thickness of 1 mm or less, with the mutation present in 6 of 7 patients dying from melanoma. In conclusion, no significant survival differences were found according to BRAF-V600 tumor mutations in patients with primary melanoma but an increasing impact of the mutational status was observed in the subgroup of patients with tumor thickness of 1 mm or less. A potential role of the mutational status as a prognostic factor especially in this subgroup needs to be investigated in larger studies.     

Discoidin domain receptors: A promising target in melanoma

The discoidin domain receptor 1 (DDR1) is a member of the receptor tyrosine kinase family that signals in response to collagen and that has been implicated in cancer progression. In the present study, we investigated the expression and role of DDR1 in human melanoma progression. Immunohistochemical staining of human melanoma specimens (n = 52) shows high DDR1 expression in melanoma lesions that correlates with poor prognosis. DDR1 expression was associated with the clinical characteristics of Clark level and ulceration and with BRAF mutations. Downregulation of DDR1 by small interfering RNA (siRNA) in vitro inhibited melanoma cells malignant properties, migration, invasion, and survival in several human melanoma cell lines. A DDR tyrosine kinase inhibitor (DDR1‐IN‐1) significantly inhibited melanoma cell proliferation in vitro, and ex vivo and in tumor xenografts, underlining the promising potential of DDR1 inhibition in melanoma.     

Focal adhesion kinase as an immunotherapeutic target

Background  Focal adhesion kinase (FAK) is a ubiquitously expressed non-receptor tyrosine kinase involved in cancer progression and metastasis that is found overexpressed in a large number of tumors such as breast, colon, prostate, melanoma, head and neck, lung and ovary. Thus, FAK could be an attractive tumor associated antigen (TAA) for developing immunotherapy against a broad type of malignancies. In this study, we determined whether predicted T cell epitopes from FAK would be able to induce anti-tumor immune cellular responses. Methods  To validate FAK as a TAA recognized by CD4 helper T lymphocytes (HTL), we have combined the use of predictive peptide/MHC class II binding algorithms with in vitro vaccination of CD4 T lymphocytes from healthy individuals and melanoma patients. Results  Two synthetic peptides, FAK_143–157 and FAK_{1,000–1,014}, induced HTL responses that directly recognized FAK-expressing tumor cells and autologous dendritic cells pulsed with FAK-expressing tumor cell lysates in an HLA class II-restricted manner. Moreover, since the FAK peptides were recognized by melanoma patient’s CD4 T cells, this is indicative that T cell precursors reactive with FAK already exist in peripheral blood of these patients. Conclusions  Our results provide evidence that FAK functions as a TAA and describe peptide epitopes that may be used for designing T cell-based immunotherapy for FAK-expressing cancers, which could be used in combination with newly developed FAK inhibitors.     

La TEP-TDM au 18FDG a-t-elle un intérêt dans la stadification ganglionnaire des mélanomes malins cutanés cervicofaciaux bénéficiant de la technique du ganglion sentinelle ? À propos de 22 cas

Background and objectivesIn N0 cutaneous head and neck melanoma, sentinel lymph node biopsy (SLNB) is less reliable and accurate than in trunk or extremities melanoma (false negative cases and spotting failure). The aim of our study was to assess the utility of PET-CT ¹⁸FDG in a specific group of N0 patients, combined with SLNB.Patients and methodsTwenty-two patients with N0 cutaneous head and neck melanoma were retrospectively reviewed. All of them had underwent PET-CT and SLNB before surgery. Average follow-up time was 17 months (1–44).ResultsAt least one sentinel lymph node (SLN) was identified in 20 patients. Ten patients (50%) had metastatic SLN. Among these 10 N+ patients, PET-CT was positive for occult nodal metastases for only two patients. During follow-up, two patients had cervical nodal recurrence, whereas SLNB was negative. PET-CT was also negative for these two patients. SLNB and PET-CT sensitivity were respectively 83 and 18%. PET-CT specificity was 84% (regarding neck sides).ConclusionIn this specific population with N0 cutaneous head and neck melanoma, PET-CT sensitivity is too low and failed to detect occult nodal metastases in two patients with false negative SLNB. Consequently, PET-CT seems to be not useful for nodal staging N0 cutaneous head and neck melanomas, in which SLNB is the most accurate technique.     

Value and prognostic significance of mitotic rate in a retrospective series of pT1 cutaneous malignant melanoma patients

Introduction: Patients affected by thin melanomas (≤1 mm) generally have a good prognosis; however, some have a recurrence and eventually die of the disease. The seventh edition of the American Joint Committee on Cancer (AJCC) melanoma staging system, introduced mitotic rate (MR) as one of the primary criteria for staging thin melanoma. Materials and methods In this study, we sought to determine the prognostic value of mitotic rate in a retrospective cohort of localized primary cutaneous melanoma patients. Results: In total, 286 cases of pT1 primary malignant melanoma occurring in the period 2003–2008 were evaluated. Mitotic counts were re-assessed on standard sections of cases without mitosis and with at least 1 mitosis at diagnosis; 5-year follow-up and recurrence-free survival were available for all patients. Of the 56 radically treated pT1b melanoma patients, 4 (7.1%) had recurrent disease. These data support the efficacy of the incorporation of mitogenicity into AJCC staging for localized cutaneous melanoma and indicate the difficulties in the accuracy and reproducibility of the mitotic count system.     

DNA methylation profiles in primary cutaneous melanomas are associated with clinically significant pathologic features

DNA methylation studies have elucidated a methylation signature distinguishing primary melanomas from benign nevi and provided new insights about genes that may be important in melanoma development. However, it is unclear whether methylation differences among primary melanomas are related to tumor pathologic features with known clinical significance. We utilized the Illumina GoldenGate Cancer Panel array to investigate the methylation profiles of 47 primary cutaneous melanomas. Arraywide methylation patterns revealed a positive association of methylation with Breslow thickness and mutated BRAF, a negative association with mitotic rate, and a weak association with ulceration. Hierarchical clustering on CpG sites exhibiting the most variable methylation (n = 235) divided the melanoma samples into three clusters, including a highly methylated cluster that was positively associated with Breslow thickness and an intermediately methylated cluster associated with Breslow thickness and mitotic rate. Our findings provide support for the existence of methylation‐defined subsets in melanomas with increased methylation associated with Breslow thickness.     

The Multivariate Prognostic Index and nuclear DNA content are independent prognostic factors in primary breast cancer patients

The predictive value of a previously described Multivariate Prognostic Index (which incorporates weighted values of the mitotic activity index, tumor size, and the axillary lymph node status), and the nuclear DNA content (DNA) was evaluated in 156 patients with primary invasive ductal breast cancer, diagnosed between 1980 and 1983. The results were analysed with respect to the occurrence of distant recurrence and survival of the patients after at least 3 yr of follow-up (range 36-73 months; median 44 months). Known prognostic factors such as lymph node status, tumor size, and the mitotic activity index correlated independently with distant recurrence. Furthermore, in respect to survival, the investigated prognostic factors (except DNA content) were significantly correlated. The results indicate that the predictive value of the Multivariate Prognostic Index (MPI) is stronger (P less than 0.001) than of the nuclear DNA content (P less than 0.005) with respect to distant recurrence. In a Cox multivariate regression analysis DNA ploidy turned out to be an independent prognostic factor once the MPI was selected. Furthermore, in Cox's analysis, DNA ploidy was the fourth selected variable after lymph node status, mitotic activity index, and tumor size in individual parameter analysis. The results of this study indicate that, with respect to breast cancer screening programs, it seems worthwhile to integrate morphometric features, the MPI, and DNA ploidy in a new prognostic model.     

Common gene variants in RAD51, XRCC2 and XPD are not associated with clinical outcome in soft-tissue sarcoma patients

BackgroundDNA repair mechanisms play a major role in cancer risk and progression. Germline variants in DNA repair genes may result in altered gene function and/or activity, thereby causing inter-individual differences in a patient's tumor recurrence capacity. In genes of the DNA repair pathway the gene variants RAD51 rs1801320 G > C, XRCC2 rs3218536 G > A and XPD rs13181 A > C have been previously related to genetic predisposition and prognosis of various cancer entities. In this study we investigated the association between these polymorphisms and time to recurrence (TTR) and overall survival (OS) in soft-tissue sarcoma (STS) patients after curative surgery.MethodsTwo hundred sixty STS patients were included in this retrospective study. Germline DNA was genotyped by 5′-exonuclease (TaqMan) technology. Kaplan Meier curves and multivariate Cox proportional models were calculated for TTR and OS.ResultsA statistically significant association was observed between tumor grade and adjuvant radiotherapy and TTR and between tumor grade and OS. No association was found between RAD51 rs1801320 G > C, XRCC2 rs3218536 G > A and XPD rs13181 A > C and TTR and OS in univariate and multivariate analysis.ConclusionOur results underline a prognostic effect of tumor grade and adjuvant radiotherapy in STS patients but indicate no association between RAD51 rs1801320 G > C, XRCC2 rs3218536 G > A and XPD rs13181 A > C and clinical outcome in STS patients after curative surgery.     

Sentinel lymph node biopsy for melanoma: experience with 234 consecutive procedures

Sentinel lymph node biopsy is increasingly used to identify occult metastases in regional lymph nodes of patients with melanoma. Selection of patients for sentinel lymph node biopsy and subsequent lymphadenectomy is an area of debate. The purpose of this study was to describe a large clinical series of these biopsies for cutaneous melanoma and to identify patients most likely to gain useful clinical information from sentinel lymph node biopsy. The Indiana University Melanoma Program computerized database was queried to identify all patients who underwent this procedure for clinically localized cutaneous melanoma. It was performed using preoperative technetium Tc 99m lymphoscintigraphy and isosulfan blue dye. Pertinent demographic, surgical, and histopathologic data were recorded. Univariate and multivariate logistic regression and classification table analyses were performed to identify clinical variables associated with sentinel node and nonsentinel node positivity. In total, 234 biopsy procedures were performed to stage 291 nonpalpable regional lymph node basins. Mean Breslow's thickness was 2.30 mm (2.08 mm for negative sentinel lymph node biopsy, 3.18 mm for positive). The mean number of sentinel nodes removed was 2.17 nodes per basin (range, 1 to 8). Forty-seven of 234 melanomas (20.1 percent) and 50 of 291 basins (17.2 percent) had a positive biopsy. Positivity correlated with AJCC tumor stage: T1, 3.6 percent; T2, 8.1 percent; T3, 27.4 percent; T4, 44 percent. By univariate logistic regression, Breslow's thickness (p = 0.003, continuous variable), ulceration (p = 0.003), mitotic index > or = 6 mitoses per high power field (p = 0.008), and Clark's level (p = 0.04) were significantly associated with sentinel lymph node biopsy result. By multivariate analysis, only Breslow's thickness (p = 0.02), tumor ulceration (p = 0.02), and mitotic index (p = 0.02) were significant predictors of biopsy positivity. Classification table analysis showed the Breslow cutpoint of 1.2 mm to be the most efficient cutpoint for sentinel lymph node biopsy result (p = 0.0004). Completion lymphadenectomy was performed in 46 sentinel node-positive patients; 12 (26.1 percent) had at least one additional positive nonsentinel node. Nonsentinel node positivity was marginally associated with the presence of multiple positive sentinel nodes (p = 0.07). At mean follow-up of 13.8 months, four of 241 sentinel node-negative basins demonstrated same-basin recurrence (1.7 percent). Sentinel lymph node biopsy is highly reliable in experienced hands but is a low-yield procedure in most thin melanomas. Patients with melanomas thicker than 1.2 mm or with ulcerated or high mitotic index lesions are most likely to have occult lymph node metastases by sentinel lymph node biopsy. Completion therapeutic lymphadenectomy is recommended after positive biopsy because it is difficult to predict the presence of positive nonsentinel nodes.     

The prognostic value of circulating tumor DNA in patients with melanoma: A systematic review and meta-analysis

BackgroundCirculating tumor DNA (ctDNA) has been investigated as a potential prognostic biomarker to evaluate the therapeutic efficacy and disease progression in melanoma patients, yet results remain inconclusive. The purpose of this study was to illustrate the prognostic value of ctDNA in melanoma.ObjectivesTo describe the clinical prognostic value of ctDNA for melanoma patients.MethodsSearched for eligible articles from Pubmed, Web of Science and Embase. Pooled hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated to evaluate the association between ctDNA at baseline or during treatment and overall survival (OS) and progression-free survival (PFS).ResultsA total of 9 articles were obtained, involving 617 melanoma patients. The pooled HRs revealed that compared with baseline undetectable ctDNA patients, detectable ctDNA was highly correlated with poor OS (HR 2.91, 95% CI: 2.22–3.82; p < 0.001) and PFS (HR 2.75, 95% CI: 1.98–3.83; p < 0.001). A meta-analysis of these adjusted HRs was performed and confirmed that ctDNA collected at baseline was associated with poorer OS/PFS (OS: HR 3.00, 95% CI 2.19–4.11, p < 0.001/PFS: HR 2.68, 95% CI 1.77–4.06, p < 0.001). During treatment, a significant association was shown between ctDNA and poorer OS/PFS (OS: HR 6.26, 95% CI 2.48–15.80, p < 0.001; PFS: HR 4.93, 95% CI 2.36–10.33, p < 0.001).ConclusionInvestigation and application of ctDNA will improve "liquid biopsy" and play a role in early prediction, monitoring disease progression and precise adjusting treatment strategies in melanoma patients.     

Can Proliferation Biomarkers Reliably Predict Recurrence in World Health Organization 2003 Defined Endometrial Stromal Sarcoma,Low Grade?

An estimated 1500–3000 invasive Endometrial Stromal Sarcomas (ESS) cases annually occur worldwide. Before 2003, ESS was divided as low and high grade ESS based on mitotic activity. In 2003 the WHO changed the names, excluded mitoses and made nuclear atypia and necrosis the essential diagnostic criteria to distinguish ESS, Low Grade (ESS-LG, recurrence-free survival >90%) and Undifferentiated Endometrial Sarcoma (UES, poor prognosis). We have evaluated in WHO2003 defined ESS-LG whether proliferation biomarkers predict recurrence. Using survival analysis, the prognostic value of classical mitosis counts (Mitotic Activity Index, MAI) in haematoxyllin-eosin (H&E) sections, and immunohistochemical proliferation biomarkers (Ki-67 and PhosphoHistone-3 (PPH3)) were examined in 24 invasive endometrial stromal sarcomas. Three of 24 (12.5%) ESS-LG recurred. The MAI, PPH3 and Ki-67 were all prognostic (P = 0.001, 0.002 and 0.03). MAI values were >3 in the recurrent cases, but never exceeded 10 (the classical threshold for low and high grade). Non-recurrent cases had 0≤MAI≤3. PPH3 and Ki67 counts can be easier to perform than MAI and therefore helpful in the diagnosis of ESS, Low Grade. In conclusion, in this small study of WHO2003 defined ESS-LG, high levels of proliferation as measured by MAI, PPH3 and Ki-67 are predictive of recurrence. Larger studies are required to confirm these results.     

Minimum acceptable sensitivity of intraoperative gamma probes used for sentinel lymph node detection in melanoma patients

The aim of this study was to determine the suspension level for the sensitivity of an intraoperative scintillation gamma probe in the detection of the sentinel lymph node (SLN) in melanoma patients.Thirty-eight consecutive patients with melanoma were enrolled in the study during a 12-month period and underwent lymphatic scintigraphy after the peritumoral intradermal administration of about 14 MBq of ^99mTc-nanocolloids. The SLNs were successfully removed during the surgical intervention about 4 h later.To identify and localize the SLN, a scintillation NaI(Tl) collimated probe was used. Predictably, the probe sensitivity decreased as the photopeak energy window was progressively narrowed, from 6.9 ± 0.7 counts per second (cps)/kBq (designated as the ‘optimum,’ or ‘OPT,’ sensitivity) to 2.5 ± 0.3 cps/kBq (LOW sensitivity) and to 1.4 ± 0.2 cps/kBq (VLOW sensitivity).Maximum lymph node count rates (cps) were determined for the foregoing energy windows prior to skin incision (PRE_OPT, PRE_LOW, PRE_VLOW, respectively) and in vivo after incision (INV_OPT, INV_LOW, INV_VLOW).Forty-three SLNs were removed with a mean source-to-detector distance of 46 ± 24 mm (min 12 mm, max 92 mm). Four SLNs could not have been detected using PRE_LOW. This figure would have decreased to 34, with nine undetectable lymph nodes, with PRE_VLOW. One SLN could not have been identified using INV_LOW and four could not have be identified using INV_VLOW.In the clinical scenario of SLN detection in melanoma patients, a system sensitivity of 2.5 cps/kBq represents a suspension level, that is, a level under which the equipment must be suspended from clinical use and the poor performance must be investigated.     

Microsomal PGE2 synthase‐1 regulates melanoma cell survival and associates with melanoma disease progression

COX‐2 and its product PGE2 enhance carcinogenesis and tumor progression, which has been previously reported in melanoma. As most COX inhibitors cause much toxicity, the downstream microsomal PGE2 synthase‐1 (mPGES1) is a consideration for targeting. Human melanoma TMAs were employed for testing mPGES1 protein staining intensity and percentage levels, and both increased with clinical stage; employing a different Stage III TMA, mPGES1 intensity (not percentage) associated with reduced patient survival. Our results further show that iNOS was also highly expressed in melanoma tissues with high mPGES1 levels, and iNOS‐mediated NO promoted mPGES1 expression and PGE2 production. An mPGES1‐specific inhibitor (CAY10526) as well as siRNA attenuated cell survival and increased apoptosis. CAY10526 significantly suppressed tumor growth and increased apoptosis in melanoma xenografts. Our findings support the value of a prognostic and predictive role for mPGES1, and suggest targeting this molecule in the PGE2 pathway as another avenue toward improving melanoma therapy.     

Optimization of the culture methods for nursing Chinese black sleeper (Bostrychus sinensis Lacépède, 1801) fry using an orthogonal array design

Chinese black sleeper (Bostrychus sinensis) is a burrow‐dwelling fish found in intertidal mudflats. As a commercially important fish in southern China, the nursing of B. sinensis fry is a critical step for its farming. The growth and survival of B. sinensis fry are closely related to the stocking density, shelter and diet. In this study, 1575 healthy fry were used to investigate the effects of three factors, namely: diet, stocking density, and shelter on the survival rate (SR), specific growth rate (SGR) and condition factor (CF). The optimal combinations of three levels of each of these three factors (A1‐3, B1‐3 and C1‐3) were also determined, using an orthogonal array design of OA₉ (3⁴). After 50 days of nursing, the results showed that: (i) the best combinations for the three factors at their optimal levels were A1B1C2 or A1B1C3; (ii) factor A was the most important factor and had significant effects (P < 0.05) on the SR and SGR of the fry, while the other factors had no significant effects (P > 0.05); (iii) the SGR, SR and CF of fry fed with commercial formulated feed (CFF) were significantly higher (P < 0.05) compared to those of fry fed with minced trash fish (MTF); and (iv) the fry fed with MTF mixed with antimicrobial peptides (MTF+AMP) showed higher SGR, SR and CF than those fed with MTF alone, which indicated that the antimicrobial peptides had positive effects on the growth and survival of the fry.     

Intérêt pronostique et prédictif de la TEP-TDM au 18F-FDG au bilan initial des mélanomes de stade IIIB-C-D et IV avant traitement par anti-PD-1

PurposeThe advent of immunotherapy by checkpoint inhibitor has profoundly changed the prognosis of patients with metastatic melanoma. The objective of our study was to evaluate the prognostic and predictive performance of 18F-FDG PET/CT of the initial extension assessment of stage IIIB-C-D and IV melanomas.MethodsWe retrospectively included 57 patients who had 18F-FDG PET/CT prior to the introduction of anti-PD-1 immunotherapy. The parameters extracted were SUVmax, SUV peak, MTV and TLG of the lesion with highest uptake (MTV LM, TLG LM), as well as MTV total and TLG total, obtained by adaptive segmentation. The18F-FDG PET/CT were dichotomized using the optimal threshold measured according to the area under the curve in the ROC (Receiver Operation Characteristic) curves. These parameters were evaluated using a Cox model. Overall survival and progression-free survival analyses were performed using the Kaplan Meier model.ResultsThe median follow-up was 25.4 months, 38 patients had progressed or recurred, and 20 patients had died. TLG LM > 132.59 (P = 0.0011), MTV total > 12 cm³ (P = 0.0139), and TLG > 94.17 (P = 0.0084) were significantly associated with a shorter progression-free survival. TLG LM > 145.92 (P = 0.0062), MTV total > 10.16 cm³ (P = 0.0051), and a metastatic spread > 2 organs (P = 0.0001) were associated with a shorter overall survival.ConclusionWe confirm the potential prognostic interest of PET-TDM at 18FDG before immunotherapy of stage IIIB-C-D and IV melanomas on progression-free survival and overall survival. The combination of these metabolic markers reflecting tumor burden with clinical and biological prognostic factors could allow early identification of patients at high risk of anti-PD-1 failure.