Multistep tumor genetic evolution and changes in immunogenicity trigger immune-mediated disease eradication in stage IV melanoma: lessons from a single case.

Durable remissions are observed in 10%-20% of treated patients with advanced metastatic melanoma but the factors associated with long-term complete clinical responses are largely unknown. Here, we report the molecular characteristics of tumor evolution during disease progression along a 9-year clinical course in a patient with advanced disseminated melanoma who received different treatments, including trametinib, ipilimumab, radiation, vemurafenib, surgical tumor debulking and a second ipilimumab course, ultimately achieving complete long-term disease remission.Longitudinal analyses of therapies-resistant metastatic tumors revealed the effects of different treatments on tumor's microenvironment and immunogenicity, ultimately creating a milieu favorable to immunotherapy response. Monitoring of the temporal dynamics of T cells by analysis of the T cell receptor (TCR) repertoire in the tumor and peripheral blood during disease evolution indicated that T-cell clones with common TCR rearrangements, present at low levels at baseline, were maintained and expanded after immunotherapy, and that TCR diversity increased. Analysis of genetic, molecular, and cellular components of the tumor depicted a multistep process in which treatment with kinase inhibitors strongly conditioned the immune microenvironment creating an inflamed milieu converting cold into hot tumors, while ipilimumab impacted and increased the TCR repertoire, a requirement for tumor rejection.Since the optimal sequencing of treatment with antibodies targeting immune checkpoints and kinase inhibitors for advanced melanoma is still clinically debated, this case indicates that immunotherapy success is possible even after progression on targeted therapy.

Impact of Cold Ischemia on the Stability of 1H-MRS-Detected Metabolic Profiles of Ovarian Cancer Specimens.

Proton magnetic resonance spectroscopy (1H-MRS) of surgically collected tumor specimens may contribute to investigating cancer metabolism and the significance of the "total choline" (tCho) peak (3.2 ppm) as malignancy and therapy response biomarker. To ensure preservation of intrinsic metabolomic information, standardized handling procedures are needed. The effects of time to freeze (cold ischemia) were evaluated in (a) surgical epithelial ovarian cancer (EOC) specimens using high-resolution (HR) 1H-MRS (9.4 T) of aqueous extracts and (b) preclinical EOC samples (xenografts in SCID mice) investigated by in vivo MRI-guided 1H-MRS (4.7 T) and by HR-1H-MRS (9.4 T) of tumor extracts or intact fragments (using magic-angle-spinning (MAS) technology). No significant changes were found in the levels of 27 of 29 MRS-detected metabolites (including the tCho profile) in clinical specimens up to 2 h cold ischemia, besides an increase in lysine and a decrease in glutathione. EOC xenografts showed a 2-fold increase in free choline within 2 h cold ischemia, without further significant changes for any MRS-detected metabolite (including phosphocholine and tCho) up to 6 h. At shorter times (≤1 h), HR-MAS analyses showed unaltered tCho components, along with significant changes in lactate, glutamate, and glutamine. Our results support the view that a time to freeze of 1 h represents a safe threshold to ensure the maintenance of a reliable tCho profile in EOC specimens.

The prognostic role of mitotic rate in cutaneous malignant melanoma: Evidence from a multicenter study on behalf of the Italian Melanoma Intergroup.

BACKGROUND: This study aimed to improve the understanding of the prognostic value of tumor mitotic rate (TMR) in cutaneous melanoma and assessed its significance as a predictor for overall, melanoma-specific, and recurrence-free survival. PATIENTS AND METHODS: This is a retrospective multicenter Italian cohort study of 13,016 patients diagnosed with and treated for invasive primary melanoma between 2005 and 2020 with median follow-up of 5.5 years. The survival probability was assessed by Kaplan-Meier method, hazard ratios (HRs), and corresponding 95% confidence interval (CI) of all-cause mortality and recurrence/death by multivariable Cox proportional hazards models. RESULTS: Higher dermal mitoses number was associated with decreased overall survival. Among patients with TMR 0/mm2 , 1/mm2 , 2/mm2 -3/mm2 , 4/mm2 -10/mm2 , and >10/mm2 , 5-year overall survival (OS) was 97.3%, 93.6%, 88.3%, 73.0%, and 60.9%, respectively. In multivariate analyses, compared to TMR of 0/mm2 , HRs for all-cause mortality were 1.35 (95% CI, 1.08-1.68), 1.70 (95% CI, 1.40-2.07), 2.04 (95% CI, 1.67-2.49), and 2.39 (95% CI, 1.90-3.00) for 1 mitoses/mm2 , 2 mitoses/mm2 -3 mitoses/mm2 , 4 mitoses/mm2 -10 mitoses/mm2 , and >10 mitoses/mm2 , respectively. A similar increase in risks was observed in melanoma-specific survival (MSS) and recurrence-free survival (RFS). The HRs for MSS and RFS for the highest compared to the lowest TMR category were 3.01 (95% CI, 2.20-4.11) and 2.26 (95% CI, 1.88-2.73), respectively. Sentinel lymph-node biopsy positivity was significantly associated with TMR increase even with adjustment for several potential confounders. CONCLUSIONS: A clear association was demonstrated between an increasing TMR and decreased OS, MSS, and RFS, suggesting a reconsideration of TMR prognostic role for future inclusion in the melanoma staging system. PLAIN LANGUAGE SUMMARY: The 8th American Joint Committee on Cancer for melanoma staging removed tumor mitotic rate (TMR) from the staging criteria for T1 melanomas, giving way to ulceration and tumor thickness as stronger prognostic predictors. However, it is still recommended that TMR should be assessed and recorded in all primary invasive melanomas. In a large retrospective multicenter study on primary invasive melanomas, we investigated the prognostic value of TMR to assess its significance as survival predictor. Our results showed a clear association between increasing TMR and decreased patients' survival, suggesting that TMR should be considered for inclusion in the melanoma staging system.

Potential risk factors, clinicopathological features and determinants of survival for multiple primary melanoma patients compared to single primary melanoma: a large single-center Italian study.

Melanoma patients have a high risk of developing subsequent primary melanomas, a condition known as multiple primary melanoma (MPM). We aimed to compare risk factors of patients with MPM and single primary melanoma (SPM). Primary MPM and SPM consecutively treated at the National Cancer Institute in Milan, Italy, from 1978 to 2021 were retrospectively investigated. Demographic and clinicopathological characteristics were analyzed. Multivariate hazard ratios and mortality were estimated using Cox proportional hazards regression models. Overall, 9122 patients with SPM and 944 with MPM were included. A total of 1437 and 85 deaths occurred in SPM and MPM group, respectively. Of these, 1315 (14.4%) within SPM patients and 60 (6.4%) in MPM group were melanoma-specific deaths (MSDs). Males had a higher risk for MPM (hazard ratio = 1.29), while age was not associated with MPM (hazard ratio = 0.98). The risk of MPM decreased by about 50% for Breslow thickness >1 mm, and by about 45 and 75% in presence of mitoses and ulceration, respectively. The multivariate hazard ratio of death for MPM compared to SPM patients was 0.85 (95% confidence interval, CI: 0.67-1.06), while considering MSD the corresponding hazard ratio was 0.93 (95% CI: 0.71-1.22). Melanoma patients should receive regular follow-up with complete skin examination to detect early subsequent primary melanoma. Patients with more advanced primary have decreased risk of MPM, while males have higher risk. Our study reported no significant difference in mortality between SPM and MPM, but the issue is still open for discussion and further studies.

Association of Excision Margin Size With Local Recurrence and Survival in Patients With T1a Melanoma at Critical Structures.

Importance: Melanoma guidelines recommend surgical excision with 10-mm margins for T1 melanoma. However, this procedure may be problematic at sites close to critical structures such as the scalp, face, external genitalia, acral, periumbilical, and perineal areas. Objective: To compare outcomes of wide (10-mm margins) vs narrow (5-mm margins) excision in patients with T1a melanoma near critical structures. Design, Setting, and Participants: This cohort study was a retrospective comparison of 1341 consecutive patients aged 18 years or older from the National Cancer Institute of Milan, Italy, diagnosed between 2001 and 2020 with T1a cutaneous melanoma close to critical structures who accepted wide excision vs narrow excision. Exposures: Local recurrence and melanoma-specific mortality (MSM) rates with 5-mm vs 10-mm excision margins. Main Outcomes and Measures: The primary aim of the study was to ascertain whether a narrower (5-mm) vs wider (10-mm) excision margin was associated with local recurrence and MSM. The secondary aim was to compare the need for reconstructive surgery in the groups defined by excision margin width. Between April 28 and August 7, 2022, associations were assessed by weighted Cox and Fine-Gray univariable and multivariable models. Results: A total of 1179 patients met the inclusion criteria (median [IQR] age, 50.0 [39.5-63.0] years; female, 610 [51.7%]; male, 569 [49.3%]). Six hundred twenty-six patients (53.1%) received a wide excision (434 [69.3%] with linear repair and 192 [30.7%] with flap or graft reconstruction) and 553 (46.9%) received a narrow excision (491 [88.8%] with linear repair and 62 [11.2%] with flap or graft reconstruction). The weighted 10-year MSM was 1.8% (95% CI, 0.8%-4.2%) in the wide group and 4.2% (95% CI, 2.2%-7.9%) in the narrow group; the weighted 10-year local recurrence rate was 5.7% (95% CI, 3.9%-8.3%) in the wide group and 6.7% (95% CI, 4.7%-9.5%) in the narrow group. Breslow thickness greater than 0.4 mm (subdistribution hazard ratio [sHR] for 0.6 vs 0.4 mm, 2.42; 95% CI, 1.59-3.68; P < .001) and mitotic rate greater than 1/mm2 (sHR for a single increment, 3.35; 95% CI, 2.59-4.32; P < .001) were associated with worse MSM. Multivariable analysis showed that acral lentiginous melanoma, lentigo maligna melanoma, and increasing Breslow thickness were associated with a higher incidence of local recurrence. Conclusions and Relevance: The study's findings suggest that local excision with 5-mm margins for T1a melanoma may not be associated with an increased risk of local recurrence. Breslow thickness greater than 0.4 mm, mitotic rate greater than 1/mm2, and acral lentiginous melanoma and lentigo maligna melanoma subtypes were associated with a higher risk of recurrence. These findings may be useful for future melanoma treatment guidelines.

Similar local recurrence and survival in patients with T1 radial growth phase melanoma on head and neck treated with 5 or 10 mm margins: A retrospective study.

BACKGROUND: Melanoma guidelines recommend surgical excision with 10 mm margins for T1 melanomas (invasive melanomas with Breslow thickness ≤1 mm), including those in radial growth phase, which are without metastatic potential; however, such margins may be problematic on head-and-neck. OBJECTIVE: We compared outcomes of wide (10 mm margins) versus narrow (5 mm margins) excisions in patients with radial growth phase T1 melanoma on head-and-neck including face. METHODS: We retrospectively examined 610 consecutive patients excised with wide versus narrow margins, from 2001 to 2018, at six European centres. In all cases, radial growth phase, and clear margins with 5 or 10 mm of clearance, were ascertained histologically. Multivariable models investigated associations of margins and other factors with overall survival and local recurrence. RESULTS: Three hundred and sixteen (51.8%) patients received wide excision, 219 (69.3%) with primary wound closure, 97 (30.7%) with reconstruction; 294 (48.2%) patients received narrow excision, 264 (89.8%) with primary wound closure, 30 (10.2%) with reconstruction (p < 0.001). Median follow-ups were 88 months (wide) and 187 months (narrow) (inter-quartile ranges 43-133 and 79-206, respectively). Ten-year overall survival (95% confidence interval) was 96.7% (94.2%-99.3%) in wide and 98.2% (96.4%-100%) in narrow patients. Ten-year local recurrence incidence was 6.4% (4.1%-10.1%) in wide and 7.8% (5.3%-11.6%) in narrow groups. Lentigo maligna melanoma subtype appeared associated with increased risk of local recurrence in narrow versus wide patients (15.0% vs. 7.5%; p = 0.190). CONCLUSIONS: Narrower excision margins for T1 radial growth phase melanoma are not associated with worse overall survival (hazard ratio 0.97, p = 0.996) or increased local recurrence (subdistribution hazard ratio: 0.87; p = 0.751) compared to wider margins, and may be safely applied to such lesions, although caution may be required in the presence of lentigo maligna melanoma.

Genetic Layout of Melanoma Lesions Is Associated with BRAF/MEK-Targeted Therapy Resistance and Transcriptional Profiles.

The genetic landscape of melanoma resistance to targeted therapy with small molecules inhibiting BRAF and MEK kinases is still largely undefined. In this study, we portrayed in detail the somatic alterations of resistant melanoma and explored the associated biological processes and their integration with transcriptional profiles. By targeted next-generation sequencing and whole-exome sequencing analyses, a list of 101 genes showing imbalance in metastatic tumors from patients with a complete/durable response or disease progression during therapy with vemurafenib or with dabrafenib and trametinib was defined. Classification of altered genes in functional categories indicated that the mutational pattern of both resistant tumors and melanoma cell lines was enriched in gene families involved in oncogenic signaling pathways and in DNA repair. Integration of genomic and transcriptomic features showed that the enrichment of mutations in gene sets associated with anabolic processes, chromatin alterations, and IFN-α response determined a significant positive modulation of the same gene signatures at the transcriptional level. In particular, MTORC1 signaling was enriched in tumors from poorly responsive patients and in resistant tumors excised from treated patients. Results indicate that genetic patterns are associated with melanoma resistance to targeted therapy and disclose the underlying key molecular pathways to define drug combinations for improved personalized therapies.

NTRK Gene Fusion Detection in Atypical Spitz Tumors.

Atypical Spitz tumors (AST) deviate from stereotypical Spitz nevi for one or more atypical features and are now regarded as an intermediate category of melanocytic tumors with uncertain malignant potential. Activating NTRK1/NTRK3 fusions elicit oncogenic events in Spitz lesions and are targetable with kinase inhibitors. However, their prevalence among ASTs and the optimal approach for their detection is yet to be determined. A series of 180 ASTs were screened with pan-TRK immunohistochemistry and the presence of NTRK fusions was confirmed using FISH, two different RNA-based NGS panels for solid tumors, and a specific real time RT-PCR panel. Overall, 26 ASTs showed pan-TRK immunostaining. NTRK1 fusions were detected in 15 of these cases showing cytoplasmic immunoreaction, whereas NTRK3 was detected in one case showing nuclear immunoreaction. Molecular tests resulted all positive in only two ASTs (included the NTRK3 translocated), RNA-based NGS and real time RT-PCR were both positive in three cases, and FISH and real time RT-PCR in another two cases. In seven ASTs NTRK1 fusions were detected only by FISH and in two cases only by real time RT-PCR. The frequency of NTRK fusions in ASTs is 9%, with a clear prevalence of NTRK1 compared to NTRK3 alterations. Pan-TRK immunohistochemistry is an excellent screening test. Confirmation of NTRK fusions may require the use of different molecular techniques.

Liquid Biopsy and Radiological Response Predict Outcomes Following Discontinuation of Targeted Therapy in Patients with BRAF Mutated Melanoma.

BACKGROUND: Outcomes of patients with metastatic melanoma discontinuing BRAF-targeted therapy for cumulative toxicity after sustained response are unknown. MATERIALS AND METHODS: This retrospective case series analysis conducted at a single Cancer Center in Italy included patients with BRAF mutated metastatic melanoma treated with a BRAF inhibitor as a single agent or in combination with a MEK inhibitor between June 1, 2011 and January 1, 2020 and interrupted treatment due to cumulative toxicity after achieving complete response (CR) or long-lasting partial response (PR; i.e. >12 months). RESULTS: We included 24 patients with a median treatment duration of 59.4 months (95% confidence interval [CI], 55.4-63.4; range, 12-88). CR and PR were achieved in 71% and 29% of patients, respectively. At a median follow-up after treatment discontinuation of 37.8 months (95% CI, 33.7-41.9), the 12-month progression-free survival after discontinuation (dPFS) rate was 70.8% (95% CI 54.8-91.6) and 24-month dPFS rate was 58.3% (95% CI, 41.6-81.8). Baseline patient and tumor characteristics as well as treatment duration and best response did not significantly impact on dPFS. Patients with CR and negative circulating tumor DNA (ctDNA) at time of discontinuation had a significantly improved dPFS compared with patients with either radiological residual disease or ctDNA positivity (p = .007). No patient in CR with undetectable ctDNA experienced progression. CONCLUSION: The risk of progression is high even in patients with sustained sensitivity to BRAF/MEK inhibitors. Integration of liquid biopsy in clinical trials investigating the optimal management of patients with sustained sensitivity to BRAF/MEK inhibitors is warranted. IMPLICATIONS FOR PRACTICE: Outcomes of patients with metastatic melanoma discontinuing BRAF-targeted therapy for cumulative toxicity are unknown. This study analyzed patients with sustained responses (median treatment duration 59.4 months). Twelve- and 24-month progression-free survival following discontinuation were 70.8% and 58.3%, respectively. Complete response and negative circulating tumor DNA at time of discontinuation are promising prognostic biomarkers in this setting.

Survival in Patients With Sentinel Node-Positive Melanoma With Extranodal Extension.

BACKGROUND: Prognostic parameters in sentinel node (SN)-positive melanoma are important indicators to identify patients at high risk of recurrence who should be candidates for adjuvant therapy. We aimed to evaluate the presence of melanoma cells beyond the SN capsule-extranodal extension (ENE)-as a prognostic factor in patients with positive SNs. METHODS: Data from 1,047 patients with melanoma and positive SNs treated from 2001 to 2020 at the Istituto Nazionale dei Tumori in Milano, Italy, were retrospectively investigated. Kaplan-Meier survival and crude cumulative incidence of recurrence curves were estimated. A multivariable logistic model was used to investigate the association between ENE and selected predictive factors. Cox models estimated the effect of the selected predictors on survival endpoints. RESULTS: Median follow-up was 69 months. The 5-year overall survival rate was 62.5% and 71.7% for patients with positive SNs with and without ENE, respectively. The 5-year disease-free survival rate was 54.0% and 64.0% for patients with positive SNs with and without ENE, respectively. The multivariable logistic model showed that age, size of the main metastatic focus in the SN, and numbers of metastatic non-SNs were associated with ENE (all P<.0001). The multivariable Cox regression models showed the estimated prognostic effects of ENE associated with age, ulceration, size of the main metastatic focus in the SN, and number of metastatic non-SNs (all P<.0001) on disease-free survival and overall survival. CONCLUSIONS: ENE was a significant prognostic factor in patients with positive-SN melanoma. This parameter may be useful in clinical practice as a selection criterion for adjuvant treatment in patients with stage IIIA disease with a tumor burden <1 mm in the SN. We recommend its inclusion as an independent prognostic determinant in future updates of melanoma guidelines.

Immune-related Bell's palsy in melanoma patients treated with immune checkpoint inhibitors.

Immune-checkpoint inhibitors (ICIs) exposed the oncology community to novel immune-related adverse events (irAEs). Here, we report on a retrospective analysis of patients with melanoma who developed an ICI-related, unilateral, acute and peripheral facial nerve paralysis (Bell's palsy).We retrospectively reviewed all the cases of ICI-related Bell's palsy in patients with melanoma treated at our institution from January 2015 to January 2020. A total of five cases of ICI-related Bell's palsy were identified. Median age was 63 years. Median time-to-onset of Bell's palsy from ICIs initiation was 15 weeks. Four patients were treated with prednisone alone, whereas one patient was treated with prednisone plus valaciclovir. All the patients completely recovered from Bell's palsy without neurological sequelae. In melanoma patients treated with ICIs, Bell's palsy is a rare, neurologic irAE with a favorable outcome following administration of oral corticosteroids.

Surgical treatment of melanoma metastases to the small bowel: A single cancer referral center real-life experience.

INTRODUCTION: Treatment of metastatic melanoma has rapidly changed during the last years, and patients often require a multidisciplinary approach to achieve effective results. We aimed to assess the survival benefit achieved through surgical approach to patients with small bowel (SB) metastases from cutaneous melanoma, to emphasize the potential role of surgery in association with novel therapies. METHODS: Ninety consecutive patients with cutaneous melanoma diagnosed as having resectable SB metastases from 1995 to 2015 were retrospectively investigated. RESULTS: Median age at surgery of melanoma metastases was 53.4 years. Among 30 patients who had a curative-intent resection, the 5- and 10-year survival rates were 61% and 54%, respectively, while among 60 patients treated with a palliative surgery the corresponding rates were both 4%. Among 29 patients, for whom the interval time between the occurrence of SB metastases and the previous surgical event on GI tract was ≥36 months, the 5-year overall survival rate was 42%; for 56 patients who had an interval time <36 months the corresponding survival rate was 14%. Within the whole series, an absence of any residual disease after surgery (R0) was a factor affecting better survival, regardless of the evidence of metastases in other organs. CONCLUSION: Our observational data showed that surgical treatment for patients with SB metastases from melanoma might increase survival, but further studies are needed to confirm this finding. In the age of novel available therapies, the increase in survival time given by surgery may offer important chances for patients to benefit from systemic therapies.

The role of sentinel lymph node status performed in melanoma patients with local recurrence or in transit metastasis.

BACKGROUND: Sentinel Node Biopsy (SNB) is routinely performed for primary melanoma, but its role in the treatment of Local Recurrence (LR) and In-Transit metastasis (IT) is controversial. This study aims to assess the role of SNB in melanoma patients who developed first loco-regional recurrence. METHODS: A series of consecutive melanoma patients who received SNB for a first IT or LR at the National Cancer Institute of Milan, Italy, from 2000 to 2015 were selected from a prospective database. Clinicopathological characteristics were analyzed. RESULTS: Seventy-two patients met selection criteria. Forty-three patients (59.7%) received SNB for LR and 29 (40.3%) for IT. The average interval between treatment of primitive melanoma and first recurrence diagnosis was 19 months (interquartile range: 6.9-49.0). SN identification rate was 97.2%. SN positivity was detected in 26 (37.1%) patients. The SN-positive ratein melanoma patients who had LR or IT was significantly higher than reported for primary tumours. Of patients with nodal involvement 17 had LR and 9 IT lesions. Disease Free Survival (DFS) was slightly higher in SN negative patients, in the absence of statistically significant differences. Overall Survival (OS) analysis showed similar values in the two groups. CONCLUSION: Since DFS and OS do not show significant differences between SN negative and positive patients, our data do not give clear indications about performing SNB in case of first LR or IT. However, we suggest submitting patients with LR to this procedure to obtain a more accurate staging and eventually candidate these patients to adjuvant treatment.

Analysis of Sentinel Node Biopsy and Clinicopathologic Features as Prognostic Factors in Patients With Atypical Melanocytic Tumors.

BACKGROUND: Atypical melanocytic tumors (AMTs) include a wide spectrum of melanocytic neoplasms that represent a challenge for clinicians due to the lack of a definitive diagnosis and the related uncertainty about their management. This study analyzed clinicopathologic features and sentinel node status as potential prognostic factors in patients with AMTs. PATIENTS AND METHODS: Clinicopathologic and follow-up data of 238 children, adolescents, and adults with histologically proved AMTs consecutively treated at 12 European centers from 2000 through 2010 were retrieved from prospectively maintained databases. The binary association between all investigated covariates was studied by evaluating the Spearman correlation coefficients, and the association between progression-free survival and all investigated covariates was evaluated using univariable Cox models. The overall survival and progression-free survival curves were established using the Kaplan-Meier method. RESULTS: Median follow-up was 126 months (interquartile range, 104-157 months). All patients received an initial diagnostic biopsy followed by wide (1 cm) excision. Sentinel node biopsy was performed in 139 patients (58.4%), 37 (26.6%) of whom had sentinel node positivity. There were 4 local recurrences, 43 regional relapses, and 8 distant metastases as first events. Six patients (2.5%) died of disease progression. Five patients who were sentinel node-negative and 3 patients who were sentinel node-positive developed distant metastases. Ten-year overall and progression-free survival rates were 97% (95% CI, 94.9%-99.2%) and 82.2% (95% CI, 77.3%-87.3%), respectively. Age, mitotic rate/mm2, mitoses at the base of the lesion, lymphovascular invasion, and 9p21 loss were factors affecting prognosis in the whole series and the sentinel node biopsy subgroup. CONCLUSIONS: Age >20 years, mitotic rate >4/mm2, mitoses at the base of the lesion, lymphovascular invasion, and 9p21 loss proved to be worse prognostic factors in patients with ATMs. Sentinel node status was not a clear prognostic predictor.

miR-146a-5p impairs melanoma resistance to kinase inhibitors by targeting COX2 and regulating NFkB-mediated inflammatory mediators.

BACKGROUND: Targeted therapy with BRAF and MEK inhibitors has improved the survival of patients with BRAF-mutated metastatic melanoma, but most patients relapse upon the onset of drug resistance induced by mechanisms including genetic and epigenetic events. Among the epigenetic alterations, microRNA perturbation is associated with the development of kinase inhibitor resistance. Here, we identified and studied the role of miR-146a-5p dysregulation in melanoma drug resistance. METHODS: The miR-146a-5p-regulated NFkB signaling network was identified in drug-resistant cell lines and melanoma tumor samples by expression profiling and knock-in and knock-out studies. A bioinformatic data analysis identified COX2 as a central gene regulated by miR-146a-5p and NFkB. The effects of miR-146a-5p/COX2 manipulation were studied in vitro in cell lines and with 3D cultures of treatment-resistant tumor explants from patients progressing during therapy. RESULTS: miR-146a-5p expression was inversely correlated with drug sensitivity and COX2 expression and was reduced in BRAF and MEK inhibitor-resistant melanoma cells and tissues. Forced miR-146a-5p expression reduced COX2 activity and significantly increased drug sensitivity by hampering prosurvival NFkB signaling, leading to reduced proliferation and enhanced apoptosis. Similar effects were obtained by inhibiting COX2 by celecoxib, a clinically approved COX2 inhibitor. CONCLUSIONS: Deregulation of the miR-146a-5p/COX2 axis occurs in the development of melanoma resistance to targeted drugs in melanoma patients. This finding reveals novel targets for more effective combination treatment. Video Abstract.

Timing of sentinel node biopsy independently predicts disease-free and overall survival in clinical stage I-II melanoma patients: A multicentre study of the Italian Melanoma Intergroup (IMI).

BACKGROUND: Sentinel lymph node biopsy (SNB) still remains a key procedure to appropriately stage melanoma patients and to select those who are candidate to novel treatments with immunotherapy and targeted therapy in the adjuvant setting. The impact of timing of SNB on disease-free survival (DFS) and overall survival (OS) is still unclear. MATERIAL AND METHODS: The study was conducted at 6 Italian Melanoma Intergroup (IMI) centres and included 8953 consecutive clinical stage I-II melanoma patients who were diagnosed, treated, and followed up between November 1997 and March 2018. All patients were prospectively included in dedicated IMI database. Multivariable Cox regression analyses were performed to investigate how baseline characteristics and time interval until SNB are related to DFS and OS. RESULTS: Considering the whole population, at multivariable analysis, after adjusting for age, gender, Breslow thickness, site, ulceration, and the SNB status, a delay in the timing of SNB was associated with a better DFS (adjusted hazard ratio [aHR, delayed versus early SNB] 0.98, 95% confidence interval [CI] 0.97-0.99, p < 0.001) and OS (aHR 0.98, 95% CI 0.97-0.99, p = 0.001). Specifically, in patients with a negative SNB status, a beneficial impact of delayed SNB (i.e. at least 32 days after primary excision) was confirmed for DFS (aHR 0.70, 95%CI 0.63-0.79, p < 0.001) and OS (aHR 0.69, 95%CI 0.61-0.78, p < 0.001), whereas in those with a positive SNB status, DFS (aHR 0.96, 95%CI 0.84-1.09, p = 0.534) and OS (aHR 0.94 95%CI 0.81-1.08, p = 0.374) were not significantly different in patients with early or delayed SNB. CONCLUSIONS: Our study does not support a strict time interval for SNB. These results may be useful for national guidelines, for counselling patients and reducing the number of high urgency referrals.

Factors Affecting Sentinel Node Metastasis in Thin (T1) Cutaneous Melanomas: Development and External Validation of a Predictive Nomogram.

PURPOSE: Thin melanomas (T1; ≤ 1 mm) constitute 70% of newly diagnosed cutaneous melanomas. Regional node metastasis determined by sentinel node biopsy (SNB) is an important prognostic factor for T1 melanoma. However, current melanoma guidelines do not provide clear indications on when to perform SNB in T1 disease and stress an individualized approach to SNB that considers all clinicopathologic risk factors. We aimed to identify determinants of sentinel node (SN) status for incorporation into an externally validated nomogram to better select patients with T1 disease for SNB. PATIENTS AND METHODS: The development cohort comprised 3,666 patients with T1 disease consecutively treated at the Istituto Nazionale Tumori (Milan, Italy) between 2001 and 2018; 4,227 patients with T1 disease treated at 13 other European centers over the same period formed the validation cohort. A random forest procedure was applied to the development data set to select characteristics associated with SN status for inclusion in a multiple binary logistic model from which a nomogram was elaborated. Decision curve analyses assessed the clinical utility of the nomogram. RESULTS: Of patients in the development cohort, 1,635 underwent SNB; 108 patients (6.6%) were SN positive. By univariable analysis, age, growth phase, Breslow thickness, ulceration, mitotic rate, regression, and lymphovascular invasion were significantly associated with SN status. The random forest procedure selected 6 variables (not growth phase) for inclusion in the logistic model and nomogram. The nomogram proved well calibrated and had good discriminative ability in both cohorts. Decision curve analyses revealed the superior net benefit of the nomogram compared with each individual variable included in it as well as with variables suggested by current guidelines. CONCLUSION: We propose the nomogram as a decision aid in all patients with T1 melanoma being considered for SNB.

The density and spatial tissue distribution of CD8+ and CD163+ immune cells predict response and outcome in melanoma patients receiving MAPK inhibitors.

BACKGROUND: Clinical response to MAPK inhibitors in metastatic melanoma patients is heterogeneous for reasons still needing to be elucidated. As the patient immune activity contributes to treatment clinical benefit, the pre-existing level of immunity at tumor site may provide biomarkers of disease outcome to therapy. Here we investigated whether assessing the density and spatial tissue distribution of key immune cells in the tumor microenvironment could identify patients predisposed to respond to MAPK inhibitors. METHODS: Pretreatment tumor biopsies from a total of 213 patients (158 for the training set and 55 for the validation set) treated with BRAF or BRAF/MEK inhibitors within the Italian Melanoma Intergroup were stained with selected immune markers (CD8, CD163, ß-catenin, PD-L1, PD-L2). Results, obtained by blinded immunohistochemical scoring and digital image analysis, were correlated with clinical response and outcome by multivariate logistic models on response to treatment and clinical outcome, adjusted for American Joint Committee on Cancer stage, performance status, lactate dehydrogenase and treatment received. RESULTS: Patients with high intratumoral, but not peritumoral, CD8+ T cells and concomitantly low CD163+ myeloid cells displayed higher probability of response (OR 9.91, 95% CI 2.23-44.0, p = 0.003) and longer overall survival (HR 0.34, 95% CI 0.16-0.72, p = 0.005) compared to those with intratumoral low CD8+ T cells and high CD163+ myeloid cells. The latter phenotype was instead associated with a shorter progression free survival (p = 0.010). In contrast, PD-L1 and PD-L2 did not correlate with clinical outcome while tumor ß-catenin overexpression showed association with lower probability of response (OR 0.48, 95% CI 0.21-1.06, p = 0.068). CONCLUSIONS: Analysis of the spatially constrained distribution of CD8+ and CD163+ cells, representative of the opposite circuits of antitumor vs protumor immunity, respectively, may assist in identifying melanoma patients with improved response and better outcome upon treatment with MAPK inhibitors. These data underline the role of endogenous immune microenvironment in predisposing metastatic melanoma patients to benefit from therapies targeting driver-oncogenic pathways.