Characteristics of long-survivor metastatic melanoma after polychemotherapy and interferon: a retrospective study

DOI: https://doi.org/https://doi.org/10.57187/s.3504

Introduction

Until the 2010s, the prognosis of patients with metastatic melanoma was dismal, with less than 10% survival at five years. The standard of care was dacarbazine, with an overall response rate of less than 20% and median survival not exceeding six months [1]. Several dacarbazine-based regimens were evaluated to prolong survival. The Dartmouth regimen (dacarbazine, carmustine, cisplatin, and tamoxifen) showed a higher response rate (40–50%) than dacarbazine alone but failed to demonstrate a survival benefit. Moreover, toxicity rates were higher in the combination arm [2]. Similarly, the triple CVD (cisplatin, vinblastine, and dacarbazine) regimen showed an increased response rate of up to 40% and a median survival of nine months [3]. The immunochemotherapy (biochemotherapy [BCT]) regimen combining CVD with double immunotherapy (interleukin [IL]-2 and interferon α-2b) increased the response rate and median progression-free survival at the cost of substantially increased toxicities and without survival benefit [4–6]. This regimen, excluding interleukin-2, which is unavailable in Switzerland, was introduced as CVD-INF in 1993 in our institution and used until 2015.

The management of metastatic melanoma has dramatically changed in the last decade with the development of B-Raf proto-oncogene serine/threonine kinase (BRAF) and mitogen-activated protein kinase (MAP2K/MEK) inhibitors and immune checkpoints inhibitors (i.e. anti-cytotoxic T-lymphocyte associated protein 4 [CTLA4] ipilimumab, followed by anti-programmed cell death 1 [PDCD1/PD1] nivolumab and pembrolizumab). Blocking signal transduction and/or enhancing the immune response leads to an unprecedented survival improvement. Nonetheless, most patients will ultimately relapse, and alternative and/or combined strategies are still needed [7, 8]. Here, we retrospectively analysed our historical cohort of patients with metastatic melanoma treated with CVD-INF. Our main aim was to describe the clinical and biological characteristics of long-term survivors, defined as patients surviving more than two years after immunochemotherapy initiation.

Methods

Patient population

We retrospectively retrieved the clinical data of patients with metastatic melanoma treated at Hôpitaux Universitaires de Genève over 22 years (from 1993 to 2015). We selected patients who received a 21-day immunochemotherapy regimen with CVD-INF (cisplatin, vinblastine, dacarbazine, and interferon α-2b). All consecutive patients were included (figure 1). The inclusion criteria were patients with metastatic melanoma who completed at least one CVD-INF cycle. The exclusion criterion was limited available clinical and pathology data. Dacarbazine was administered at 800 mg/m² on day 1, cisplatin at 20 mg/m² daily (days 2–5), vinblastine at 1.6 mg/m² daily (days 1–5), and interferon α-2b at 5 mio UI daily (days 1–5), adapted from [4, 5]. This study was conducted according to the declaration of Helsinki and the Swiss Law (HRA Art.34/HRO) that authorises the reuse of clinical and biological samples. The research protocol was approved by the Ethics Committee of Geneva (CCER 14-268) and amended in 2016 to allow genomic and immunohistochemistry (IHC) analyses. The Ethics Committee approved a waiver of informed consent due to the death of most patients.

Figure 1Flowchart of patient selection. HUG: Hôpitaux Universitaires de Genève.

Data collection

Clinical and treatment characteristics were extracted from the medical records of eligible patients at the Hôpitaux Universitaires de Genève. Clinical characteristics included age, sex, site of the primary tumour, site and number of metastases, American Joint Committee on Cancer (AJCC) 8^th edition stage, performance status estimated with the Eastern Cooperative Oncology Group (ECOG) scale, serum lactate dehydrogenase (LDH) levels, and toxicities.

Outcome measures

Tumour response was evaluated with contrast-enhanced computed tomography or magnetic resonance imaging. Clinical tumour response was assessed according to the Response Evaluation Criteria in Solid Tumours (RECIST, version 1.1) criteria as follows: a complete response was defined as the disappearance of disease evidence, a partial response as a more than 30% decrease in tumour size without the appearance of new disease, progressive disease as a more than 20% increase in tumour size or the appearance of a new lesion, and stable disease as neither a partial response nor progressive disease [9]. Toxicity was graded according to the National Cancer Institute Common Toxicity Criteria (CTCAE, version 5.0). The clinical characteristics of long-term survivors, defined as surviving more than two years after immunochemotherapy initiation, were analysed.

Immunohistochemistry

Tumour infiltration by CD8⁺ T cells (DAKO; cloneC8/144B, 1:50 dilution) and tumour cell expression of PD-L1 Cell Signaling Technology; clone E1L3N, 1:200 dilution) were immunohistochemically determined in 28 patients with available biopsies: 8 long-survivors (≥2 years) and 20 non-long survivors (<2 years). The tumour immune phenotype was determined based on the spatial distribution of CD8⁺ T cells in the tumour core and stroma (inflamed, excluded, or desert [10]). Inflamed tumours were defined by CD8⁺ T cells in the tumour core. Excluded tumours were defined by CD8⁺ T cells exclusively in the stroma adjacent to or within the tumour. Desert tumours were defined by a low prevalence of CD8⁺ T cells. PD-L1 positivity was defined as at least 5% of tumour cells showing PD-L1 staining of any intensity on their surface [11]. BRAF V600E status was available for 47 patients (Ventana, clone VE1).

Genomic analyses

Next-generation sequencing of a custom panel of 443 genes was performed in three long-survivors.

Statistical analyses

This ad hoc study’s primary aim was to describe the clinical and biological characteristics of long-term survivors, defined as patients surviving more than 2 years after immunochemotherapy initiation. Overall survival was measured from immunochemotherapy initiation to death or last follow-up. Progression-free survival was defined as the interval from immunochemotherapy initiation to clinical or radiological progression or death. Long-term survivors were defined as patients surviving more than 2 years after immunochemotherapy initiation. Survival was modelled with the Cox proportional hazards model, and the association of different variables with survival was tested with the log-rank and log-ratio tests. Univariate analysis of overall survival was conducted for the following variables: age, sex, serum LDH level, BRAF V600E status, primary tumour site, central nervous system metastases, and number of metastatic sites. Variables significantly associated with survival in the univariate analyses were included in a multivariate Cox regression. The Kaplan-Meier method was used to plot survival curves and calculate median survival and follow-up loss. The proportional hazard assumption was confirmed by testing the Schoenfeld residuals against transformed time with the cox.zph() function. P-values <0.05 at a two-tailed alpha were considered statistically significant. All analyses were conducted with the R statistical software (version 4.0.2).

Results

Patients’ demographic and clinical characteristics

We identified 90 patients with metastatic melanoma who received at least one cycle of CVD-INF between 1993 and 2015 (median = 3, range = 1–6). Fifty-five patients (61%) were male. The median age at metastatic melanoma diagnosis was 55 years (20–75). The primary tumour site was cutaneous in 63 patients (70%), mucosal in 6 patients (7%), and acral in 2 patients (2%). Nineteen patients (21%) had melanoma with unknown primary (MUP). The BRAF V600E mutational status was available for 47 patients, of whom 59.6% had the mutation. Most patients presented with a good performance status: 83 (92%) had an ECOG score of ≤1. Almost all patients (88/90) were in stage IV. The number of metastatic sites was 1 in 15 patients (17%), 2–3 in 46 patients (51%), and ≥4 in 29 patients (32%). Twenty-nine patients (32%) had central nervous system metastases at diagnosis. Almost two-thirds of the patients (59%) had elevated serum LDH. Patients’ demographic and clinical characteristics are summarised in table 1.

Survival analyses

The median follow-up was 102.6 months, the median progression-free survival was 2.8 months, and the median overall survival was 7.2 months (figure 2A). The study period was defined as the interval (in years) between the start of the data collection period, corresponding to the first patient, and the inclusion of each successive patient. Since data collection spanned two decades, this variable was established to examine the evolution of the treatment effect over the studied period. Overall survival did not vary as a function of the study period (hazard ratio [HR]: 1.00; 95% confidence interval [CI]: 0.96–1.04; p = 0.89). Univariate analysis of overall survival revealed that young age (<50 years), elevated LDH, central nervous system metastases, and multiple metastatic sites (≥3) were associated with worse prognosis (table 2). None of the variables associated with shorter overall survival changed over the study period (age, performance status, LDH, central nervous system metastases, and multiple metastases; all p >0.1). The paradoxical increase in hazard observed in younger patients was fully explained in the multivariate analysis by the presence of central nervous system metastases. Indeed, central nervous system metastases were more common in patients aged <50 years (45%) than those aged ≥50 years (25%), with a trend toward statistical significance (Fisher’s test: odds ratio: 0.39; p = 0.06; see table S1 in the appendix). In the multivariate analysis, elevated LDH, multiple metastases, and central nervous system metastases were independently associated with shorter survival (figures 2B and 2C; table 2). 

Figure 2Overall survival of patients with metastatic melanoma treated with CVD-interferon α-2b. (A) Overall survival of the entire cohort. (B) Overall survival by the presence of central nervous system (CNS) metastases. (C) Overall survival by lactate dehydrogenase (LDH) level. (D) Overall survival by the number of metastatic sites.

CVD: cisplatin, vinblastine, dacarbazine.

Long survivors

Eleven patients (~12%) survived more than 2 years. Their median survival was not reached at the end of the study period. Their clinical characteristics are summarised in table 3. Eight long-survivors achieved a complete response, one had stable disease, and two had progressive disease after CVD-INF. The 8 patients who achieved a complete response had cutaneous melanoma (n = 4) or MUP (n = 4). Only one patient among the 11 had previously received immune checkpoint inhibitors, and they progressed before the introduction of immunochemotherapy. One patient who achieved a complete response relapsed after 18 years and received a tyrosine kinase inhibitor (TKI). One of the two patients who had progressive disease after immunochemotherapy did not receive further standard therapy. They were lost to follow-up for 13 years and reappeared during the COVID-19 outbreak in complete response. They disclosed having been treated by a traditional healer.

None of the long-survivors had central nervous system metastases (p = 0.014, Fisher’s test), and 75% had normal LDH levels (p = 0.052, Fisher’s test). Eight of the nine long-survivors (89%) had the BRAF V600E mutation compared to 11 of the 26 non-long-survivors (42%) (table 4). Of the patients without central nervous system metastases at baseline (n = 61), five (8%) survived at least 8 years, and six were censored. Three long-survivors developed vitiligo after immunochemotherapy.

Genomic alterations of tumours from long-survivors

We performed next-generation sequencing of a panel of 443 genes in biopsies from 3 long-survivors. All three patients had the BRAF V600E Glu mutation (confirmed by IHC), and two out of three had pathogenic ARID1A mutations (see table S2 in the appendix).

Tumour immune phenotype in long-survivors

We compared the spatial distribution pattern of CD8⁺ T cells in tumour compartments (core, stroma and margins) [10] in long-survivors (n = 8) and short-survivors (n = 20). We found that most long-survivors (75%; 6/8) had an inflamed pattern compared to 25% of short-survivors (5/20; p = 0.029, Fisher’s test) (table 4 and figure 3). Two of the eight long-survivors expressed PD-L1 (≥5%) compared to none of the short-survivors (p = 0.074, Fisher’s test) (table 4).

Figure 3The spatial distribution pattern of CD8+ T cells. (A) A representative IHC CD8+ image of the fully-inflamed phenotype. (B) A representative IHC CD8+ image of the immune excluded phenotype. Magnification: 200×.

Subsequent therapies

Forty-four patients received at least one subsequent therapy, mainly for central nervous system metastases. Specifically, 30 received temozolomide, 19 received radiation therapy, 4 received TKIs, two received recombinant IL-2, two patients were included in a clinical trial of vaccine combined with recombinant IL-2, one received dacarbazine, and one received thalidomide.

Toxicity

Overall, 89% of patients had adverse events of any grade, and 50 (56%) had severe (grade 3–4) adverse events. The most frequent severe adverse events were haematological: neutropenia (40%), thrombopenia (18%), anaemia (9%), febrile neutropenia (20%), and fatigue (12%).

Discussion

This retrospective study described a cohort of patients with metastatic melanoma treated with the CVD-INF immunochemotherapy regimen between 1993 and 2015 at a single institution. Central nervous system metastases, multiple metastases, and elevated LDH were independently associated with shorter survival, consistent with the literature [12, 13]. These findings are consistent with those of a recent study describing a large cohort of patients with metastatic melanoma treated with chemotherapy [14]. We observed that central nervous system metastases were more common in young patients (aged <50 years), an observation consistent with a large AJCC study showing that decreasing age at diagnosis was an independent predictive factor for the occurrence of central nervous system metastases at relapse for patients with stage III melanoma [15]. In the context of our cohort, this increased frequency of central nervous system metastases may also reflect selection bias. Elderly patients with central nervous system metastases might have been considered unfit for chemotherapy and excluded from our cohort.

Interestingly, 12% of patients survived at least two years, which was achieved without exposure to TKIs or immunotherapy (except for two patients). The proportion of patients alive at two years was 18% (11 of 61) in those without baseline central nervous system metastases at diagnosis. This proportion is in the same range as the 20%-survival plateau observed with anti-CTLA4 ipililumab in large randomised trials that excluded patients with central nervous system metastases [11, 16]. In addition, retrospective studies have observed a survival plateau with ipililumab only in patients without elevated LDH [17]. Our cohort included many patients with central nervous system metastases (32%) and/or elevated LDH (73%), which are associated with particularly poor outcomes with any type of therapy [18, 19]. This finding indicates that the survival plateau we observed after polychemotherapy and interferon is not due to the selection of patients with melanoma with a good prognosis.

The long survival obtained with CVD-INF in a subset of patients and the appearance of vitiligo in three of them may suggest that this regimen has an immunomodulating effect [20]. Therefore, we explored the tumour immune phenotype of long-survivors. CD8⁺ T cells are key effectors mediating tumour rejection [21, 22]. Three classes of tumour immune microenvironment were defined based on the spatial distribution of CD8⁺ T cells within tumour compartments (core, stroma, and invasive margins) [10, 23–25]: (a) “Immune-inflamed” or “hot” tumours are characterised by high CD8⁺ T cell infiltration, increased interferon-γ signalling, and PD-L1 expression; (b) “Immune-excluded” or “cold” tumours are characterised by T cells localised in the tumour margin along the border of the tumour which are prevented from infiltrating the tumour core by myeloid cells [23]; (c) “Immune desert” tumours are characterised by few or no CD8⁺ T cells. Immune-excluded and desert tumours are associated with poor outcomes [24, 26–29] and reduced response to immune checkpoint blockade [21, 30–33]. We found that most long-survivors had an inflamed tumour immune phenotype. This pattern has been associated with a better response to immune checkpoint blockade [32, 33] and chemotherapy [34] in metastatic cancer. It would be interesting to explore the combination of immune checkpoint blockade and chemotherapy in patients with such a phenotype.

An intriguing observation was the proportion of long-survivors with MUP (4 of 11), which represents 3% of newly diagnosed melanomas and 18% of stage IV melanomas [35]. The exact aetiology of MUP is unclear. It was suggested that MUPs are due to the regression of the primary lesion secondary to a robust immune response. They are more frequent in older patients, and males are disproportionally represented [36]. Stage IV MUPs have better outcomes than melanoma with known primary [35, 37] and may benefit more from immune checkpoint inhibitors [38]. Their mutational spectrum is similar to cutaneous melanoma with frequent mutations in BRAF and telomerase reverse transcriptase (TERT) [36, 39, 40]. Data on the tumour microenvironment of MUPs are scarce, and they need further investigation.

The current frontline treatment for metastatic melanoma is immune checkpoint blockade or tyrosine kinase inhibitors, and these breakthrough therapies have led to unprecedented improvement in survival [41]. Nonetheless, half of patients with metastatic melanoma will progress and die from the disease within five years of diagnosis [41]. Adoptive T cell therapy with tumour-infiltrating lymphocytes is a new option for patients with metastatic melanoma resistant to PD1 blockade [42]. However, survival benefit is observed only in a subset of these patients. Alternative therapies are urgently needed. Combining chemotherapy with immunotherapy, such as immune checkpoint inhibitor blockade (reviewed in [43]) or intralesional injections of oncolytic virus, is currently being explored [44].

Our study suggests that a subset of patients with metastatic melanoma treated with immunochemotherapy who have a tumour-inflamed pattern show prolonged survival. These patients with a favourable immune phenotype are highly likely to respond well to any treatment. Indeed, the inflamed phenotype has been associated with better outcomes in other cancers treated with various therapeutic approaches [10, 45]. Whether chemotherapy may potentiate the benefit of immunotherapy should be explored in well-designed clinical studies, including in-depth translational analysis.

Availability of materials and data

All data analysed in this study have been included in the manuscript (and its supplementary information files).

Acknowledgments

Author contributions: Conceptualisation: CP, SILG and PYD; methodology: PT and SILG; data collection: CP, GK and CDV; writing: CP, PT, SILG and PYD. All the authors reviewed and edited the manuscript. Author contributions: Conceptualisation: CP, SILG and PYD; methodology: PT and SILG; data collection: CP, GK and CDV; writing: CP, PT, SILG and PYD. All the authors reviewed and edited the manuscript.

References

1. Middleton MR, Grob JJ, Aaronson N, Fierlbeck G, Tilgen W, Seiter S, et al. Randomized phase III study of temozolomide versus dacarbazine in the treatment of patients with advanced metastatic malignant melanoma. J Clin Oncol. 2000 Jan;18(1):158–66. 10.1200/JCO.2000.18.1.158 

2. Chapman PB, Einhorn LH, Meyers ML, Saxman S, Destro AN, Panageas KS, et al. Phase III multicenter randomized trial of the Dartmouth regimen versus dacarbazine in patients with metastatic melanoma. J Clin Oncol. 1999 Sep;17(9):2745–51. 10.1200/JCO.1999.17.9.2745 

3. Legha SS, Ring S, Papadopoulos N, Plager C, Chawla S, Benjamin R. A prospective evaluation of a triple-drug regimen containing cisplatin, vinblastine, and dacarbazine (CVD) for metastatic melanoma. Cancer. 1989 Nov;64(10):2024–9. 10.1002/1097-0142(19891115)64:10<2024::AID-CNCR2820641010>3.0.CO;2-V 

4. Eton O, Legha SS, Bedikian AY, Lee JJ, Buzaid AC, Hodges C, et al. Sequential biochemotherapy versus chemotherapy for metastatic melanoma: results from a phase III randomized trial. J Clin Oncol. 2002 Apr;20(8):2045–52. 10.1200/JCO.2002.07.044 

5. Atkins MB, Hsu J, Lee S, Cohen GI, Flaherty LE, Sosman JA, et al.; Eastern Cooperative Oncology Group. Phase III trial comparing concurrent biochemotherapy with cisplatin, vinblastine, dacarbazine, interleukin-2, and interferon alfa-2b with cisplatin, vinblastine, and dacarbazine alone in patients with metastatic malignant melanoma (E3695): a trial coordinated by the Eastern Cooperative Oncology Group. J Clin Oncol. 2008 Dec;26(35):5748–54. 10.1200/JCO.2008.17.5448 

6. Bajetta E, Del Vecchio M, Nova P, Fusi A, Daponte A, Sertoli MR, et al. Multicenter phase III randomized trial of polychemotherapy (CVD regimen) versus the same chemotherapy (CT) plus subcutaneous interleukin-2 and interferon-alpha2b in metastatic melanoma. Ann Oncol. 2006 Apr;17(4):571–7. 10.1093/annonc/mdl007 

7. Wolchok JD, Chiarion-Sileni V, Gonzalez R, Rutkowski P, Grob JJ, Cowey CL, et al. Overall Survival with Combined Nivolumab and Ipilimumab in Advanced Melanoma. N Engl J Med. 2017 Oct;377(14):1345–56. 10.1056/NEJMoa1709684 

8. Robert C, Karaszewska B, Schachter J, Rutkowski P, Mackiewicz A, Stroiakovski D, et al. Improved overall survival in melanoma with combined dabrafenib and trametinib. N Engl J Med. 2015 Jan;372(1):30–9. 10.1056/NEJMoa1412690 

9. Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer. 2009 Jan;45(2):228–47. 10.1016/j.ejca.2008.10.026 

10. Desbois M, Udyavar AR, Ryner L, Kozlowski C, Guan Y, Dürrbaum M, et al. Integrated digital pathology and transcriptome analysis identifies molecular mediators of T-cell exclusion in ovarian cancer. Nat Commun. 2020 Nov;11(1):5583. 10.1038/s41467-020-19408-2 

11. Larkin J, Chiarion-Sileni V, Gonzalez R, Grob JJ, Cowey CL, Lao CD, et al. Combined Nivolumab and Ipilimumab or Monotherapy in Untreated Melanoma. N Engl J Med. 2015 Jul;373(1):23–34. 10.1056/NEJMoa1504030 

12. Balch CM, Gershenwald JE, Soong SJ, Thompson JF, Atkins MB, Byrd DR, et al. Final version of 2009 AJCC melanoma staging and classification. J Clin Oncol. 2009 Dec;27(36):6199–206. 10.1200/JCO.2009.23.4799 

13. Gershenwald JE, et al. Melanoma staging: Evidence-based changes in the American Joint Committee on Cancer eighth edition cancer staging manual. CA Cancer J Clin, 2017. 67(6): p. 472-492. 

14. Goldinger SM, Buder-Bakhaya K, Lo SN, Forschner A, McKean M, Zimmer L, et al. Chemotherapy after immune checkpoint inhibitor failure in metastatic melanoma: a retrospective multicentre analysis. Eur J Cancer. 2022 Feb;162:22–33. 10.1016/j.ejca.2021.11.022 

15. Haydu LE, et al. Cumulative Incidence and Predictors of CNS Metastasis for Patients With American Joint Committee on Cancer 8th Edition Stage III Melanoma. J Clin Oncol, 2020. 38(13): p. 1429-1441. 

16. Schachter J, Ribas A, Long GV, Arance A, Grob JJ, Mortier L, et al. Pembrolizumab versus ipilimumab for advanced melanoma: final overall survival results of a multicentre, randomised, open-label phase 3 study (KEYNOTE-006). Lancet. 2017 Oct;390(10105):1853–62. 10.1016/S0140-6736(17)31601-X 

17. Kelderman S, Heemskerk B, van Tinteren H, van den Brom RR, Hospers GA, van den Eertwegh AJ, et al. Lactate dehydrogenase as a selection criterion for ipilimumab treatment in metastatic melanoma. Cancer Immunol Immunother. 2014 May;63(5):449–58. 10.1007/s00262-014-1528-9 

18. Long GV, Grob JJ, Nathan P, Ribas A, Robert C, Schadendorf D, et al. Factors predictive of response, disease progression, and overall survival after dabrafenib and trametinib combination treatment: a pooled analysis of individual patient data from randomised trials. Lancet Oncol. 2016 Dec;17(12):1743–54. 10.1016/S1470-2045(16)30578-2 

19. Gutzmer R, Vordermark D, Hassel JC, Krex D, Wendl C, Schadendorf D, et al. Melanoma brain metastases - Interdisciplinary management recommendations 2020. Cancer Treat Rev. 2020 Sep;89:102083. 10.1016/j.ctrv.2020.102083 

20. Teulings HE, Limpens J, Jansen SN, Zwinderman AH, Reitsma JB, Spuls PI, et al. Vitiligo-like depigmentation in patients with stage III-IV melanoma receiving immunotherapy and its association with survival: a systematic review and meta-analysis. J Clin Oncol. 2015 Mar;33(7):773–81. 10.1200/JCO.2014.57.4756 

21. Tumeh PC, Harview CL, Yearley JH, Shintaku IP, Taylor EJ, Robert L, et al. PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature. 2014 Nov;515(7528):568–71. 10.1038/nature13954 

22. Schumacher TN, Schreiber RD. Neoantigens in cancer immunotherapy. Science. 2015 Apr;348(6230):69–74. 10.1126/science.aaa4971 

23. Binnewies M, Roberts EW, Kersten K, Chan V, Fearon DF, Merad M, et al. Understanding the tumor immune microenvironment (TIME) for effective therapy. Nat Med. 2018 May;24(5):541–50. 10.1038/s41591-018-0014-x 

24. Gruosso T, Gigoux M, Manem VS, Bertos N, Zuo D, Perlitch I, et al. Spatially distinct tumor immune microenvironments stratify triple-negative breast cancers. J Clin Invest. 2019 Apr;129(4):1785–800. 10.1172/JCI96313 

25. Hegde PS, Karanikas V, Evers S. The Where, the When, and the How of Immune Monitoring for Cancer Immunotherapies in the Era of Checkpoint Inhibition. Clin Cancer Res. 2016 Apr;22(8):1865–74. 10.1158/1078-0432.CCR-15-1507 

26. Azimi F, Scolyer RA, Rumcheva P, Moncrieff M, Murali R, McCarthy SW, et al. Tumor-infiltrating lymphocyte grade is an independent predictor of sentinel lymph node status and survival in patients with cutaneous melanoma. J Clin Oncol. 2012 Jul;30(21):2678–83. 10.1200/JCO.2011.37.8539 

27. Harlin H, Meng Y, Peterson AC, Zha Y, Tretiakova M, Slingluff C, et al. Chemokine expression in melanoma metastases associated with CD8+ T-cell recruitment. Cancer Res. 2009 Apr;69(7):3077–85. 10.1158/0008-5472.CAN-08-2281 

28. Kakavand H, Vilain RE, Wilmott JS, Burke H, Yearley JH, Thompson JF, et al. Tumor PD-L1 expression, immune cell correlates and PD-1+ lymphocytes in sentinel lymph node melanoma metastases. Mod Pathol. 2015 Dec;28(12):1535–44. 10.1038/modpathol.2015.110 

29. Cabrita R, Lauss M, Sanna A, Donia M, Skaarup Larsen M, Mitra S, et al. Tertiary lymphoid structures improve immunotherapy and survival in melanoma. Nature. 2020 Jan;577(7791):561–5. 10.1038/s41586-019-1914-8 

30. Ji RR, Chasalow SD, Wang L, Hamid O, Schmidt H, Cogswell J, et al. An immune-active tumor microenvironment favors clinical response to ipilimumab. Cancer Immunol Immunother. 2012 Jul;61(7):1019–31. 10.1007/s00262-011-1172-6 

31. Huang AC, Orlowski RJ, Xu X, Mick R, George SM, Yan PK, et al. A single dose of neoadjuvant PD-1 blockade predicts clinical outcomes in resectable melanoma. Nat Med. 2019 Mar;25(3):454–61. 10.1038/s41591-019-0357-y 

32. Jiang P, Gu S, Pan D, Fu J, Sahu A, Hu X, et al. Signatures of T cell dysfunction and exclusion predict cancer immunotherapy response. Nat Med. 2018 Oct;24(10):1550–8. 10.1038/s41591-018-0136-1 

33. Sun R, Limkin EJ, Vakalopoulou M, Dercle L, Champiat S, Han SR, et al. A radiomics approach to assess tumour-infiltrating CD8 cells and response to anti-PD-1 or anti-PD-L1 immunotherapy: an imaging biomarker, retrospective multicohort study. Lancet Oncol. 2018 Sep;19(9):1180–91. 10.1016/S1470-2045(18)30413-3 

34. Moretto R, Corallo S, Belfiore A, Rossini D, Boccaccino A, Lonardi S, et al. Prognostic impact of immune-microenvironment in colorectal liver metastases resected after triplets plus a biologic agent: A pooled analysis of five prospective trials. Eur J Cancer. 2020 Aug;135:78–88. 10.1016/j.ejca.2020.04.045 

35. Lee CC, Faries MB, Wanek LA, Morton DL. Improved survival for stage IV melanoma from an unknown primary site. J Clin Oncol. 2009 Jul;27(21):3489–95. 10.1200/JCO.2008.18.9845 

36. De Andrade JP, Wong P, O’Leary MP, Parekh V, Amini A, Schoellhammer HF, et al. Multidisciplinary Care for Melanoma of Unknown Primary: Experience in the Era of Molecular Profiling. Ann Surg Oncol. 2020 Dec;27(13):5240–7. 10.1245/s10434-020-09112-2 

37. Bae JM, Choi YY, Kim DS, Lee JH, Jang HS, Lee JH, et al. Metastatic melanomas of unknown primary show better prognosis than those of known primary: a systematic review and meta-analysis of observational studies. J Am Acad Dermatol. 2015 Jan;72(1):59–70. 10.1016/j.jaad.2014.09.029 

38. Gambichler T, Chatzipantazi M, Schröter U, Stockfleth E, Gedik C. Patients with melanoma of unknown primary show better outcome under immune checkpoint inhibitor therapy than patients with known primary: preliminary results. OncoImmunology. 2019 Oct;8(12):e1677139. 10.1080/2162402X.2019.1677139 

39. Egberts F, Bergner I, Krüger S, Haag J, Behrens HM, Hauschild A, et al. Metastatic melanoma of unknown primary resembles the genotype of cutaneous melanomas. Ann Oncol. 2014 Jan;25(1):246–50. 10.1093/annonc/mdt411 

40. Egberts F, Krüger S, Behrens HM, Bergner I, Papaspyrou G, Werner JA, et al. Melanomas of unknown primary frequently harbor TERT-promoter mutations. Melanoma Res. 2014 Apr;24(2):131–6. 10.1097/CMR.0000000000000048 

41. Ugurel S, Röhmel J, Ascierto PA, Becker JC, Flaherty KT, Grob JJ, et al. Survival of patients with advanced metastatic melanoma: the impact of MAP kinase pathway inhibition and immune checkpoint inhibition - Update 2019. Eur J Cancer. 2020 May;130:126–38. 10.1016/j.ejca.2020.02.021 

42. Rohaan MW, Borch TH, van den Berg JH, Met Ö, Kessels R, Geukes Foppen MH, et al. Tumor-Infiltrating Lymphocyte Therapy or Ipilimumab in Advanced Melanoma. N Engl J Med. 2022 Dec;387(23):2113–25. 10.1056/NEJMoa2210233 

43. Eggermont AM, Crittenden M, Wargo J. Combination Immunotherapy Development in Melanoma. Am Soc Clin Oncol Educ Book. 2018 May;38(38):197–207. 10.1200/EDBK_201131 

44. Soliman H, Hogue D, Han H, Mooney B, Costa R, Lee MC, et al. Oncolytic T-VEC virotherapy plus neoadjuvant chemotherapy in nonmetastatic triple-negative breast cancer: a phase 2 trial. Nat Med. 2023 Feb;29(2):450–7. 10.1038/s41591-023-02210-0 

45. Hammerl D, Martens JW, Timmermans M, Smid M, Trapman-Jansen AM, Foekens R, et al. Spatial immunophenotypes predict response to anti-PD1 treatment and capture distinct paths of T cell evasion in triple negative breast cancer. Nat Commun. 2021 Sep;12(1):5668. 10.1038/s41467-021-25962-0 

Appendix: supplementary tables