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In the past decade, immunotherapy with checkpoint inhibitors has revolutionized the field of oncology. Checkpoint inhibitors have been approved for several types of cancer and thousands of patients in Sweden now receive oncological immunotherapy annually. Immune-related side effects are common and can occur in almost any organ. These side effects are different from those that occur with traditional oncological treatments. The side effects are usually mild, but can be serious and even lethal. In a short time, health care providers have had to readjust to be able to handle these side effects. Early and correct diagnosis of immune-related side effects, proper management and a multidisciplinary approach is crucial. Here, we give an overview of the presentation, diagnosis and treatment of immune-related side effects, with emphasis of those occurring in gastrointestinal organs, lungs and kidneys.

The aim of this study was to estimate the impact on survival of NRAS and BRAF mutations and activation of Akt and extracellular signal-regulated kinase (ERK) in primary melanomas. A cohort of 57 primary cutaneous T1-2 melanoma tumors was analyzed. Mutation frequency for both genes was 61% (NRAS 26% and BRAF 39%). In a univariate analysis, shorter overall survival was associated with the presence of ulceration (P=0.001) and BRAF exon 15 mutations (P=0.005) as well as the absence of nuclear activation of Akt (P=0.022) and of cytoplasmic activation of ERK (P=0.003). Unexpectedly, ulceration was a significant adverse prognostic factor only in melanomas with BRAF mutations, whereas there was no effect of ulceration on overall survival in tumors with wild-type BRAF. A multivariate analysis showed that significant independent adverse survival prognostic markers were absence of cytoplasmic activation of ERK (P=0.007) and ulceration (P=0.008), whereas BRAF exon 15 mutation status showed a nonsignificant trend (P=0.066). The absence of cytoplasmic ERK activation in poor prognosis T1-2 melanomas may be associated with activation of some other uncharacterized pathway leading to tumor progression and adverse outcome. Immunohistochemical analysis of cytoplasmic phosphorylated ERK could be used as a prognostic marker in primary melanomas if confirmed in another data set.

To the Editor Germline aberrations in the CDKN2A gene are observed in some melanoma-prone families and represent high penetrance mutations (Hussussian et al., 1994; Kamb et al., 1994). INK4A (p16) and ARF (p14) are two distinct proteins encoded by the CDKN2A locus. Loss of INK4A has been associated with unrestricted cell cycle progression through retinoblastoma (RB) protein inactivation, while loss of ARF has been linked to p53 inactivation with subsequent malfunction in cell cycle regulation, apoptosis and DNA repair (Chin et al., 2006). The NRAS and BRAF genes are commonly mutated in sporadic primary cutaneous melanomas, with mutation frequencies between 4–50% (Platz et al., 2008) and 25–80% (Platz et al., 2008), respectively. Codon 61 is the most common position of NRAS alterations in melanoma, with frequent glutamine changes to either lysine, Q61K (c.181C>A), or arginine, Q61R (c.182A>G) (Omholt et al., 2002). Mutations in this residue lock the Ras protein in the GTP-bound state with subsequent continuous activation of its downstream effectors (Platz et al., 2008) through the Ras-Raf-MEK-ERK and Ras-PI3K-Akt pathways. Approximately 90% of reported BRAF mutations occur at residue 600, which is located in the activation domain of this kinase (Thomas, 2006). Current results from melanoma cohorts show that mutations in these genes are almost always mutually exclusive (Edlundh-Rose et al., 2006; Omholt et al., 2003; Platz et al., 2008). Moreover, a high rate of BRAF mutation is found also in nevi, suggesting a role in early stages of the neoplastic process (Pollock et al., 2003). Previously, a high frequency of NRAS mutations (95%) has been reported in Swedish familial melanoma cases with germline CDKN2A alterations (Eskandarpour et al., 2003). The association of BRAF somatic mutations with MC1R germline variants indicates an influence of constitutive genotype on the preferential acquisition of specific mutations during melanoma development (Landi et al., 2006). In line with this, cooperation between RAS and CDKN2A has been shown in animal models of melanoma (Chin et al., 1997). Since there is limited information on NRAS and BRAF mutations in familial melanoma we sought to assess their mutation frequency in melanomas from patients with different CDKN2A germline alterations. The study was performed on formalin-fixed, paraffin-embedded (FFPE) primary familial cutaneous melanomas originating from Brisbane, Australia (16 samples from 15 patients) and Genoa, Italy (3 patients/samples). Clinical and pathological characteristics are shown in Table 1. One patient with a CDKN2A L32P mutation had two melanomas originating from the trunk and upper extremity, respectively, while a single melanoma was analyzed from each of the other patients. Eight different germline CDKN2A mutations were present in the patients from whom the melanomas were analyzed (Table 2). Laser capture microdissection, DNA extraction, polymerase chain reaction (PCR) amplification, single strand conformation polymorphism (SSCP) and nucleotide sequence analyses of NRAS exon 2 and BRAF exon 15 were carried out as previously described (Jovanovic et al., 2008; Omholt et al., 2002; Omholt et al., 2003). Each mutation was confirmed by two independent PCR/SSCP analyses followed by sequence analysis performed in both directions. The study was approved by the Ethics Committees of the Queensland Institute of Medical Research, University of Genoa and Karolinska Institutet. Table 1 Patient and tumor characteristics Table 2 NRAS and BRAF genotypes in familial melanomas harboring CDKN2A germline mutations Three (16%) samples had NRAS residue 61 alterations (one Q61R and two Q61K substitutions) while 7 (37%) samples had valine to glutamic acid changes in amino acid 600 of BRAF (V600E; Table 2). The NRAS mutation frequency in this study was lower than we reported previously in melanomas from Swedish families with germline CDKN2A mutations (Eskandarpour et al., 2003). The reason for this is unclear. It could possibly be attributed to different origins of studied cohorts (Sweden versus Australia/Italy) and to different CDKN2A germline alterations in these two studies (the 112Argdup founder mutation is predominant in Swedish families, which is in contrast to genotypes reported here). However, we cannot exclude that technical factors, possibly related to the fragmented nature of DNA extracted from FFPE samples, may play a role in different rates of mutation detection. Intriguingly, all 3 tumors with NRAS mutations also had BRAF V600E mutations. The presence of both NRAS and BRAF V600E mutations in the same lesions is contrary to the current consensus that such mutations are almost always mutually exclusive in melanomas and other tumor types (Davies et al., 2002; Omholt et al., 2003; Thomas et al., 2007). However, Pollock et al (2003) observed concomitant NRAS and BRAF V600E mutations in 9% of nevi and suggested this might be due to different clonal nests of cells within these tumors carrying distinct mutations, a possibility that could also explain our findings. Although intriguingly, the joint presence of both NRAS and BRAF mutations was found only in tumors from patients with CDKN2A L32P mutations. In two of these tumors, NRAS and BRAF mutations (NRAS/BRAF: Q61K/V600E and Q61R/V600E) were found in two different DNA extracts while in one case, alterations of both genes (NRAS/BRAF: Q61K/V600E) were identified in the same DNA extract. Thus far, it is recognized that a mutation in either NRAS or BRAF is sufficient for activation of the Ras-Raf-MEK-ERK pathway, with mutant RAS having a 50-fold higher activation effect than mutant BRAF (Davies et al., 2002). Although we do not have any evidence that the NRAS and BRAF mutations found in the same DNA extract were coexisting in the same cells, it is possible that the L32P mutation in CDKN2A somehow permits cellular tolerance of these dual mutations. In conclusion, the NRAS and BRAF mutation rates we observed in familial melanomas were generally lower than most previous reports in sporadic melanoma but equal to those reported for primary melanomas of similar thickness (Goel et al., 2006; Shinozaki et al., 2004). Samples that harbored INK4A L32P substitutions also had high frequency of coexisting mutations in both NRAS and BRAF. This suggests that in some instances constitutional CDKN2A mutations affect the occurrence of somatic mutations in NRAS and BRAF, although further work is needed to substantiate this hypothesis.