Mismatch repair deficiency (dMMR) with microsatellite instability (MSI) is frequent in cancer, particularly in gastrointestinal and endometrial malignancies. The increased tumor mutational burden renders dMMR/MSI tumors suitable targets for immune checkpoint inhibitors—provided the regulatory genetic defect can be detected. dMMR and MSI are considered equally effective predictors of the efficacy of ICIs; however, while dMMR testing is based on detection of missing MMR proteins in immunohistochemistry (IHC), MSI polymerase chain reaction (PCR) testing focuses on the consequences of dMMR at the genomic level. A retrospective analysis was carried out in a large cancer cohort (n = 1306). dMMR was tested by four IHC reactions (MLH1, PMS2, MSH2, MSH6), and MSI was assessed by pentaplex PCR (BAT-25, BAT-26, MONO-27, NR-21, NR-24) in 703 cases. In 64 cases (5

Background The tumor-agnostic indication of immune checkpoint inhibitors to treat cancers with mismatch repair deficiency (dMMR)/microsatellite instability (MSI) increased the demand for such tests beyond Lynch syndrome. International guideline recommendations accept immunohistochemistry (IHC) for dMMR or molecular techniques (PCR or NGS) for MSI status determinations considering the two tests are equal, although there are scattered reports contradicting to this presumption. Materials and methods Here we have directly compared four protein MMR immunohistochemistry (IHC) to MSI Pentaplex PCR test in a large cancer patient cohort (n = 1306) of our diagnostic center where the two tests have been run parallel in 703 cases. Results In this study we have found a high discrepancy rate (19.3%) of the two tests which was independent of the tumor types. The MSI PCR sensitivity for MMR IHC status was found to be very low resulting in a relatively low positive and negative predicting values. As a consequence, the correlation of the two tests was low (kappa < 0.7). During analysis of the possible contributing factors of this poor performance, we have excluded low tumor percentage of the samples, but identified dMMR phenotypes (classic versus non-classic or unusual) as possible contributors. Conclusion Although our cohort did not include samples with identified technical errors, our data strongly support previous reports that unidentified preanalytical factors might have the major influence on the poor performance of the MSI PCR and MMR IHC. Furthermore, the case is open whether the two test types are equally powerful predictive markers of immunotherapies.

Molecular epidemiology of mismatch repair deficiency (dMMR)/microsatellite instability (MSI) are different in various ethnic groups; accordingly, our aim was to test this in a large single-center Hungarian cancer patient cohort. We have found that dMMR/MSI incidence correlates well with TCGA data in case of colorectal, gastric and endometrial cancers. We have also observed that immunohistochemistry- based dMMR incidences are higher as compared to MSI. We suggest that the testing guidelines must be fine-tuned for immune-oncology indications. Nádorvári ML, Kiss A, Barbai T, Rásó E, Tímár J. Molecular epidemiology of mismatch repair deficiency, microsatellite instability in a large single diagnostic center cancer cohort.

We have followed the genomic progression of cutaneous melanoma in visceral metastases using genome-wide copy number analysis. We have detected an increased chromosomal instability due to the loss of several DNA repair genes. Furthermore, we found co-amplifications of HGF and MET genes in metastases. The most interesting finding was gene amplifications of several, mostly IFN-regulated immune cell genes in lung metastases. Next we have defined a type I IFN resistance gene expression signature (GES) using human cell lines, several elements of which were proved to be stable in vitro and in vivo as well. The components of this GES have been detected in TCGA as well as in publicly available datasets of immunotherapy-treated melanoma cases. In case of samples from previously IFN-treated melanoma cases we have identified treatment-specific genomic alterations (predominantly amplifications) which were most characteristic for brain metastases.

Background: Head and neck squamous cell carcinomas (HNSCCs) are among the most abundant malignancies worldwide. Patients with recurrent/metastatic disease undergo combination chemotherapy containing cetuximab, the monoclonal antibody used against the epidermal growth factor receptor (EGFR). Cetuximab augments the effect of chemotherapy; however, a significant number of patients show therapy resistance. The mechanism of resistance is yet to be unveiled, although extracellular alterations of the receptor have been reported, and their role in cetuximab failure has been proposed. Aims: Here, we investigate possible effects of the multi-exon deletion variant (EGFRvIII), and the single nucleotide polymorphism EGFR R521K on cetuximab efficacy. Results: Our results show that in HNSCC patients, the EGFRvIII allele frequency is under 1%; therefore, it cannot lead to common resistance. EGFR R521K, present in 42% of the patients, is investigated in vitro in four HNSCC cell lines (two wild-type and two heterozygous for EGFR R521K). While no direct effect is found to be related to the EGFR status, cells harboring R521K show a reduced sensitivity in ADCC experiments and in vivo xenograft experiments. However, this preclinical difference is not reflected in the progression-free or overall survival of HNSCC patients. Furthermore, NK cell and macrophage presence in tumors is not related to EGFR R521K. Discussion: Our results suggest that EGFR R521K, unlike reported previously, is unable to cause cetuximab resistance in HNSCC patients; therefore, its screening before therapy selection is not justifiable.

We developed a human melanoma model using the HT168-M1 cell line to induce IFN-α2 resistance in vitro (HT168-M1res), which was proven to be maintained in vivo in SCID mice. Comparing the mRNA profile of in vitro cultured HT168-M1res cells to its sensitive counterpart, we found 79 differentially expressed genes (DEGs). We found that only a 13-gene core of the DEGs was stable in vitro and only a 4-gene core was stable in vivo. Using an in silico cohort of IFN-treated melanoma tissues, we validated a differentially expressed 9-gene core of the DEGs. Furthermore, using an in silico cohort of immune checkpoint inhibitor (ICI)-treated melanoma tissues, we tested the predictive power of the DEGs for the response rate. Analysis of the top four upregulated and top four downregulated genes of the DEGs identified WFDC1, EFNA3, DDX10, and PTBP1 as predictive genes, and analysis of the “stable” genes of DEGs for predictive potential of ICI response revealed another 13 genes, out of which CDCA4, SOX4, DEK, and HSPA1B were identified as IFN-regulated genes. Interestingly, the IFN treatment associated genes and the ICI-therapy predictive genes overlapped by three genes: WFDC1, BCAN, and MT2A, suggesting a connection between the two biological processes.

Background: Anti-EGFR antibody therapy is still one of the clinical choices in head and neck squamous cell carcinoma (HNSCC) patients, but the emergence of cetuximab resistance questioned its effectiveness and reduced its applicability. Although several possible reasons of resistance against the antibody treatment and alternative therapeutic proposals have been described (EGFR alterations, activation of other signaling pathways), there is no method to predict the effectiveness of anti-EGFR antibody treatments and to suggest novel therapeutics. Our study investigated the effect of EGFR R521K alteration on efficiency of cetuximab therapy of HNSCC cell lines and tried to find alternative therapeutic approaches against the resistant cells. Methods: After genetic characterization of HNSCC cells, we chose one wild type and one R521K+ cell line for in vitro proliferation and apoptosis tests, and in vivo animal models using different therapeutic agents. Results: Although the cetuximab treatment affected EGFR signalization in both cells, it did not alter in vitro cell proliferation or apoptosis. In vivo cetuximab therapy was also ineffective on R521K harboring tumor xenografts, while blocked the tumor growth of EGFR-wild type xenografts. Interestingly, the cetuximab-resistant R521K tumors were successfully treated with c-MET tyrosine kinase inhibitor SU11274. Conclusion: Our results suggest that HNSCC cell line expressing the R521K mutant form of EGFR does not respond well to cetuximab treatment in vitro or in vivo, but hopefully might be targeted by c-MET tyrosine kinase inhibitor treatment.

Background: KRAS mutation is the most common genetic alteration in lung adenocarcinoma (LADC) in Western countries and is associated with worse outcome in bone-metastatic cases. Yet, to date, no effective treatment guidelines were developed for these patients. Accordingly, our aim was to investigate the impact of KRAS mutation on bisphosphonate (BTx) and radiation therapy (RTx) in bone-metastatic LADC patients. Methods: Clinicopathological variables of 134 consecutive LADC patients with bone metastases at diagnosis and known KRAS status were retrospectively analyzed. The effects of BTx, RTx and KRAS mutation on overall survival (OS) were investigated. Results: Of the total cohort, 93 patients were identified as KRAS wild-type (WT) (69.4%) and 41 (30.6%) as KRAS mutant patients. The presence of KRAS mutation was associated with significantly reduced median OS (5.1 vs. 10.2 months in KRAS WT patients; P=0.008). Irrespective of KRAS mutational status both BTx (P=0.007) and RTx (P=0.021) conferred a significant benefit for OS. Notably, however, when analyzing the patients with KRAS-mutant and KRAS WT tumors separately, the benefit from BTx and RTx on OS remained statistically significant only in KRAS WT patients (P=0.032 and P=0.031, respectively). Conclusions: KRAS mutation is a strong negative prognostic factor in bone-metastatic LADC patients. Both BTx and RTx can increase the OS with a pronounced benefit for patients with KRAS WT tumors. Altogether, KRAS mutational status should be considered during therapeutic decision making in bone metastatic LADC patients.

Head and neck squamous cell carcinomas (HNSCC) take many lifes worldwide. Patients with recurrent/metastatic disease receive combination chemotherapy containing anti-EGFR antibody cetuximab. However, resistance often hurdles therapy. The mechanism is yet to unveil, although EGFR extracellular alterations and activity of c-Met signaling were accused. We investigated the effects of EGFR-vIII and EGFR-R521K on cetuximab efficacy in HNSCC in cellular, xenograft, and clinical setup. Furthermore, we investigated the efficacy of c-Met inhibition in HNSCC in vitro and in vivo. We showed that EGFR-vIII is very rare in HNSCC, while the common R521K polymorphism abolishes antibody-dependent cellular cytotoxicity and in vivo antitumor effect of cetuximab. This selectivity was not reflected in immunophenotype or survival data of HNSCC patients, suggesting a more complex mechanism behind. Interestingly, c-Met inhibitor SU11274 was more effective in cetuximab-resistant, EGFR R521K heterozygous cells and xenografts, raising the possible importance of simultaneous targeting of the two receptors.

The therapeutic impact of KRAS mutations remains controversial in bone metastatic lung adenocarcinoma (LADC). Therefore, our aim was to investigate the effects of KRAS mutational status on overall survival (OS) in these patients according to bisphosphonate therapy (BTx) and radiation therapy (RTx). In total, 134 LADC patients diagnosed with simultaneous bone metastasis were included in this study. The results of the univariate (p=0.008) and multivariate (p=0.004) survival analyses indicated that KRAS mutation is a negative prognostic factor. Both BTx and RTx can increase the OS with a pronounced benefit for patients with KRAS wild-type tumors. Importantly, the concomitant use of BTx and RTx might increase the OS irrespective of KRAS status compared to BTx or RTx alone. In summary, our results might contribute to the development of new therapeutic approaches with regards to KRAS mutational status in bone metastatic LADC.

Negative predictive markers of the anti-EGFR antibody therapies are RAS or BRAF mutations, while left sidedness can be considered as a positive predictor. Here we analyzed 97 wild type RAS metastatic colorectal cancers looking for the prognostic and predictive roles of EGFR protein expression. We found that right-sided colorectal cancers are characterized by significantly higher EGFR protein expression as compared to left-sided ones, irrespective of the primary or metastatic tissue analysis. Furthermore, tumors with multiple organ involvement are characterized by significantly higher EGFR protein expression as compared to single organ ones. In the homogenous cetuximab treated cohort (n=90) we have found that lower than the applied EGFR protein expression cut-off was associated with favorable survival. In the multivariate analysis only sidedness proved to be a strong independent predictor, however sidedness is an EGFR-dependent predictor of anti-EGFR therapy.

Zinc as an essential trace metal is a ubiquitous component of various molecules of the cell. Studies indicated that it may modulate functions of various cancer cell types, and can even inhibit metastasis formation in experimental models. In melanoma, zinc was shown to affect melanin production and to induce apoptosis. Using human melanoma cell lines, we have tested the effects of ZnSO 4 on cell proliferation, survival, migration as well as in vivo on experimental liver colony formation. We have found that ZnSO 4 has antiproliferative and proapoptotic effects in vitro. In SCID mice intraperitoneal administration of ZnSO 4 specifically inhibited liver colony formation without affecting primary tumor growth. To reveal the molecular mechanisms of action of zinc in human melanoma, we have tested mRNA expression of zinc finger transcription factors and found a strong inhibitory effect on HIF1α, as compared to WT1 whereas HIF2α and MTF1 expression was unaffected. Immunohistochemical detection of HIF1α protein in liver metastases confirmed its decreased nuclear expression after in vivo ZnSO 4 treatment. These data indicate that in human melanoma zinc administration may have an antimetastatic effect due to a selective downregulation of HIF1α.

One of the mechanisms potentially explaining the discrepancy between the number of human genes and the functional complexity of organisms is generating alternative splice variants, an attribute of the vast majority of multi-exon genes. Members of the RAS family, such as NRAS, KRAS and HRAS, all of which are of significant importance in cancer biology, are no exception. The structural and functional differences of these splice variants, particularly if they contain the canonical (and therefore routinely targeted for diagnostic purposes)hot spotmutations, pose a significant challenge for targeted therapies. We must therefore consider whether these alternative splice variants constitute a minor component as originally thought and how therapies targeting the canonical isoforms affect these alternative splice variants and their overall functions.

Langerhans cell histiocytosis (LCH) is characterized by mutations of the RAS-RAF-MAPK signaling pathway. We analyzed MAP2K1, NRAS and KIT mutation incidence in skin lesions of BRAF wild-type (wt) LCH patients. We evaluated the occurrence of MAP2K1, NRAS and KIT mutations in seven LCH and one indeterminate cell histiocytosis (ICH) patients. MAP2K1 mutation frequency was found to be 3/7 (42.9%) in LCH and also found in ICH. Similarly, the KIT mutation frequency was found to be equally prevalent (4/7, 57.1%) in LCH and also occurred in ICH. Involvement of KIT exons in LCH-ICH indicated that exon 9/11/18 were equally prevalent followed by exon 13. This exploratory analysis on BRAF-wt LCH revealed a KIT mutation rate comparable to MAP2K1. Although the detected KIT mutations are different from activating mutations found in other KIT-dependent neoplasms, our data suggest that KIT-inhibitors might have a role in treating BRAF-wt LCH patients.

The selection of colorectal cancer patients for anti-epidermal growth factor receptor (EGFR) antibody therapy is based on the determination of their RAS mutation status-a strongly negative predictive factor-since the protein target, EGFR, is not a reliable predictor of therapeutic response. In this study, we revisited the EGFR protein issue using a cohort of 90 patients with KRAS exon2 wild-type colorectal cancer who have been treated with cetuximab therapy. Twenty-nine of these patients had metastatic tissue available for analysis. The level of EGFR protein expression in the patients was determined by immunohistochemistry and evaluated by H-score (HS) methodology. Progression-free survival (PFS) and overall survival (OS) of the patients were determined according to the EGFR-HS ranges of both the primary and metastatic tissues using Kaplan-Meyer statistics. In the case of primary tumors, EGFR scores lower than HS = 200 were associated with significantly longer OS. In the case of metastatic tissues, all levels lower than the EGFR-HS range chosen were associated with significantly longer OS. These results are explained by the fact that metastatic tissues rarely maintained the expression levels of the primary tumors. On the other hand, high EGFR expression levels in either primary tumors or metastatic tissues were associated with multiple metastatic disease. This suggests a negative prognostic role of EGFR expression. However, in a multivariate analysis, one-sidedness remained a strong independent predictive factor of survival. Previous studies demonstrated that the EGFR expression level depends on sidedness. Therefore, a subgroup analysis of the left- and right-sided cases was performed on both primary and metastatic tissues. In the case of metastic tissues, an analysis confirmed a better OS in low EGFR protein-expressing cases than in high EGFR protein-expressing cases. Collectively, these data suggest that EGFR protein expression is another negative predictive factor of the efficacy of cetuximab therapy of KRAS exon2 wild-type colorectal cancer.

Following publication of the original article [1], the authors reported the family name of the second author was incorrectly published.

Data on the KIT mutation rate in melanoma in the central European region is missing. Accordingly, in a cohort of 79 BRAF/NRAS double wild type cutaneous melanoma and 17 mucosal melanoma KIT mutation was assessed by Sanger sequencing of exons 9,11,13,17 and 18. In this cutaneous melanoma cohort KIT mutation frequency was found to be 34/79 (43.04%) with a significantly higher rate in acrolentiginous melanoma (ALM) as compared to UV-induced common variants (20/34, 58.8% versus 14/45, 31.1%, p = 0.014). In the double wild type mucosal melanoma cohort the KIT mutation frequency was found to be comparable (41.2%). The actual frequency of KIT mutation in the original 227 patient cutaneous melanoma cohort was 34/227, 14.9%. Exon 11 was the most frequent mutation site (44.7%) followed by exon 9 (21.1%) equally characterizing UV-induced common histotypes and ALM tumors. In mucosal melanoma exon 9 was the most frequently involved exon followed by exon 13 and 17. KIT mutation hotspots were identified in exon 9 (c482/491/492), in exon 11 (c559,c572, c570), in exon 13 (c642), in exon 17 (c822) and in exon 18 (c853). The relatively high KIT mutation rate in cutaneous melanoma in this central-European cohort justifies regular testing of this molecular target in this entity, not only in mucosal variants.

Thirty-one cases of low-grade renal cell carcinoma (RCC) with clear cells and tubulopapillary/papillary architecture were analyzed retrospectively with immunohistochemical and genetic markers to gain more experience with the differential diagnosis of such cases. All samples coexpressed CK7 and CA9; the TFE3 or TFEB reactions were negative; the CD10 and the AMACR stainings were negative in 27 cases and 30 cases, respectively. The FISH assays for papillary RCC, available in 27 cases, and deletion of chromosome 3p, available in 29 cases, gave negative results. The results for 3p deletion, VHL gene mutation or VHL gene promoter region hypermethylation testing, along with the diffuse CD10-positivity in 2 cases confirmed 21 cases as clear cell papillary RCC (CCPRCC; CK7+, CA9+; no 3p loss, no VHL abnormality) and 10 cases as clear cell RCC (CCRCC; CK7+, CA9+; no 3p loss, VHL abnormality mutation/hypermethylation present). In CCPRCCs, the representative growth pattern was branching tubulo-acinar, commonly accompanied by cyst formation. The linear nuclear arrangement or cup-shaped staining of CA9 did not necessarily indicate CCPRCC, and the absence of these did not exclude the diagnosis of CCPPRC. One tumor infiltrated the renal sinus; the others exhibited pT1 stage; and metastatic outcome was not recorded. The CCRCC cases were in pT1 stage; 6 exhibited cup-shaped staining of CA9, and 1 displayed lymph node metastasis at the time of surgery. Distant metastatic disease was not observed. In summary, the VHL abnormalities distinguished the subset of CCRCC with diffuse CK7-positivity and no 3p loss from cases of CCPRCC.

Bevacizumab, combined with platinum-based chemotherapy, has been widely used in the treatment of advanced-stage lung adenocarcinoma (LADC). Although KRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) mutation is the most common genetic alteration in human LADC and its role in promoting angiogenesis has been well established, its prognostic and predictive role in the above setting remains unclear. The association between KRAS exon 2 mutational status and clinicopathological variables including progression-free survival and overall survival (PFS and OS, respectively) was retrospectively analyzed in 501 Caucasian stage IIIB-IV LADC patients receiving first-line platinum-based chemotherapy (CHT) with or without bevacizumab (BEV). EGFR (epidermal growth factor receptor)-mutant cases were excluded. Of 247 BEV/CHT and 254 CHT patients, 95 (38.5%) and 75 (29.5%) had mutations in KRAS, respectively. KRAS mutation was associated with smoking (p = 0.008) and female gender (p = 0.002) in the BEV/CHT group. We found no difference in OS between patients with KRAS-mutant versus KRAS wild-type tumors in the CHT-alone group (p = 0.6771). Notably, patients with KRAS-mutant tumors demonstrated significantly shorter PFS (p = 0.0255) and OS (p = 0.0186) in response to BEV/CHT compared to KRAS wild-type patients. KRAS mutation was an independent predictor of shorter PFS (hazard ratio, 0.597; p = 0.011) and OS (hazard ratio, 0.645; p = 0.012) in the BEV/CHT group. G12D KRAS-mutant patients receiving BEV/CHT showed significantly shorter PFS (3.7 months versus 8.27 months in the G12/13x group; p = 0.0032) and OS (7.2 months versus 16.1 months in the G12/13x group; p = 0.0144). In this single-center, retrospective study, KRAS-mutant LADC patients receiving BEV/CHT treatment exhibited inferior PFS and OS compared to those with KRAS wild-type advanced LADC. G12D mutations may define a subset of KRAS-mutant LADC patients unsuitable for antiangiogenic therapy with BEV.