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Pancreatic adenocarcinoma is one of the deadliest forms of cancer with no effective therapeutic options. A KRAS mutation can be found in up to 90% of all pancreatic tumors, making it a promising therapeutic target. The introduction of new KRAS inhibitors has been a milestone in the history of KRAS mutant tumors; however, therapeutic resistance limits their efficacy. Thus, new therapeutic options, including combination therapies, are urgently needed. Recently, we have shown that KRAS G12C inhibitors in combination with farnesyl-transferase inhibitors exert synergistic antitumor effects. Here, we provide evidence for the feasibility of this combinational approach to break down resistance in KRAS G12D mutant pancreatic cancer. Although we have shown that the 3D environment dramatically sensitizes cells to MRTX1133 treatment, the synergistic effect of this drug combination is present in both 2D and 3D in the PANC1 pancreatic adenocarcinoma model, which showed high resistance to MRTX1133 in 2D. The effects of the combination treatment show an association with the inhibition of farnesylated regulatory proteins, including HRAS and RHEB, along with the expression level of KRAS. Our study warrants further investigation for the potential applicability of KRAS G12D inhibitors in combination with farnesyl-transferase inhibitors for the treatment of KRAS mutant pancreatic adenocarcinoma., Competing Interests: Author MB was employed by the company Kineto Lab Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Molnár, Baranyi, Szigeti, Hegedűs, Bordás, Gábriel, Petényi, Tóvári, Hegedűs and Tímár.)

Malignant melanoma is a difficult-to-treat skin cancer with increasing incidence worldwide. Although type-I interferon (IFN) is no longer part of guidelines, several melanoma patients are treated with type-I interferon (IFN) at some point of the disease, potentially affecting its genetic progression. We run genome-wide copy number variation (CNV) analysis on previously type-I IFN-treated (n = 17) and control (n = 11) visceral metastases of melanoma patients. Results were completed with data from the TCGA and MM500 databases. We identified metastasis- and brain metastasis-specific gene signatures mostly affected by CN gains. Some cases were genetically resistant to IFN showing characteristic gene alterations (e.g. ABCA4 or ZEB2 gain and alterations of DNA repair genes). Analysis of a previously identified type-I IFN resistance gene set indicates that only a proportion of these genes was exclusive for the IFN-treated metastases reflecting a possible selective genomic pressure of endogenous IFNs during progression. Our data suggest that previous type-I IFN treatment and/or endogenous IFN production by immune response affect genomic progression of melanoma which may have clinical relevance, potentially influence immune checkpoint regulation in the tumor microenvironment., (© 2024. The Author(s).)

Background: 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., Materials and Methods: 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%), technical failures (mostly poor preanalytical fixation) prevented dMMR/MSI testing. Tumors were colorectal (CRC; n: 978), cancer of unknown primary (n: 126), endometrial (n: 39), pancreatic (n: 36), and gastric (n: 33). dMMR was diagnosed as classical, nonclassical, or unusual, depending on IHC., Results: The MSI-high incidence was 12.1% overall and similar in the CRC subcohort. Interestingly, the dMMR incidence was higher in the total cohort (20.3%) and similar in the CRC subcohort. The incidences of proficient MMR (pMMR) and microsatellite stability (MSS) were similar in the total cohort and in the CRC subcohort. A 19.3% discrepancy was found between MMR IHC and MSI PCR for the entire cohort, independent of tumor types. In the case of pMMR, the discrepancy rate for MSS/MSI-low was low (2.0%; entire cohort and the CRC subcohort). However, the discrepancy between dMMR and MSI-high was high within the entire cohort (60.9%) and in the CRC (58.6%) and non-CRC subcohorts (68%). This high discrepancy was not due to tumors with a low T/N ratio. Regarding dMMR phenotypes, classical dMMR had a ~ 60% correlation with MSI-high status, while non-classical dMMR had a much lower and unusual dMMR a very low (< 10%) correlation with MSI-high in the entire cohort and in the CRC subcohort. Overall, the MSI PCR sensitivity for MMR IHC status was very low., Conclusion: dMMR and MSI-high likely result in an increased rate of structurally altered proteins, i.e., neoantigens, and the efficacy of cancer immunotherapies is thus expected to be higher. We compared MMR IHC to MSI PCR in a large cohort of cancer patients to study how PCR test results correlate to MMR IHC. Our data imply that preanalytical factors strongly influence the results of MMR IHC and MSI PCR and may question the current dogma that dMMR phenotype and genetic MSI status are equivalent predictive markers for immunotherapies., Competing Interests: Declarations. Conflict of interest: A. Kiss, M.L. Nádorvári, J. Kulka, T. Barbai, E. Rásó, I. Kenessey, G. Lotz, and J. Tímár declare that they have no competing interests. The retrospective analysis of this cohort was approved by the local Ethics Committee of the Semmelweis University (RKEB62/2023). The author(s) declare that no financial support was receivedfor the research, authorship, and/or publication of this article. The supplement containing this article is not sponsored by industry., (© 2024. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

Current clinical guidelines recommend mismatch repair (MMR) protein immunohistochemistry (IHC) or molecular microsatellite instability (MSI) tests as predictive markers of immunotherapies. Most of the pathological guidelines consider MMR protein IHC as the gold standard test to identify cancers with MMR deficiency and recommend molecular MSI tests only in special circumstances or to screen for Lynch syndrome. However, there are data in the literature which suggest that the two test types may not be equal. For example, molecular epidemiology studies reported different rates of deficient MMR (dMMR) and MSI in various cancer types. Additionally, direct comparisons of the two tests revealed relatively frequent discrepancies between MMR IHC and MSI tests, especially in non-colorectal and non-endometrial cancers and in cases with unusual dMMR phenotypes. There are also scattered clinical data showing that the efficacy of immune checkpoint inhibitors is different if the patient selection was based on dMMR versus MSI status of the cancers. All these observations question the current dogma that dMMR phenotype and genetic MSI status are equal predictive markers of the immunotherapies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Nádorvári, Lotz, Kulka, Kiss and Tímár.)

Background: Inhibition of mutant KRAS challenged cancer research for decades. Recently, allele-specific inhibitors were approved for the treatment of KRAS-G12C mutant lung cancer. However, de novo and acquired resistance limit their efficacy and several combinations are in clinical development. Our study shows the potential of combining G12C inhibitors with farnesyl-transferase inhibitors., Methods: Combinations of clinically approved farnesyl-transferase inhibitors and KRAS G12C inhibitors are tested on human lung, colorectal and pancreatic adenocarcinoma cells in vitro in 2D, 3D and subcutaneous xenograft models of lung adenocarcinoma. Treatment effects on migration, proliferation, apoptosis, farnesylation and RAS signaling were measured by histopathological analyses, videomicroscopy, cell cycle analyses, immunoblot, immunofluorescence and RAS pulldown., Results: Combination of tipifarnib with sotorasib shows synergistic inhibitory effects on lung adenocarcinoma cells in vitro in 2D and 3D. Mechanistically, we present antiproliferative effect of the combination and interference with compensatory HRAS activation and RHEB and lamin farnesylation. Enhanced efficacy of sotorasib in combination with tipifarnib is recapitulated in the subcutaneous xenograft model of lung adenocarcinoma. Finally, combination of additional KRAS G1C and farnesyl-transferase inhibitors also shows synergism in lung, colorectal and pancreatic adenocarcinoma cellular models., Discussion: Our findings warrant the clinical exploration of KRAS-G12C inhibitors in combination with farnesyl-transferase inhibitors., (© 2024. The Author(s).)

Mutated genes may lead to cancer development in numerous tissues. While more than 600 cancer-causing genes are known today, some of the most widespread mutations are connected to the RAS gene; RAS mutations are found in approximately 25% of all human tumors. Specifically, KRAS mutations are involved in the three most lethal cancers in the U.S., namely pancreatic ductal adenocarcinoma, colorectal adenocarcinoma, and lung adenocarcinoma. These cancers are among the most difficult to treat, and they are frequently excluded from chemotherapeutic attacks as hopeless cases. The mutated KRAS proteins have specific three-dimensional conformations, which perturb functional interaction with the GAP protein on the GAP-RAS complex surface, leading to a signaling cascade and uncontrolled cell growth. Here, we describe a gluing docking method for finding small molecules that bind to both the GAP and the mutated KRAS molecules. These small molecules glue together the GAP and the mutated KRAS molecules and may serve as new cancer drugs for the most lethal, most difficult-to-treat, carcinomas. As a proof of concept, we identify two new, drug-like small molecules with the new method; these compounds specifically inhibit the growth of the PANC-1 cell line with KRAS mutation G12D in vitro and in vivo. Importantly, the two new compounds show significantly lower IC 50 and higher specificity against the G12D KRAS mutant human pancreatic cancer cell line PANC-1, as compared to the recently described selective G12D KRAS inhibitor MRTX-1133.

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., (© 2024. The Author(s).)

KRAS mutant lung cancer is the most prevalent molecular subclass of adenocarcinoma (LUAD), which is a heterogenous group depending on the mutation-type which affects not only the function of the oncogene but affects the biological behavior of the cancer as well. Furthermore, KRAS mutation affects radiation sensitivity but leads also to bevacizumab and bisphosphonate resistance as well. It was highly significant that allele specific irreversible inhibitors have been developed for the smoking associated G12C mutant KRAS (sotorasib and adagrasib). Based on trial data both sotorasib and adagrasib obtained conditional approval by FDA for the treatment of previously treated advanced LUAD. Similar to other target therapies, clinical administration of KRASG12C inhibitors (sotorasib and adagrasib) resulted in acquired resistance due to various genetic changes not only in KRAS but in other oncogenes as well. Recent clinical studies are aiming to increase the efficacy of G12C inhibitors by novel combination strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Moldvay and Tímár.)

In silico studies raised the possibility that farnesyltransferase inhibitors (FTIs) may have antitumoral effects on KRAS mutant cancer cells. Accordingly, we have tested FTIs (tipifarnib and lonafarnib) in G12C mutant human cancer cell lines in vitro and in vivo. We have discovered that the combination of the two drugs has a synergistic antitumoral effect. Next, we have tested FTIs on G12D mutant human cancer cell lines and found that the combination has antitumoral effect in various preclinical cancer models. At last, we have also tested FTIs on G12V mutant human cancer cells and again we have detected antitumoral effects. We suggest that FTIs may have clinical relevance outside the HRAS mutant cancers.

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.

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.

Cancer plasticity is now a recognized new hallmark of cancer which is due to disturbances of cell differentiation programs. It is manifested not only in various forms like the best-known epithelial-mesenchymal transition (EMT) but also in vasculogenic and megakaryocytic mimicries regulated by EMT-specific or less-specific transcription factors such as HIF1a or STAT1/2. Studies in the past decades provided ample data that cancer plasticity can be manifested also in the expression of a vast array of immune cell genes; best-known examples are PDL1/CD274, CD47, or IDO, and we termed it immunogenic mimicry (IGM). However, unlike other types of plasticities which are epigenetically regulated, expression of IGM genes are frequently due to gene amplifications. It is important that the majority of the IGM genes are regulated by interferons (IFNs) suggesting that their protein expressions are regulated by the immune microenvironment. Most of the IGM genes have been shown to be involved in immune escape of cancers broadening the repertoire of these mechanisms and offering novel targets for immunotherapeutics., (© 2023. The Author(s).)

The concept of precision medicine is based on the identification of hallmarks of cancer to exploit them as drug targets. The basic idea was that in this way the therapeutic modalities will be more effective and the side effects will be less. Since the majority of these novel modalities are not specific for a cancer-related biological process or a cancer-specific (mutant) target protein, it is not a surprise that we had to learn new type of side effects, because these therapeutics also affect physiological or pathological processes. Even more, in cases of some of these novel therapies we were able to discover new molecular mechanisms of physiological and pathological processes. Identification of the on-target side effects of targeted drugs can help to prevent the development of them or better manage the patients when emerge during cancer therapy., Competing Interests: JT is the editor-in-chief of Pathology and Oncology Research. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tímár and Uhlyarik.)

Skin melanoma became one of the most frequent malignancies of the skin mainly due to the increased exposure to environmental UV irradiation. However, this did not lead to the increase of mortality, mainly due to the efficient early diagnostics as well as to the development of new therapies which were based on the identification of the mutation patterns and understanding of the immunobiology. Molecular markers are important to differentiate malignant tumors from precancerous ones but also routinely available to define prognosis. Immunotherapy can be used in any molecular class of melanoma, while target therapy is only available in case of BRAF mutant form. Meanwhile these novel therapies induce new resistance mechanisms based on the development of acquired genetic alterations. Due to these facts the initial molecular profiling of the resected primary tumors must gradually be replaced by continuous monitoring.

Similar to other malignancies, TCGA network efforts identified the detailed genomic picture of skin melanoma, laying down the basis of molecular classification. On the other hand, genome-wide association studies discovered the genetic background of the hereditary melanomas and the susceptibility genes. These genetic studies helped to fine-tune the differential diagnostics of malignant melanocytic lesions, using either FISH tests or the myPath gene expression signature. Although the original genomic studies on skin melanoma were mostly based on primary tumors, data started to accumulate on the genetic diversity of the progressing disease. The prognostication of skin melanoma is still based on staging but can be completed with gene expression analysis (DecisionDx). Meanwhile, this genetic knowledge base of skin melanoma did not turn to the expected wide array of target therapies, except the BRAF inhibitors. The major breakthrough of melanoma therapy was the introduction of immune checkpoint inhibitors, which showed outstanding efficacy in skin melanoma, probably due to their high immunogenicity. Unfortunately, beyond BRAF , KIT mutations and tumor mutation burden, no clinically validated predictive markers exist in melanoma, although several promising biomarkers have been described, such as the expression of immune-related genes or mutations in the IFN-signaling pathway. After the initial success of either target or immunotherapies, sooner or later, relapses occur in the majority of patients, due to various induced genetic alterations, the diagnosis of which could be developed to novel predictive genetic markers.

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.