Comparison of standard mismatch repair deficiency and microsatellite instability tests in a large cancer series.

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.

Identification of a Tumor Cell Associated Type I IFN Resistance Gene Expression Signature of Human Melanoma, the Components of Which Have a Predictive Potential for Immunotherapy.

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.

[Prognostic and predictive markers of malignant melanoma]. / A malignus melanóma prognosztikus és prediktív markerei.

Malignant melanoma biologically can be divided into non-metastatic and metastatic forms which cannot be predicted precisely using classical clinicopathological parameters, therefore studies on novel genetic or protein markers are abundant in the literature. These studies did not result in clinically useful markers because mostly ignored the results of studies on the genetic basis of metastatic potential of malignant melanoma. Accordingly, the list of promising novel markers is short (BCL2, CDK2, MART-1, OPN). Similar to other solid malignancies, introduction of targeted therapy into clinical practice of melanoma turned the attention toward the genetic basis of resistance to chemo- and targeted therapies. These novel data could lead to the development of molecular diagnostics which can help in designing more effective therapeutic strategies of malignant melanoma.

[Molecular epidemiology of microsatellite instability/mismatch repair deficiency in our institute - methodical aspects]. / A mikroszatellita-instabilitás/mismatch repair deficiencia genetikai hibák molekuláris epidemiológiája intézetünkben: módszertani megfontolások.

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.

Liver alterations and detection of SARS-CoV-2 RNA and proteins in COVID-19 autopsies.

The most severe alterations in Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) infection are seen in the lung. However, other organs also are affected. Here, we report histopathologic findings in the liver and detection of viral proteins and RNA in COVID-19 autopsies performed at the Semmelweis University (Budapest, Hungary). Between March 2020 through March 2022, 150 autopsies on patients who died of COVID-19 were analyzed. Cause-of-death categories were formed based on the association with SARS-CoV-2 as strong, contributive, or weak. Samples for histopathologic study were obtained from all organs, fixed in formalin, and embedded in paraffin (FFPE). Immunohistochemical study (IHC) to detect SARS-CoV-2 spike protein and nucleocapsid protein (NP), CD31, claudin-5, factor VIII, macrosialin (CD68), and cytokeratin 7, with reverse transcriptase polymerase chain reaction (RT-PCR), and in situ hybridization (ISH, RNAscope®) for SARS-CoV-2 RNA were conducted using FFPE samples of livers taken from 20 autopsies performed ≤ 2 days postmortem. All glass slides were scanned; the digital images were evaluated by semiquantitative scoring and scores were analyzed statistically. Steatosis, single-cell and focal/zonal hepatocyte necrosis, portal fibrosis, and chronic inflammation were found in varying percentages. Sinusoidal ectasia, endothelial cell disruption, and fibrin-filled sinusoids were seen in all cases; these were assessed semiquantitatively for severity (SEF scored). SEF scores did not correlate with cause-of-death categories (p = 0.92) or with severity of lung alterations (p = 0.96). SARS-CoV-2 RNA was detected in 13/20 cases by PCR and in 9/20 by ISH, with IHC demonstration of spike protein in 4/20 cases and NP in 15/20. Viral RNA and proteins were located in endothelial and Kupffer cells, and in portal macrophages, but not in hepatocytes and cholangiocytes. In conclusion, endothelial damage (SEF scores) was the most common alteration in the liver and was a characteristic, but not specific alteration in COVID-19, suggesting an important role in the pathogenesis of COVID-19-associated liver disease. Detection of SARS-CoV-2 RNA and viral proteins in liver non-parenchymal cells suggests that while the most extended primary viral cytotoxic effect occurs in the lung, viral components are present in other organs too, as in the liver. The necrosis/apoptosis and endothelial damage associated with viral infection in COVID-19 suggest that those patients who survive more severe COVID-19 may face prolonged liver repair and accordingly should be followed regularly in the post-COVID period.

Characteristics of CD44 alternative splice pattern in the course of human colorectal adenocarcinoma progression.

BACKGROUND: CD44 is considered as 'a' metastasis associated gene, despite the fact that it is an umbrella term for a group of molecules produced from a single gene by alternative splicing. However, little consideration is given to the above in the literature of colorectal carcinomas as well as other tumour types, leading to confusion and contradictory results about its possible role in tumour progression. METHODS: We compared the CD44 alternative splice pattern (ASP) of three genetically different human colorectal cancer cell lines (HT25, HT29, HCT116) using a series of PCR reactions and next- generation sequencing method, as well as identified a colorectal adenocarcinoma specific CD44 ASP. This ASP was further investigated in terms of its qualitative and quantitative stability in our experimental iso- and xenograft mouse models for colorectal cancer progression. A complex preclinical experimental set-up was established to separately test the different steps of tumour progression and the role of tumour microenvironment, respectively, focusing on the role of 'CD44' in this process. RESULTS: We managed to present a colorectal cancer-specific CD44 ASP, which remained unchanged from cell lines throughout primary tumour formation and metastatic progression. Furthermore, we report a unique roster of all expressed CD44 variant isoforms characteristic to colorectal cancer. Finally, on quantitative assessment of the variable exons v3 and v6, higher co-expression levels were found to be characteristic to metastatically potent tumour cells. CONCLUSION: Particular CD44 variant isoforms seem to act as "metastasis genes" via tumour microenvironment-driven shifts in v3 and v6 expressions. However, this function may just affect a minority of tumour subclones. This fact and the huge potential number of different CD44 splice variants that can contain v3 and v6 domains can explain incoherence of clinical studies regarding functional asessment of CD44 variants, as well as diminish the chances of using CD44 variants for predictive purpose.

[Effect of KRAS mutation status on the efficiency of Avastin therapy of colorectal cancer]. / A kolorektális rákok KRAS-mutációs státuszának hatása az Avastin-terápia hatékonyságára.

Anti-angiogenic therapy became a standard care of advanced colorectal cancer. Since the most frequent genetic alteration of colorectal cancer is KRAS mutation we have analyzed its effect on the efficacy of Avastin treatment. Since 2008 we have determined the KRAS status of 575 patients with colorectal carcinoma using a sensitive screening method and sequencing. In our database the frequency of KRAS mutation in colorectal cancer is 37% (codon 12: 31% followed by codon 13: 6%). We have examined the effect of KRAS status on the efficacy of Avastin treatment in 35 patients. Progression-free survival of KRAS mutant patients was highly similar to that of wild-type patients using log-rank test (9.2+/-5.5 months versus 8.7+/-5.7 months, respectively). Our data support those observations that KRAS status of colorectal cancer does not interfere with the efficacy of Avastin treatment.

EGFR Alterations Influence the Cetuximab Treatment Response and c-MET Tyrosine-Kinase Inhibitor Sensitivity in Experimental Head and Neck Squamous Cell Carcinomas.

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.

[Predictive relevance of KRAS mutational status in bone metastatic lung adenocarcinoma treated with bisphosphonate therapy]. / A KRAS-mutációs státusz prediktív szerepe biszfoszfonáttal kezelt, csontáttétet képzo tüdo-adenokarcinómában.

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.

The effects of bisphosphonate and radiation therapy in bone-metastatic lung adenocarcinoma: the impact of KRAS mutation.

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.

KRAS Mutations Predict Response and Outcome in Advanced Lung Adenocarcinoma Patients Receiving First-Line Bevacizumab and Platinum-Based Chemotherapy.

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.

Phenotype of bone metastases of non-small cell lung cancer: epidermal growth factor receptor expression and K-RAS mutational status.

Bone metastasis is a frequent complication of lung cancer progression, however, studies on bone metastatic tissues are scanty. Here we have collected a small cohort of 11 non-small cell lung cancer cases where primary tumors and corresponding bone metastases were available for pathological analysis. We have tested two molecular markers: EGFR protein expression and K-RAS mutation at codon 12 using immunohistochemistry and RFLPPCR, respectively. We have shown that using improved protocols, EGFR protein (both the extracellular as well as the cytoplasmic domain) is readily detectable in decalcified bone tissue. We found that the EGFR expression status is highly similar in bone metastases compared to the primary tumors, although the expression levels may change. Individual comparison of corresponding primary and metastatic NSCLC tissues indicated that downregulation of EGFR was a rare event (2/11) compared to upregulation (4/11) in bone metastases. On the other hand, our data indicate that the K-RAS mutational status of the primary tumor does not predict the status of the bone metastatic tissue of NSCLC, since we have observed both emergence of mutant clones in metastases from wild-type (wt) primary tumors and loss of mutant clones in metastases from mutant primaries in addition to the maintained mutant status. Our data support that at least two progression models occur in NSCLC, the samegene as well as the clonal selection one. It is noteworthy that in NSCLC cases with wt- or mutant KRAS, downregulation of EGFR expression was a rare event although upregulation in bone metastases was observed more frequently in wt K-RAS cases.

Aquaporin 1 protein expression is associated with BRAF V600 mutation and adverse prognosis in cutaneous melanoma.

Despite experimental findings suggesting the prognostic significance of Aquaporin 1 (AQP1) in human melanoma, no published clinical data are available. We studied the expression of AQP1 protein in cutaneous melanoma, correlated our findings with standard histological and genetic markers, and long-term clinical follow-up. Our study evaluated the AQP1 protein expression in 78 melanoma patients, representing two predefined risk cohorts using the immune labeling technique with commercially available anti-AQP1 antibodies on routinely formalin-fixed and paraffin-embedded tumor tissue samples. BRAF V600E mutation analyses were carried out successfully in 70 patients using PCR and restriction fragment length polymorphism analyses, followed by confirmatory analysis with the Sanger sequencing technique. AQP1-expressing melanoma cells were found in 52 cases (66.7%, median H-score=124.24). Significantly higher AQP1 H-scores (P=0.047) were found in the 'high-risk' patients. No correlations were found with the established histological markers, such as mitotic index (P=0.42), Clark level (P=0.95), and Breslow thickness (P=0.51). BRAF V600 mutation analyses were successful in 89%, and showed a two times higher mutation frequency in the 'high-risk' group. The BRAF V600 mutations were significantly associated with AQP1 expression (P=0.014). Long-term follow-up indicated a reduced progression-free survival (P=0.036) and overall survival (P=0.017) for the AQP1-positive cutaneous melanoma patients. AQP1 expression is likely to be associated with an adverse prognosis in cutaneous melanoma.

The importance of microenvironment: the role of CCL8 in metastasis formation of melanoma.

We have attempted to characterize the changes occurring on the host side during the progression of human melanoma. To investigate the role of tumor microenvironment, we set up such an animal model, which was able to isolate the host related factors playing central role in metastasis formation. One of these 'factors', CCL12, was consequently selected and its behavior was examined alongside its human homologue (CCL8). In our animal model, metastasis forming primary melanoma in the host exhibited increased level of CCL12 mRNA expression. In clinical samples, when examining the tumor and the host together, the cumulative (tumor and host) CCL8 expression was lower in the group in which human primary melanoma formed lung metastasis compared to non-metastatic primary tumors. We could not detect significant difference in CCL8 receptor (CCR1) expression between the two groups. Increased migration of the examined tumor cell lines was observed when CCL8 was applied as a chemoattractant. The tumor cells and their interactions can be influenced the expression of CCL8 by dermal fibroblasts, as a significant change in the metastatic microenvironment. Furthermore, we examined changes in miRNA profile resulted by CCL8 and miR146a appears to be a promising prognostic marker for following this process.

Sensitivity of Melanoma Cells to EGFR and FGFR Activation but Not Inhibition is Influenced by Oncogenic BRAF and NRAS Mutations.

BRAF and NRAS are the two most frequent oncogenic driver mutations in melanoma and are pivotal components of both the EGF and FGF signaling network. Accordingly, we investigated the effect of BRAF and NRAS oncogenic mutation on the response to the stimulation and inhibition of epidermal and fibroblast growth factor receptors in melanoma cells. In the three BRAF mutant, two NRAS mutant and two double wild-type cell lines growth factor receptor expression had been verified by qRT-PCR. Cell proliferation and migration were determined by the analysis of 3-days-long time-lapse videomicroscopic recordings. Of note, a more profound response was found in motility as compared to proliferation and double wild-type cells displayed a higher sensitivity to EGF and FGF2 treatment when compared to mutant cells. Both baseline and induced activation of the growth factor signaling was assessed by immunoblot analysis of the phosphorylation of the downstream effectors Erk1/2. Low baseline and higher inducibility of the signaling pathway was characteristic in double wild-type cells. In contrast, oncogenic BRAF or NRAS mutation did not influence the response to EGF or FGF receptor inhibitors in vitro. Our findings demonstrate that the oncogenic mutations in melanoma have a profound impact on the motogenic effect of the activation of growth factor receptor signaling. Since emerging molecularly targeted therapies aim at the growth factor receptor signaling, the appropriate mutational analysis of individual melanoma cases is essential in both preclinical studies and in the clinical trials and practice.

Distinct Epidemiology and Clinical Consequence of Classic Versus Rare EGFR Mutations in Lung Adenocarcinoma.

INTRODUCTION: Although classic sensitizing mutations of epidermal growth factor receptor (EGFR) are positive predictive markers for EGFR tyrosine kinase inhibitors (TKIs) in lung adenocarcinoma, there are rare EGFR mutations with unknown epidemiology and influence on prognosis and TKI response. METHODS: Eight hundred and fourteen lung adenocarcinoma patients with KRAS and/or EGFR mutation analyses for TKI therapy indication were identified. Six hundred and forty-five patients were included in the epidemiological analysis. The clinical outcome was analyzed in 419 advanced-stage patients with follow-up data. RESULTS: Four hundred and eighty (59%) KRAS/EGFR double wild-type, 216 (27%) KRAS mutant, 42 (5%) classic, 49 (6%) rare, and 27 (3%) synonymous EGFR mutant cases were identified. Twenty previously unpublished non-synonymous mutations were found. Rare EGFR mutations were significantly associated with smoking (vs. classic EGFR mutations; p = 0.0062). Classic EGFR mutations but not rare ones were independent predictors of increased overall survival (hazard ratios, 0.45; 95% confidence intervals, 0.25-0.82; p = 0.009). TKI therapy response rate of patients harboring classic EGFR mutations was significantly higher (vs. rare EGFR mutations; 71% vs. 37%; p = 0.039). Patients with classic or sensitizing rare (G719x and L861Q) EGFR mutations had significantly longer progression-free survival when compared with the remaining rare mutation cases (12 vs. 6.2 months; p = 0.048). CONCLUSIONS: The majority of rare EGFR mutations was associated with smoking, shorter overall survival, and decreased TKI response when compared with classic EGFR mutations. However, studies characterizing the TKI sensitizing effect of individual rare mutations are indispensable to prevent the exclusion of patients with sensitizing rare EGFR mutations who may benefit from anti-EGFR therapy.

Subtype-specific KRAS mutations in advanced lung adenocarcinoma: a retrospective study of patients treated with platinum-based chemotherapy.

BACKGROUND: Platinum-based chemotherapy is the most common treatment in advanced-stage lung adenocarcinoma. Because the clinical significance of KRAS mutational status in this setting has not yet been clearly determined, a mutation subtype-specific analysis was performed in the so far largest cohort of Caucasian patients with KRAS mutant advanced-stage lung adenocarcinoma treated with platinum-based chemotherapy. METHODS: 505 Caucasian stage III-IV lung adenocarcinoma patients with known amino acid substitution-specific KRAS mutational status and treated with platinum-based chemotherapy were included. The correlations of subtype-specific KRAS mutations with smoking status, progression-free and overall survival (PFS and OS, respectively) and therapeutic response were analysed. RESULTS: Among 338 KRAS wild-type, 147 codon 12 mutant and 20 codon 13 mutant patients, there were no mutation-related significant differences in PFS or OS (P values were 0.534 and 0.917, respectively). Eastern Cooperative Oncology Group (ECOG) status and clinical stage were significant independent prognostic factors. KRAS mutation showed a significant correlation with smoking status (P=0.018). Importantly, however, G12V KRAS mutant patients were significantly more frequent among never-smokers than all other codon 12 KRAS mutant (G12x) subtypes (P=0.016). Furthermore, this subgroup tended to have a higher response rate (66% versus 47%; P=0.077). A modestly longer median PFS was also found in the G12V mutant cohort (233days; versus 175days in the G12x group; P=0.145). CONCLUSIONS: While KRAS mutation status per se is neither prognostic nor predictive in stage III-IV lung adenocarcinoma, subtype-specific analysis may indeed identify clinically relevant subgroups of patients that may ultimately influence treatment decisions.

Demonstration of a melanoma-specific CD44 alternative splicing pattern that remains qualitatively stable, but shows quantitative changes during tumour progression.

The role of CD44 in the progression of human melanoma has mostly been characterised by qualitative changes in expression of its individual variable exons. These exons however, may be expressed to form a number of molecules, the alternative splice variants of CD44, which may be structurally and functionally different. Using real-time PCR measurements with variable exon specific primers we have determined that all are expressed in human melanoma. To permit comparison between different tumours we identified a stable CD44 variable exon (CD44v) expression pattern, or CD44 'fingerprint'. This was found to remain unchanged in melanoma cell lines cultured in different matrix environments. To evaluate evolution of this fingerprint during tumour progression we established a scid mouse model, in which the pure expression pattern of metastatic primary tumours, circulating cells and metastases, non-metastatic primary tumours and lung colonies could be studied. Our analyses demonstrated, that although the melanoma CD44 fingerprint is qualitatively stable, quantitative changes are observed suggesting a possible role in tumour progression.

EGFR autophosphorylation but not protein score correlates with histologic and molecular subtypes in lung adenocarcinoma.

Established clinicopathologic characteristics of non-small cell lung cancer patients define a subgroup responding better to EGFR-TK inhibitors: adenocarcinoma histology, ethnicity, sex, smoking status, presence of activating EGFR mutation, and/or K-RAS wild type. However, EGFR mutation does not automatically lead to increased activity of the protein influenced by several factors. As adenocarcinoma can be further divided into histologic subclasses, we compared adenocarcinomas without lepidic growth pattern (NLAC) to those characterized by pure or predominant lepidic growth (LAC) for EGFR protein expression and autophosphorylation activity (Y1173), as determined by immunohistochemistry. This pretarget therapy cohort comprised a total of 110 surgically operated patients of stage I non-small cell lung cancer: 49 NLAC and 61 LAC variants. The LAC group had a significantly better prognosis and the incidence of phospho-EGFR-positive tumors was significantly higher compared with NLAC. Patient sex did not influence EGFR activity, but the incidence of pEGFR-positive tumors was significantly lower among smoker patients. There was no statistically significant difference in EGFR or KRAS mutation frequencies between the 2 groups. In NLAC, pEGFR-positive tumors occurred exclusively among EGFR-mutant/K-RAS wild-type tumors. On the contrary, in LAC tumors, pEGFR-positive tumors were similarly frequent in the EGFR or K-RAS mutant groups indicating an interesting feedback activation of EGFR signaling in K-RAS mutant tumors. Our data also indicate that EGFR mutation leads to EGFR autophosphorylation only in a small fraction of adenocarcinoma patients, which might have clinical significance.

Molecular characteristics of fibrolamellar hepatocellular carcinoma.

Fibrolamellar hepatocellular carcinoma (FLC) occurs in non-cirrhotic liver and the etiopathogenesis is still obscure. Both hepatocellular and cholangiocellular markers are expressed in the tumor, however, molecular alterations and altered pathways playing role in the tumor pathogenesis are not clearly identified. The purpose of the present study was to compare the expression level of EGFR, syndecan-1 and ß-catenin in FLC, conventional hepatocellular carcinoma (cHCC) and cholangiocellular carcinoma (CCC) and to investigate the possibility of mutation both in EGFR and K-RAS. Eight FLCs were compared with 7 cHCCs, 7 CCCs and 5 normal liver samples. Cytokeratins 7, 8, 18, 19, HepPar1 (HSA), EGFR, syndecan-1 (CD138) and ß-catenin were detected by immunohistochemistry. In addition EGFR, ß-catenin and syndecan-1 were evaluated by digital morphometry and K-RAS, EGFR mutations in FLC cases using paraffin-embedded samples. All FLCs were positive for HepPar1 (HSA) and cytokeratins 7, 8, 18, but negative for cytokeratin 19 by immunohistochemistry. EGFR was significantly overexpressed in all three tumor types, being highest in FLCs (p = 0,0001). EGFR, K-RAS mutation analyses revealed no mutations in exons studied in FLCs. Our findings proved that expression of EGFR is higher in FLC than in other types of primary malignant hepatic tumors and no K-RAS mutation can be detected, so FLC is a good candidate for anti-EGFR treatment.