Multiplexed RNA-FISH-guided Laser Capture Microdissection RNA Sequencing Improves Breast Cancer Molecular Subtyping, Prognostic Classification, and Predicts Response to Antibody Drug Conjugates.

On a retrospective cohort of 1,082 FFPE breast tumors, we demonstrated the analytical validity of a test using multiplexed RNA-FISH-guided laser capture microdissection (LCM) coupled with RNA-sequencing (mFISHseq), which showed 93% accuracy compared to immunohistochemistry. The combination of these technologies makes strides in i) precisely assessing tumor heterogeneity, ii) obtaining pure tumor samples using LCM to ensure accurate biomarker expression and multigene testing, and iii) providing thorough and granular data from whole transcriptome profiling. We also constructed a 293-gene intrinsic subtype classifier that performed equivalent to the research based PAM50 and AIMS classifiers. By combining three molecular classifiers for consensus subtyping, mFISHseq alleviated single sample discordance, provided near perfect concordance with other classifiers (κ > 0.85), and reclassified 30% of samples into different subtypes with prognostic implications. We also use a consensus approach to combine information from 4 multigene prognostic classifiers and clinical risk to characterize high, low, and ultra-low risk patients that relapse early (< 5 years), late (> 10 years), and rarely, respectively. Lastly, to identify potential patient subpopulations that may be responsive to treatments like antibody drug-conjugates (ADC), we curated a list of 92 genes and 110 gene signatures to interrogate their association with molecular subtype and overall survival. Many genes and gene signatures related to ADC processing (e.g., antigen/payload targets, endocytosis, and lysosome activity) were independent predictors of overall survival in multivariate Cox regression models, thus highlighting potential ADC treatment-responsive subgroups. To test this hypothesis, we constructed a unique 19-feature classifier using multivariate logistic regression with elastic net that predicted response to trastuzumab emtansine (T-DM1; AUC = 0.96) better than either ERBB2 mRNA or Her2 IHC alone in the T-DM1 arm of the I-SPY2 trial. This test was deployed in a research-use only format on 26 patients and revealed clinical insights into patient selection for novel therapies like ADCs and immunotherapies and de-escalation of adjuvant chemotherapy.

Osimertinib with or without Chemotherapy in EGFR-Mutated Advanced NSCLC.

BACKGROUND: Osimertinib is a third-generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) that is selective for EGFR-TKI-sensitizing and EGFR T790M resistance mutations. Evidence suggests that the addition of chemotherapy may extend the benefits of EGFR-TKI therapy. METHODS: In this phase 3, international, open-label trial, we randomly assigned in a 1:1 ratio patients with EGFR-mutated (exon 19 deletion or L858R mutation) advanced non-small-cell lung cancer (NSCLC) who had not previously received treatment for advanced disease to receive osimertinib (80 mg once daily) with chemotherapy (pemetrexed [500 mg per square meter of body-surface area] plus either cisplatin [75 mg per square meter] or carboplatin [pharmacologically guided dose]) or to receive osimertinib monotherapy (80 mg once daily). The primary end point was investigator-assessed progression-free survival. Response and safety were also assessed. RESULTS: A total of 557 patients underwent randomization. Investigator-assessed progression-free survival was significantly longer in the osimertinib-chemotherapy group than in the osimertinib group (hazard ratio for disease progression or death, 0.62; 95% confidence interval [CI], 0.49 to 0.79; P<0.001). At 24 months, 57% (95% CI, 50 to 63) of the patients in the osimertinib-chemotherapy group and 41% (95% CI, 35 to 47) of those in the osimertinib group were alive and progression-free. Progression-free survival as assessed according to blinded independent central review was consistent with the primary analysis (hazard ratio, 0.62; 95% CI, 0.48 to 0.80). An objective (complete or partial) response was observed in 83% of the patients in the osimertinib-chemotherapy group and in 76% of those in the osimertinib group; the median response duration was 24.0 months (95% CI, 20.9 to 27.8) and 15.3 months (95% CI, 12.7 to 19.4), respectively. The incidence of grade 3 or higher adverse events from any cause was higher with the combination than with monotherapy - a finding driven by known chemotherapy-related adverse events. The safety profile of osimertinib plus pemetrexed and a platinum-based agent was consistent with the established profiles of the individual agents. CONCLUSIONS: First-line treatment with osimertinib-chemotherapy led to significantly longer progression-free survival than osimertinib monotherapy among patients with EGFR-mutated advanced NSCLC. (Funded by AstraZeneca; FLAURA2 ClinicalTrials.gov number, NCT04035486.).

Alcoholic Liver Disease-Related Hepatocellular Carcinoma: Characteristics and Comparison to General Slovak Hepatocellular Cancer Population.

Hepatocellular carcinoma (HCC) has multiple molecular classes that are associated with distinct etiologies and, besides particular molecular characteristics, that also differ in clinical aspects. We aim to characterize the clinical aspects of alcoholic liver disease-related HCC by a retrospective observational study that included all consequent patients diagnosed with MRI or histologically verified HCC in participating centers from 2010 to 2016. A total of 429 patients were included in the analysis, of which 412 patients (96%) had cirrhosis at the time of diagnosis. The most common etiologies were alcoholic liver disease (ALD) (48.3%), chronic hepatitis C (14.9%), NAFLD (12.6%), and chronic hepatitis B (10%). Patients with ALD-related HCC were more commonly males, more commonly had cirrhosis that was in more advanced stages, and had poorer performance status. Despite these results, no differences were observed in the overall (median 8.1 vs. 8.5 months) and progression-free survival (median 4.9 vs. 5.7 months). ALD-HCC patients within BCLC stage 0-A less frequently received potentially curative treatment as compared to the control HCC patients (62.2% vs. 87.5%, p = 0.017); and in patients with ALD-HCC liver function (MELD score) seemed to have a stronger influence on the prognosis compared to the control group HCC. Systemic inflammatory indexes were strongly associated with survival in the whole cohort. In conclusion, alcoholic liver disease is the most common cause of hepatocellular carcinoma in Slovakia, accounting for almost 50% of cases; and patients with ALD-related HCC more commonly had cirrhosis that was in more advanced stages and had poorer performance status, although no difference in survival between ALD-related and other etiology-related HCC was observed.

Inflammatory Indexes as Prognostic Factors of Survival in Geriatric Patients with Hepatocellular Carcinoma: A Case Control Study of Eight Slovak Centers.

Background and Aims: Hepatocellular cancer (HCC) often occurs in geriatric patients. The aim of our study was to compare overall survival and progression-free survival between geriatric patients (>75 years) and patients younger than 75 years and to identify predictive factors of survival in geriatric patients with HCC. Material and Methods: We performed a retrospective analysis of patients with HCC diagnosed in Slovakia between 2010−2016. Cases (HCC patients ≥75 years) were matched to controls (HCC patients <74 years) based on the propensity score (gender, BCLC stage and the first-line treatment). Results: We included 148 patients (84 men, 57%) with HCC. There were no differences between cases and controls in the baseline characteristics. The overall survival in geriatric patients with HCC was comparable to younger controls (p = 0.42). The one-, two-, and three-year overall survival was 42% and 31%, 19% and 12%, and 12% and 9% in geriatric patients and controls, respectively (p = 0.2, 0.4, 0.8). Similarly, there was no difference in the one- and two-year progression-free survival: 28% and 18% vs. 10% and 7% in geriatric HCC patients and controls, respectively (p = 0.2, 1, -). There was no case−control difference between geriatric HCC patients and younger HCC controls in the overall survival in the subpopulation of patients with no known comorbidities (p = 0.5), one and two comorbidities (p = 0.49), and three or more comorbidities (p = 0.39). Log (CRP), log (NLR), log (PLR), and log (SII) were all associated with the three-year survival in geriatric HCC patients in simple logistic regression analyses. However, this time, only log (NLR) remained associated even after controlling for the age and BCLC confounding (OR 5.32, 95% CI 1.43−28.85). Conclusions. We found no differences in overall survival and progression-free survival between older and younger HCC patients. Parameters of subclinical inflammation predict prognosis in geriatric patients with HCC. A limitation of the study is small number of the treated patients; therefore, further investigation is warranted.

Autologous dendritic cell-based immunotherapy (DCVAC/LuCa) and carboplatin/paclitaxel in advanced non-small cell lung cancer: A randomized, open-label, phase I/II trial.

PURPOSE: To investigate the efficacy and safety of an active cellular immunotherapy (DCVAC/LuCa) and chemotherapy in patients with stage IV non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: SLU01 was a multicenter, open-label, parallel-group, randomized, phase I/II trial. NSCLC patients were randomized in a ratio of 1:1:1 to receive: DCVAC/LuCa and chemotherapy (carboplatin and paclitaxel; Group A); DCVAC/LuCa, chemotherapy, pegylated interferon-α2b, and hydroxychloroquine (Group B); or chemotherapy alone (Group C). DCVAC/LuCa was administered subcutaneously every 3-6 weeks (up to 15 doses). The primary endpoint was overall survival (OS). During the study, enrollment into Group B was discontinued for strategic reasons. RESULTS: Forty-five patients were randomized to Group A, 29 patients to Group B, and 38 patients to Group C. The median OS in the modified intention-to-treat (mITT) population was 3.7 months longer in Group A than in Group C (15.5 vs. 11.8 months; p = 0.0179; hazard ratio = 0.54; 95% confidence interval: 0.32-0.91). This OS effect was consistent across subgroups of the mITT population (females, males, current smokers, former smokers, and patients with non-squamous and squamous cell histology). The most common treatment-emergent adverse events of any grade reported in Groups A, B, and C, respectively, were neutropenia (50.0%, 29.6%, and 20.6%), fatigue (40.0%, 18.5%, and 20.6%), anemia (35.0%, 44.4%, and 32.4%), paresthesia (27.5%, 25.9%, and 17.6%), and alopecia (25.0%, 29.6%, and 41.2%). CONCLUSION: DCVAC/LuCa in combination with carboplatin and paclitaxel extended OS and was well tolerated.

Nonalcoholic Fatty Liver Disease-Related Hepatocellular Carcinoma: Clinical Patterns, Outcomes, and Prognostic Factors for Overall Survival-A Retrospective Analysis of a Slovak Cohort.

OBJECTIVE: To compare NAFLD-related HCC and other etiology-related HCC and to describe predictive factors for survival in patients with NAFLD-related HCC independent of the BCLC staging system. METHODS: We performed a multicenter longitudinal retrospective observational study of patients diagnosed with HCC during the period from 2010 through 2016. RESULTS: 12.59% of patients had NAFLD-related HCC, and 21.91% had either NAFLD or cryptogenic etiology. NAFLD-related HCC patients were younger (p = 0.0007), with a higher proportion of women (p < 0.001) compared to other etiology-related HCC patients. The NAFLD group had a significantly lower proportion of patients with liver cirrhosis at the time of HCC diagnosis (p < 0.0001), and they were more frequently diagnosed with both diabetes and metabolic syndrome when compared to other etiology-related HCC (p < 0.0001). We did not find any difference in the overall survival or in the progression-free survival between NAFLD-related and other etiology-related HCC patients staged as BCLC B and BCLC C. NAFLD-related HCC patients with three or more liver lesions had a shorter overall survival when compared to patients with one or two liver lesions (p = 0.0097), while patients with baseline CRP values of ≥5 mg/L or with PLR ≥ 150 had worse overall survival (p = 0.012 and p = 0.0028, respectively). ALBI Grade 3 predicted worse overall survival compared to ALBI Grade 1 or 2 (p = 0.00021). In NAFLD-related HCC patients, PLR and ALBI remained significant predictors of overall survival even after adjusting for BCLC. CONCLUSION: NAFLD-related HCC patients have a similar prognosis when compared to other etiology-related HCC. In NAFLD-related HCC patients, ALBI and PLR are significant predictors of the overall survival independent of the BCLC staging system.

Proteomic analysis of the vitamin C effect on the doxorubicin cytotoxicity in the MCF-7 breast cancer cell line.

PURPOSE: Doxorubicin is an anthracycline drug which inhibits the growth of breast cancer cell lines. However, a major factor limiting its use is a cumulative, dose-dependent cardiotoxicity, resulting in a permanent loss of cardiomyocytes. Vitamin C was found to potentiate the cytotoxic effects of a variety of chemotherapeutic drugs including doxorubicin. The aim of the study was to describe the changes in protein expression and proliferation of the MCF-7 cells induced by the vitamin C applied with doxorubicin. METHODS: Label-free quantitative proteomics and real-time cell analysis methods were used to search for proteome and cell proliferation changes. These changes were induced by the pure DOX and by DOX combined with vitamin C applied on the MCF-7 cell line. RESULTS: From the real-time cell analysis experiments, it is clear that the highest anti-proliferative effect occurs with the addition of 200 µM of vitamin C to 1 µM of doxorubicin. By applying both the label-free protein quantification method and total ion current assay, we found statistically significant changes (p ≤ 0.05) of 26 proteins induced by the addition of vitamin C to doxorubicin on the MCF-7 cell line. These differentially expressed proteins are involved in processes such as structural molecule activity, transcription and translation, immune system process and antioxidant, cellular signalling and transport. CONCLUSION: The detected proteins may be capable of predicting response to DOX therapy. This is a key tool in the treatment of breast cancer, and the combination with vit C seems to be of particular interest due to the fact that it can potentiate anti-proliferative effect of DOX.