Long-Term Complete Response and Survival in Metastatic Extramammary Paget's Disease Treated With Trastuzumab Plus Paclitaxel: A Case Report.

Extramammary Paget's disease is a rare skin cancer that usually arises from the secretory cells of the apocrine glands. In most cases, an extramammary Paget's tumor occurs as a single intraepithelial form not associated with another cancer, although rarely, it may be associated with other loco-regional or distant cancer. It is generally slow-growing and diagnosed in situ. Most often, surgical excision with wide margins is curative, with the local recurrence rate being lower after the Mohs micrographic surgery technique. Nonetheless, relapses are frequent. In the metastatic setting, there are no treatment guidelines or standard therapies; additionally, the experience is limited to a few individual cases, and the efficacy of conventional chemotherapies is not well-defined. Moreover, chemotherapy can also have serious side effects; therefore, there is a need to identify more effective and less toxic therapies. In this case report, we have observed a long-lasting complete response with anti-HER2 plus paclitaxel.

Early Changes in LIPI Score Predict Immune-Related Adverse Events: A Propensity Score Matched Analysis in Advanced Non-Small Cell Lung Cancer Patients on Immune Checkpoint Blockade.

In advanced cancer patients undergoing immune checkpoint blockade, the burden of immune-related adverse events (irAEs) is high. The need for reliable biomarkers for irAEs remains unfulfilled in this expanding therapeutic field. The lung immune prognostic index (LIPI) is a noninvasive measure of systemic inflammation that has consistently shown a correlation with survival in various cancer types when assessed at baseline. This study sought to determine whether early changes in the LIPI score could discriminate the risk of irAEs and different survival outcomes in advanced non-small cell lung cancer (NSCLC) patients receiving PD-(L)1 blockade-based therapies. We included consecutive patients diagnosed with metastatic NSCLC who received pembrolizumab, nivolumab, or atezolizumab as second-line therapy following platinum-based chemotherapy, or first-line pembrolizumab either alone or in combination with platinum-based chemotherapy. The LIPI score relied on the combined values of derived neutrophil/lymphocyte ratio (dNLR) and lactate dehydrogenase. Their assessment at baseline and after two cycles of treatment allowed us to categorize the population into three subgroups with good (LIPI-0), intermediate (LIPI-1), and poor (LIPI-2) prognosis. Between April 2016 and May 2023, we enrolled a total of 345 eligible patients, 165 (47.8%) and 180 (52.2%) of whom were treated as first- and second-line at our facility, respectively. After applying propensity score matching, we considered 83 relevant patients in each cohort with a homogeneous distribution of all characteristics across the baseline LIPI subgroups. There was a noticeable change in the distribution of LIPI categories due to a significant decrease in dNLR values during treatment. Although no patients shifted to a worse prognosis category, 20 (24.1%) transitioned from LIPI-1 to LIPI-0, and 7 (8.4%) moved from LIPI-2 to LIPI-1 (p < 0.001). Throughout a median observation period of 7.3 (IQR 3.9-15.1) months, a total of 158 irAEs (63.5%) were documented, with 121 (48.6%) and 39 (15.7%) patients experiencing mild to moderate and severe adverse events, respectively. Multivariate logistic regression analysis showed that the classification and changes in the LIPI score while on treatment were independent predictors of irAEs. The LIPI-0 group was found to have significantly increased odds of experiencing irAEs. Following a median follow-up period of 21.1 (95% CI 17.9-25.8) months, the multivariable Cox model confirmed LIPI categorization at any given time point as a significant covariate with influence on overall survival, irrespective of the treatment line. These findings suggest that reassessing the LIPI score after two cycles of treatment could help pinpoint patients particularly prone to immune-related toxicities. Those who maintain a good LIPI score or move from the intermediate to good category would be more likely to develop irAEs. The continuous assessment of LIPI provides prognostic insights and could be useful for predicting the benefit of PD-(L)1 checkpoint inhibitors.

Herpes zoster after the third dose of SARS-CoV-2 mRNA-BNT162b2 vaccine in actively treated cancer patients: a prospective study.

Several concerns have been raised about a causal relationship between COVID-19 mRNA-based vaccines and the development of herpes zoster (HZ). We performed a prospective analysis of the Vax-On-Third-Profile study to investigate the incidence of HZ after the third dose of mRNA-BNT162b2 (tozinameran) and its correlation with immune responses. Patients who had received a booster dose and had been actively treated for at least 8 weeks were eligible. Serologic assessment was performed before the third dose of tozinameran (timepoint-1) and 4 weeks later (timepoint-2). We also assessed the incidence of SARS-CoV-2 breakthrough infections at predefined time points. The current analysis included 310 patients, of whom 109 (35.2%) and 111 (35.8%) were being treated with targeted therapies and cytotoxic chemotherapy, respectively. All participants received a third dose of tozinameran between September 26 and October 30, 2021. After a mean follow-up of 17.3 (IQR 15.1-18.4) months, HZ occurred in 8 recipients, for a cumulative incidence of 2.6%, and an incidence rate of 0.310 per person-year (95% CI 0.267-0.333). All HZ cases occurred within 30 days of booster dosing (range 5-29 days), with a median time to onset of 15 (IQR 9-22) days. Among the 7 patients (2.2%) who also contracted a SARS-CoV-2 infection, all cases preceded COVID-19 outbreaks. No instances of complicated HZ were reported. In multivariate analysis, impaired T helper and T cytotoxic cell counts independently correlated with HZ occurrence. These findings provide the first evidence that cancer patients on active treatment have a not negligible risk of developing HZ within 30 days after the third dose of tozinameran. The favorable clinical outcome of all observed cases confirms that protective effects of boosters in reducing the risk of severe COVID-19 outweigh the potential risk of HZ occurrence.

Immune responses and clinical outcomes following the third dose of SARS-CoV-2 mRNA-BNT162b2 vaccine in advanced breast cancer patients receiving targeted therapies: a prospective study.

Purpose: Metastatic breast cancer patients are the most prevalent oncology population with advanced disease facing COVID-19 pandemic. Immune responses after mRNA-based vaccination during treatment with CDK4/6 inhibitors or HER2-directed agents remain unclear. We conducted a prospective analysis to elucidate changes in antibody titers and lymphocyte counts following full course of mRNA-BNT162b2 (tozinameran) vaccination in recipients undergoing these targeted therapies. Methods: Patients who had received a booster dosing and had been treated for at least 6 months were eligible. Antibody titers against SARS-CoV-2 spike protein were measured at four subsequent time points. Immunophenotyping of circulating lymphocytes was performed before the third dose of tozinameran and four weeks later to quantify the absolute counts of CD3+CD4+ T-helper cells, CD3+CD8+ T-cytotoxic cells, CD19+ B cells, and CD56+CD16+ NK cells. We also assessed the incidence of breakthrough infections and investigated whether immune changes affect time-to-treatment failure (TTF) after booster vaccination. Results: The current analysis included 69 patients, of whom 38 (55%) and 31 (45%) were being treated with CDK4/6 inhibitors and HER2-targeted therapies, respectively. All participants received a third dose of tozinameran between September 23 and October 7, 2021. Multivariate analysis revealed that CDK4/6 inhibition predicted a significantly impaired humoral response after the booster dose. This detrimental effect was also evident for T-helper cell counts before the third immunization, but it disappeared in the subsequent evaluation. After a median follow-up of 22.3 months, we observed 19 (26%) cases of COVID-19 outbreaks, all experiencing favorable clinical outcomes. Univariate analysis showed a significant association between the onset of SARS-CoV-2 infections and the use of CDK4/6 inhibitors, as well as with an impaired antibody and T-helper cell response. Only the last two covariates remained independent predictors after multivariate testing. Dynamic variations in antibody titers and T-helper cell counts did not affect TTF in multivariate regression analysis. Conclusions: Our results confirm that the immune response to tozinameran is impaired by CDK4/6 inhibitors, increasing the odds of breakthrough infections despite the third vaccine dose. Current evidence recommends maintaining efforts to provide booster immunizations to the most vulnerable cancer patients, including those with advanced breast cancer undergoing CDK4/6 inhibition.

Effects of Acetaminophen Exposure on Outcomes of Patients Receiving Immune Checkpoint Inhibitors for Advanced Non-Small-Cell Lung Cancer: A Propensity Score-Matched Analysis.

(1) Background: Several studies have investigated potential interactions between immune checkpoint inhibitors (ICIs) and commonly prescribed medications. Although acetaminophen (APAP) has not been considered susceptible to interaction with ICIs, recent research has shown that detectable plasma levels of this drug can hinder the efficacy of PD-1/PD-L1 blockade therapies. A reliable assessment of the potential interaction between APAP and ICIs in advanced non-small cell lung cancer (NSCLC) patients would be worthwhile since it is often prescribed in this condition. We sought to evaluate the impact of the concomitant use of APAP in patients with advanced NSCLC on PD-1/PD-L1 blockade using real-world evidence. (2) Methods: This study included consecutive patients with histologically proven stage IV NSCLC who underwent first-line therapy with pembrolizumab as a single agent or in combination with platinum-based chemotherapy, or second-line therapy with pembrolizumab, nivolumab, or atezolizumab. The intensity of APAP exposure was classified as low (therapeutic intake lasting less than 24 h or a cumulative intake lower than 60 doses of 1000 mg) or high (therapeutic intake lasting more than 24 h or a total intake exceeding 60 doses of 1000 mg). The favorable outcome of anti-PD-1/PD-L1 therapies was defined by durable clinical benefit (DCB). Progression-free survival (PFS) and overall survival (OS) were relevant to our efficacy analysis. Propensity score matching (PSM) methods were applied to adjust for differences between the APAP exposure subgroups. (3) Results: Over the course of April 2018 to October 2022, 80 patients were treated with first-line pembrolizumab either as single-agent therapy or in combination with platinum-based chemotherapy. During the period from June 2015 to November 2022, 145 patients were given anti-PD-1/PD-L1 blockade therapy as second-line treatment. Subsequent efficacy analyses relied on adjusted PSM populations in both treatment settings. Multivariate testing revealed that only the level of APAP and corticosteroid intake had an independent effect on DCB in both treatment lines. Multivariate Cox regression analysis confirmed high exposure to APAP and immunosuppressive corticosteroid therapy as independent predictors of shorter PFS and OS in both treatment settings. (4) Conclusions: Our findings would strengthen the available evidence that concomitant intake of APAP blunts the efficacy of ICIs in patients with advanced NSCLC. The detrimental effects appear to depend on the cumulative dose and duration of exposure to APAP. The inherent shortcomings of the current research warrant confirmation in larger independent series.

Changes in Peripheral Immune Cells after the Third Dose of SARS-CoV-2 mRNA-BNT162b2 Vaccine and Disease Outcomes in Cancer Patients Receiving Immune Checkpoint Inhibitors: A Prospective Analysis of the Vax-on-Third-Profile Study.

BACKGROUND: Anti-SARS-CoV-2 mRNA vaccines can deeply affect cell-mediated immune responses in immunocompromised recipients, including cancer patients receiving active treatments. The clinical implications of changes in peripheral blood lymphocyte subsets following the third dose of mRNA-BNT162b2 vaccination (tozinameran) in patients on immune checkpoint blockade are not fully understood. We conducted a prospective analysis of the Vax-On-Third-Profile study to evaluate the impact of circulating lymphocyte dynamics on disease outcomes in this subgroup of patients. METHODS: Recipients of booster dosing who had received before vaccination at least one course of an anti-PD-1/PD-L1 treatment for an advanced solid tumor were eligible. Immunophenotyping of peripheral blood was performed before the third dose of tozinameran (timepoint-1) and four weeks later (timepoint-2) to quantify the absolute counts of lymphocyte subpopulations, including CD3+CD4+ T cells, CD3+CD8+ T cells, B cells, and NK cells. Logistic regression was used to analyze the relationship between lymphocyte subsets and durable clinical benefit (DCB). The log-rank test and Cox regression model were applied to evaluate the relationship between lymphocyte subpopulations and both vaccine-related time-to-treatment failure (V-TTF) and overall survival (OS). RESULTS: We included a total of 56 patients with metastatic disease who were given a third dose of tozinameran between 23 September and 7 October 2021 (median age: 66 years; male: 71%). Most recipients had a diagnosis of lung cancer and were being treated with pembrolizumab or nivolumab. Compared to baseline, the third immunization resulted in an incremental change in the median counts of all lymphocyte subpopulations, which was statistically significant only for NK cells (p < 0.001). A significant correlation was found between NK cell counts and DCB at timepoint-2 (p < 0.001). Multivariate logistic regression analysis of DCB confirmed the predictive significance of high-level NK cell counts (p = 0.020). In multivariate Cox regression analysis, high-level NK cell counts independently predicted longer V-TTF [HR 0.34 (95% CI 0.14-0.80), p = 0.014] and OS [HR 0.36 (95% CI 0.15-0.89), p = 0.027]. CONCLUSIONS: Our data suggest expansion of NK cell counts as the most noteworthy change in circulating lymphocytes after the third dose of tozinameran in cancer patients receiving PD-1/PD-L1-targeted agents. This change correlated with enhanced therapeutic efficacy, improving the rate of disease control, and prolonging survival outcomes. Similar findings have not been previously reported, implying that they have proof-of-concept value and warrant further confirmation.

Immune-related adverse events and disease outcomes after the third dose of SARS-CoV-2 mRNA-BNT162b2 vaccine in cancer patients receiving immune checkpoint inhibitors.

BACKGROUND: The clinical implications of the third dose of coronavirus disease 2019 (COVID-19) vaccines in patients receiving immune checkpoint inhibitors are currently unknown. We performed a prospective analysis of the Vax-On-Third study to investigate the effects of antibody response on immune-related adverse events (irAEs) and disease outcomes. METHODS: Recipients of the booster dose of SARS-CoV-2 mRNA-BNT162b2 vaccine who had received at least one course of an anti-PD-1/PD-L1 treatment before vaccination for an advanced solid malignancy were eligible. RESULTS: The current analysis included 56 patients with metastatic disease (median age: 66 years; male: 71%), most of whom had a lung cancer diagnosis and were being treated with pembrolizumab- or nivolumab-based regimens. The optimal cut-point antibody titer of 486 BAU/mL allowed a dichotomization of recipients into low-responders (Low-R, < 486 BAU/mL) or high-responders (High-R, ≥ 486 BAU/mL). After a median follow-up time of 226 days, 21.4% of patients experienced moderate to severe irAEs without any recrudescence of immune toxicities preceding the booster dose. The frequencies of irAE before and after the third dose did not differ, but an increase in the cumulative incidence of immuno-related thyroiditis was observed within the High-R subgroup. On multivariate analysis, an enhanced humoral response correlated with a better outcome in terms of durable clinical benefit, which resulted in a significant reduction in the risk of disease control loss but not mortality. CONCLUSIONS: Our findings would strengthen the recommendation not to change anti-PD-1/PD-L1 treatment plans based on current or future immunization schedules, implying that all these patients should be closely monitored.

Co-feeding strategy alleviates hypoxic stress in large-scale bioreactor for optimal production of secretory recombinant proteins in Pichia pastoris.

The loss of mixing efficiency inherent to bioreactor process operated at large-scale yields to the formation of concentration gradient and thus to heterogeneous culture conditions. For processes operated with methanol feeding, P. pastoris faces oscillatory culture conditions that significantly affect the cell ability to produce secretory recombinant proteins at high yield. Extended cell residence time in microenvironments of high methanol concentration and low oxygen availability that are typically found in the upper part of the bioreactor near the feeding point, triggers the unfolded protein response (UPR) and thus impairs proper protein secretion. Methanol co-feeding with sorbitol was shown herein to reduce the UPR response and to restore productivity of secreted protein.

Effects of Antibody Response after Booster Vaccination on SARS-CoV-2 Breakthrough Infections and Disease Outcomes in Advanced Cancer Patients: A Prospective Analysis of the Vax-on-Third Study.

(1) Background: The clinical implications of COVID-19 outbreaks following SARS-CoV-2 vaccination in immunocompromised recipients are a worldwide concern. Cancer patients on active treatment remain at an increased risk of developing breakthrough infections because of waning immunity and the emergence of SARS-CoV-2 variants. There is a paucity of data on the effects of COVID-19 outbreaks on long-term survival outcomes in this population. (2) Methods: We enrolled 230 cancer patients who were on active treatment for advanced disease and had received booster dosing of an mRNA-BNT162b2 vaccine as part of the Vax-On-Third trial between September 2021 and October 2021. Four weeks after the third immunization, IgG antibodies against the spike receptor domain of SARS-CoV-2 were tested in all patients. We prospectively evaluated the incidence of breakthrough infections and disease outcomes. The coprimary endpoints were the effects of antibody titers on the development of breakthrough infections and the impact of COVID-19 outbreaks on cancer treatment failure. (3) Results: At a median follow-up of 16.3 months (95% CI 14.5-17.0), 85 (37%) patients developed SARS-CoV-2 infection. Hospitalization was required in 11 patients (12.9%) and only 2 (2.3%) deaths related to COVID-19 outbreaks were observed. Median antibody titers were significantly lower in breakthrough cases than in non-cases (291 BAU/mL (95% CI 210-505) vs. 2798 BAU/mL (95% CI 2323-3613), p < 0.001). A serological titer cut-off below 803 BAU/mL was predictive of breakthrough infection. In multivariate testing, antibody titers and cytotoxic chemotherapy were independently associated with an increased risk of outbreaks. Time-to-treatment failure after booster dosing was significantly shorter in patients who contracted SARS-CoV-2 infection (3.1 months (95% CI 2.3-3.6) vs. 16.2 months (95% CI 14.3-17.0), p < 0.001) and had an antibody level below the cut-off (3.6 months (95% CI 3.0-4.5) vs. 14.6 months (95% CI 11.9-16.3), p < 0.001). A multivariate Cox regression model confirmed that both covariates independently had a worsening effect on time-to-treatment failure. (4) Conclusions: These data support the role of vaccine boosters in preventing the incidence and severity of COVID-19 outbreaks. Enhanced humoral immunity after the third vaccination significantly correlates with protection against breakthrough infections. Strategies aimed at restraining SARS-CoV-2 transmission in advanced cancer patients undergoing active treatment should be prioritized to mitigate the impact on disease outcomes.

Is heterogeneity in large-scale bioreactors a real problem in recombinant protein synthesis by Pichia pastoris?

Culture medium heterogeneity is inherent in industrial bioreactors. The loss of mixing efficiency in a large-scale bioreactor yields to the formation of concentration gradients. Consequently, cells face oscillatory culture conditions that may deeply affect their metabolism. Herein, cell response to transient perturbations, namely high methanol concentration combined with hypoxia, has been investigated using a two stirred-tank reactor compartiments (STR-STR) scale-down system and a Pichia pastoris strain expressing the gene encoding enhanced green fluorescent protein (eGFP) under the control of the alcohol oxidase 1 (AOX1) promoter. Cell residence times under transient stressing conditions were calculated based on the typical hydraulic circulation times of bioreactors of tens and hundreds cubic metres. A significant increase in methanol and oxygen uptake rates was observed as the cell residence time was increased. Stressful culture conditions impaired biomass formation and triggered cell flocculation. More importantly, both expression levels of genes under the control of pAOX1 promoter and eGFP specific fluorescence were higher in those oscillatory culture conditions, suggesting that those a priori unfavourable culture conditions in fact benefit to recombinant protein productivity. Flocculent cells were also identified as the most productive as compared to ovoid cells. KEY POINTS: • Transient hypoxia and high methanol trigger high level of recombinant protein synthesis • In Pichia pastoris, pAOX1 induction is higher in flocculent cells • Medium heterogeneity leads to morphological diversification.

Engineering of Chinese hamster ovary cells for co-overexpressing MYC and XBP1s increased cell proliferation and recombinant EPO production.

Improving the cellular capacity of Chinese hamster ovary (CHO) cells to produce large amounts of therapeutic proteins remains a major challenge for the biopharmaceutical industry. In previous studies, we observed strong correlations between the performance of CHO cells and expression of two transcription factors (TFs), MYC and XBP1s. Here, we have evaluated the effective of overexpression of these two TFs on CHO cell productivity. To address this goal, we generated an EPO-producing cell line (CHOEPO) using a targeted integration approach, and subsequently engineered it to co-overexpress MYC and XBP1s (a cell line referred to as CHOCXEPO). Cells overexpressing MYC and XBP1s increased simultaneously viable cell densities and EPO production, leading to an enhanced overall performance in cultures. These improvements resulted from the individual effect of each TF in the cell behaviour (i.e., MYC-growth and XBP1s-productivity). An evaluation of the CHOCXEPO cells under different environmental conditions (temperature and media glucose concentration) indicated that CHOCXEPO cells increased cell productivity in high glucose concentration. This study showed the potential of combining TF-based cell engineering and process optimisation for increasing CHO cell productivity.

Dynamic changes in peripheral lymphocytes and antibody response following a third dose of SARS-CoV-2 mRNA-BNT162b2 vaccine in cancer patients.

The aim of this study was to evaluate the association of circulating lymphocytes profiling with antibody response in cancer patients receiving the third dose of COVID-19 mRNA-BNT162b2 vaccine. Immunophenotyping of peripheral blood was used to determine absolute counts of lymphocyte subsets, alongside detection of IgG antibodies against receptor-binding-domain (RBD) of the SARS-CoV-2 Spike protein (S1) before booster dosing (timepoint-1) and four weeks afterward (timepoint-2). An IgG titer ≥ 50 AU/mL defined a positive seroconversion response. An IgG titer ≥ 4446 AU/mL was assumed as a correlate of 50% vaccine efficacy against symptomatic infections. A total of 258 patients on active treatment within the previous six months were enrolled between September 23 and October 7, 2021. The third dose resulted in an exponential increase in median anti-RBD-S1 IgG titer (P < 0.001), seroconversion rates (P < 0.001), and 50% vaccine efficacy rates (P < 0.001). According to ROC curve analysis, T helper and B cells were significantly associated with seroconversion responses at timepoint-1, whereas only B cells were relevant to 50% vaccine efficacy rates at timepoint-2. A positive linear correlation was shown between anti-RBD-S1 IgG titers and these lymphocyte subset counts. Multivariate analysis ruled out a potential role of T helper cells but confirmed a significant interaction between higher B cell levels and improved antibody response. These findings suggest that peripheral counts of B cells correlate with humoral response to the third dose of mRNA-BNT162b2 vaccine in actively treated cancer patients and could provide insights into a more comprehensive assessment of vaccination efficacy.

Enhanced recombinant protein production in CHO cell continuous cultures under growth-inhibiting conditions is associated with an arrested cell cycle in G1/G0 phase.

Low temperature and sodium butyrate (NaBu) are two of the most used productivity-enhancing strategies in CHO cell cultures during biopharmaceutical manufacturing. While these two approaches alter the balance in the reciprocal relationship between cell growth and productivity, we do not fully understand their mechanisms of action beyond a gross cell growth inhibition. Here, we used continuous culture to evaluate the differential effect of low temperature and NaBu supplementation on CHO cell performance and gene expression profile. We found that an increase in cell-productivity under growth-inhibiting conditions was associated with the arrest of cells in the G1/G0 phase. A transcriptome analysis revealed that the molecular mechanisms by which low temperature and NaBu arrested cell cycle in G1/G0 differed from each other through the deregulation of different cell cycle checkpoints and regulators. The individual transcriptome changes in pattern observed in response to low temperature and NaBu were retained when these two strategies were combined, leading to an additive effect in arresting the cell cycle in G1/G0 phase. The findings presented here offer novel molecular insights about the cell cycle regulation during the CHO cell bioprocessing and its implications for increased recombinant protein production. This data provides a background for engineering productivity-enhanced CHO cell lines for continuous manufacturing.

Real-Life Experience of the Prognostic Significance of the Primary Tumor Location on the Timing of Colorectal Liver Metastases: A Retrospective Analysis.

Background Numerous research studies have looked into how the primary tumor location (PTL) affects patients' prognosis for colorectal cancer (CRC). Our research aimed to investigate the prognostic effects of PTL in patients with synchronous (SM) and metachronous (MM) colorectal cancer liver metastases (CRCLM). Material and methods From 2016 to 2021, we looked back at the records of patients at our institute who were affected by CRCLM. Results 109 patients were included, of whom 21.1% received CRCLM resection (R0=73.9%), with 57.7% having left-sided colon cancer (LCC) and 42.2% having right-sided colon cancer (RCC). SM predominated (69.7%). The median duration of follow-up was 21,3 months (95%CI=15,4-25,2). ≥5 hepatic metastases prevailed in the SM group (N=61; 83.5%). 21% of all patients underwent CRCLM resection (R0=78.2%). We observed a double rate of patients unresponsive to standard systemic antineoplastic treatments in the SM group (35.8% vs. 17.9% of the MM group) (p=0.27). We found a significantly longer median overall survival (OS) in patients with MM-LCC compared with the other groups (27.7 months; HR=0.3797; 95%CI=0.19-0.74; p=0.0205). The median OS, regardless of PTL, was higher in the MM group (16,5 months vs. 16,1 months; HR=0,29; 95%CI=0,13-0,67; p=0.0038) as well as progression-free survival (PFS) (11 months vs. 10,2 months; HR=0,61; 95%CI=0,33-1,12; p=0.11). Finally, in patients undergoing liver surgery, a noteworthy median OS was shown to be significantly in favor of patients with metachronous liver metastases from the primary left tumor (37.0 months; HR=0.47; 95%CI=0.11-1.96; p=0.0041). Conclusions Our real-life study demonstrated that patients with LCC, particularly MM-LCC, have the highest survival and that the timing of CRCLM should be a prognostic factor.

Role of Dissimilative Pathway of Komagataella phaffii (Pichia pastoris): Formaldehyde Toxicity and Energy Metabolism.

Komagataella phaffii (aka Pichia pastoris) is a yeast able to grow in methanol as the sole carbon and energy source. This substrate is converted into formaldehyde, a toxic intermediary that can either be assimilated to biomass or dissimilated to CO2 through the enzymes formaldehyde dehydrogenase (FLD) and formate dehydrogenase, also producing energy in the form of NADH. The dissimilative pathway has been described as an energy producing and a detoxifying route, but conclusive evidence has not been provided for this. In order to elucidate this theory, we generated mutants lacking the FLD activity (Δfld1) and used flux analysis to evaluate the metabolic impact of this disrupted pathway. Unexpectedly, we found that the specific growth rate of the Δfld1 strain was only slightly lower (92%) than the control. In contrast, the sensitivity to formaldehyde pulses (up to 8mM) was significantly higher in the Δfld1 mutant strain and was associated with a higher maintenance energy. In addition, the intracellular flux estimation revealed a high metabolic flexibility of K. phaffii in response to the disrupted pathway. Our results suggest that the role of the dissimilative pathway is mainly to protect the cells from the harmful effect of formaldehyde, as they were able to compensate for the energy provided from this pathway when disrupted.

Dynamic Changes of Peripheral NK Cells Predict Outcome in Patients with PD-L1 Positive Non-small-cell Lung Cancer Undergoing Immune Checkpoint Inhibitors as Second-line Therapy.

We evaluated immune cell frequencies in peripheral blood samples of 41 NSCLC patients before and after second-line therapy with anti-PD-1/PD-L1 agents. Changes in lymphocyte subsets and their correlation with clinical response, progression-free survival (PFS), and overall survival (OS) were analyzed. We observed an increase in median values of all lymphocyte subsets, being significant only for NK cells. A correlation was retrieved between higher post-treatment NK cell level and clinical benefit. On multivariate analysis, PD-L1 tumor proportion score ≥1% and higher post-treatment NK cell counts were predictive of longer PFS and OS. Co-presence of these factors was characterized by longer survival.

Peripheral lymphocyte subset counts predict antibody response after SARS-CoV-2 mRNA-BNT162b2 vaccine in cancer patients: Results from the Vax-On-Profile study.

BACKGROUND: The adaptive immune response following COVID-19 vaccination is essential for humoral immunogenicity and clinical protection against symptomatic infections. We present the results of circulating lymphocyte profiling and their correlation with antibody response in cancer patients tested serologically six months after receiving a two-dose schedule of mRNA-BNT162b2 vaccine. METHODS: Absolute counts of lymphocyte subsets were determined using peripheral blood immunophenotyping. We collected samples for flow cytometry analysis alongside quantitative detection of IgG antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein (S1). An IgG titer ≥ 50 AU/mL defined a positive seroconversion response. RESULTS: 311 patients were evaluable for lymphocyte profiling and serologic testing. A preliminary multivariate analysis revealed that cytotoxic chemotherapy was the most consistent factor associated with lower counts of all lymphocyte subpopulations. T helper and B cells were found to be useful in predicting the occurrence of a positive seroconversion response using ROC curve analysis. A significant positive linear correlation was shown when anti-RBD-S1 IgG titers were compared to these lymphocyte subset counts. Univariate analysis indicated that antibody titers and seroconversion rates were significantly improved in the high-level T and B cell subgroups. Multivariate analysis confirmed these significant interactions, as well as the negative predictive value of immunosuppressive corticosteroid therapy. CONCLUSIONS: These findings suggest that simple and widely available peripheral counts of T helper and B cells correlate with humoral response to mRNA-BNT162b2 vaccine in actively treated cancer patients. Upon validation, our results could provide additional insights into the predictive assessment of vaccination efficacy.

Advances in Cell Engineering of the Komagataella phaffii Platform for Recombinant Protein Production.

Komagataella phaffii (formerly known as Pichia pastoris) has become an increasingly important microorganism for recombinant protein production. This yeast species has gained high interest in an industrial setting for the production of a wide range of proteins, including enzymes and biopharmaceuticals. During the last decades, relevant bioprocess progress has been achieved in order to increase recombinant protein productivity and to reduce production costs. More recently, the improvement of cell features and performance has also been considered for this aim, and promising strategies with a direct and substantial impact on protein productivity have been reported. In this review, cell engineering approaches including metabolic engineering and energy supply, transcription factor modulation, and manipulation of routes involved in folding and secretion of recombinant protein are discussed. A lack of studies performed at the higher-scale bioreactor involving optimisation of cultivation parameters is also evidenced, which highlights new research aims to be considered.

Immune-related Thyroid Dysfunction (irTD) in Non-small Cell Lung Cancer (NSCLC) Correlates With Response and Survival.

Background: There is no clear information on the proportion of patients who need therapy for immune-related thyroid dysfunction (irTD) or who need to delay, omit, or discontinue immunotherapy. Furthermore, it is not well known whether irTD correlates with better outcomes or not. Patients and Methods: We conducted a retrospective study in patients with metastatic non-small cell lung cancer (NSCLC) treated with anti-PD1 or anti-PD-L1. Results: Our study enrolled 75 patients, 25.3% of them developed immune-related thyroid dysfunction. Three patients delayed a course of immunotherapy due to irTD, 2 patients omitted a course and 1 patient permanently discontinued. In patients with irTD compared with those without irTD the ORR was 42.1% vs. 7.1% (p<0.001), DCR was 78.9% vs. 32.1% (p<0.001); mPFS was 15.7 vs. 3.6 months (p<0.001) and mOS was 18.6 months vs. 5.1 months (p<0.001). Conclusion: Immune-related thyroid dysfunction has a mild impact on the immunotherapy treatment program. The occurrence of irTD correlates with more favorable response and survival.

Recombinant protein production in Pichia pastoris: from transcriptionally redesigned strains to bioprocess optimization and metabolic modelling.

Pichia pastoris is one of the most widely used host for the production of recombinant proteins. Expression systems that rely mostly on promoters from genes encoding alcohol oxidase 1 or glyceraldehyde-3-phosphate dehydrogenase have been developed together with related bioreactor operation strategies based on carbon sources such as methanol, glycerol, or glucose. Although, these processes are relatively efficient and easy to use, there have been notable improvements over the last twenty years to better control gene expression from these promoters and their engineered variants. Methanol-free and more efficient protein production platforms have been developed by engineering promoters and transcription factors. The production window of P. pastoris has been also extended by using alternative feedstocks including ethanol, lactic acid, mannitol, sorbitol, sucrose, xylose, gluconate, formate or rhamnose. Herein, the specific aspects that are emerging as key parameters for recombinant protein synthesis are discussed. For this purpose, a holistic approach has been considered to scrutinize protein production processes from strain design to bioprocess optimization, particularly focusing on promoter engineering, transcriptional circuitry redesign. This review also considers the optimization of bioprocess based on alternative carbon sources and derived co-feeding strategies. Optimization strategies for recombinant protein synthesis through metabolic modelling are also discussed.