Immunotherapy for patients with advanced non-small cell lung cancer harboring oncogenic driver alterations other than EGFR: a multicenter real-world analysis.

Background: The administration of immune checkpoint inhibitors (ICIs) in advanced non-small cell lung cancer (NSCLC) with oncogenic driver alterations other than epidermal growth factor receptor (EGFR) aroused a heated discussion. We thus aimed to evaluate ICI treatment in these patients in real-world routine clinical practice. Methods: A multicenter, retrospective study was conducted for NSCLC patients with at least one gene alteration (KRAS, HER2, BRAF, MET, RET, ALK, ROS1) receiving ICI monotherapy or combination treatment. The data regarding clinicopathologic characteristics, clinical efficacy, and safety were investigated. Results: A total of 216 patients were included, the median age was 60 years, 72.7% of patients were male, and 46.8% had a smoking history. The molecular alterations involved KRAS (n=95), HER2 (n=42), BRAF (n=22), MET (n=21), RET (n=14), ALK (n=14), and ROS1 (n=8); 56.5% of patients received immunotherapy in the first-line, and the rest 43.5% were treated as a second-line and above. For the entire cohort who received immunotherapy-based regimens in the first-line, the median progression-free survival (PFS) was 7.5 months and the median overall survival (OS) was 24.8 months. For the entire cohort who received immunotherapy-based regimens in the second-line and above, the median PFS was 4.7 months and median OS was 17.1 months. KRAS mutated NSCLC treated with immunotherapy-based regimens in the first-line setting had a median PFS and OS were 7.8 and 26.1 months, respectively. Moreover, the median PFS and OS of immunotherapy-based regimens for KRAS-mutant NSCLC that progressed after chemotherapy were 5.9 and 17.1 months. Programmed death ligand 1 (PD-L1) expression level was not consistently associated with response to immunotherapy across different gene alteration subsets. In the KRAS group, PD-L1 positivity [tumor proportion score (TPS) ≥1%] was associated with better PFS and OS according to the multivariate Cox analysis. No statistically significant association was found for smoking status, age, or gender with clinical efficacy in any gene group analyses. Conclusions: KRAS-mutant NSCLC could obtain clinical benefits from ICIs either for treatment-naive patients or those who have experienced progression after chemotherapy, and PD-L1 positive expression (TPS >1%) may be a potential positive predictor. For NSCLC with ALK, RET and ROS1 rearrangement, MET exon 14 skipping mutation, or BRAF V600E mutation, effectiveness of single or combined ICI therapy remains limited, therefore, targeted therapies should be considered prior to immunotherapy regimens. Future studies should address the investigation of better predictive biomarkers for immunotherapy response in oncogene-driven NSCLC.

Comparative analysis of 10X Chromium vs. BD Rhapsody whole transcriptome single-cell sequencing technologies in complex human tissues.

The development of single-cell omics tools has enabled scientists to study the tumor microenvironment (TME) in unprecedented detail. However, each of the different techniques may have its unique strengths and limitations. Here we directly compared two commercially available high-throughput single-cell RNA sequencing (scRNA-seq) technologies - droplet-based 10X Chromium vs. microwell-based BD Rhapsody - using paired samples from patients with localized prostate cancer (PCa) undergoing a radical prostatectomy. Although high technical consistency was observed in unraveling the whole transcriptome, the relative abundance of cell populations differed. Cells with low mRNA content such as T cells were underrepresented in the droplet-based system, at least partly due to lower RNA capture rates. In contrast, microwell-based scRNA-seq recovered less cells of epithelial origin. Moreover, we discovered platform-dependent variabilities in mRNA quantification and cell-type marker annotation. Overall, our study provides important information for selection of the appropriate scRNA-seq platform and for the interpretation of published results.

Intravenous iron-induced hypophosphatemia and kidney stone disease.

Patients with Crohn's disease are at increased risk for symptomatic nephrolithiasis. Stones in these patients are most commonly composed of calcium oxalate monohydrate or mixed calcium-oxalate and calcium-phosphate. Precipitation of both minerals depends on urinary pH, calcium, phosphate and oxalate excretion. The present manuscript reports on two patients with Crohn's disease and bowel resection, in whom the onset of symptomatic urolithiasis occurred after repeated infusions of ferric carboxymaltose - a drug, which is known to cause hyperphosphaturia. The present study shows that ferric carboxymaltose-induced hyperphosphaturia can be associated with kidney stone formation and symptomatic urolithiasis, especially in patients treated with calcitriol. Calcitriol has been shown to mitigate ferric carboxymaltose-induced secondary hyperparathyroidism and hyperphosphaturia, but is known to increase urinary calcium excretion. Chemical analysis of recovered stones revealed that they were mixed calcium oxalate and phosphate stones. Ring-like deposition of iron detected by spatially resolved elemental analysis using laser ablation-inductively coupled plasma mass spectrometry, showed that the stones also contained iron. Based on our findings, we propose that patients with inflammatory bowel disease requiring intravenous iron therapy should be carefully monitored for the development of hypophosphatemia and urolithiasis. If hypophosphatemia occurs in such patients, calcitriol should be used with caution.

The impact and relevance of techniques and fluids on lung injury in machine perfusion of lungs.

Primary graft dysfunction (PGD) is a common complication after lung transplantation. A plethora of contributing factors are known and assessment of donor lung function prior to organ retrieval is mandatory for determination of lung quality. Specialized centers increasingly perform ex vivo lung perfusion (EVLP) to further assess lung functionality and improve and extend lung preservation with the aim to increase lung utilization. EVLP can be performed following different protocols. The impact of the individual EVLP parameters on PGD development, organ function and postoperative outcome remains to be fully investigated. The variables relate to the engineering and function of the respective perfusion devices, such as the type of pump used, functional, like ventilation modes or physiological (e.g. perfusion solutions). This review reflects on the individual technical and fluid components relevant to EVLP and their respective impact on inflammatory response and outcome. We discuss key components of EVLP protocols and options for further improvement of EVLP in regard to PGD. This review offers an overview of available options for centers establishing an EVLP program and for researchers looking for ways to adapt existing protocols.

Case report: Dissolving carotid plaque associated to Lorlatinib-related dyslipidemia.

We present a case with prolonged Lorlatinib-related dyslipidemia causing internal carotid artery stenosis, putting the patient at risk of cerebrovascular events. Through intensified lipid-lowering treatment and dose reduction of Lorlatinib, LDL-C levels decreased markedly. Surprisingly, the left sided internal carotid artery stenosis dissolved accordingly. Due to the high efficacy of the new selective tyrosine kinase inhibitors and resulting long-term treatment, it is essential to carefully follow-up and include drug specific side effect monitoring. This case emphasizes that Loraltinib-related dyslipidemia has to be taken seriously and treatment should be initiated as promptly as possible. We conclude that in cases were lipid dysregulation remains and Lorlatinib treatment has to be continued, cerebrovascular appraisal through ultrasound should be considered and, if stenosis is evident, intensified treatment regimen of dyslipidemia or dose reduction of Lorlatinib should be discussed in an interdisciplinary setting.

The multivisceral landscape of colorectal cancer metastasis: implications for targeted therapies.

Colorectal cancer (CRC) is among the most common cancer types and the second deadliest malignancy for both sexes. Metastatic disease poses substantial therapeutic challenges, and peritoneal spread, in particular, reduces quality of life and has a dismal outcome. In this issue of the JCI, Berlin and authors have made considerable advancements in understanding the cellular and molecular composition of multivisceral CRC metastasis in a sophisticated murine orthotopic organoid model and in humans. The study provides unprecedented insights into the complex biology of the disease and points toward the development of compartmentalized immune-therapeutic strategies.

Beyond binary: bridging neutrophil diversity to new therapeutic approaches in NSCLC.

Neutrophils represent the most abundant myeloid cell subtype in the non-small-cell lung cancer (NSCLC) tumor microenvironment (TME). By anti- or protumor polarization, they impact multiple aspects of tumor biology and affect sensitivity to conventional therapies and immunotherapies. Single-cell RNA sequencing (scRNA-seq) analyses have unraveled an extensive neutrophil heterogeneity, helping our understanding of their pleiotropic role. In this review we summarize recent data and models on tumor-associated neutrophil (TAN) biology, focusing on the diversity that evolves in response to tumor-intrinsic cues. We categorize available transcriptomic profiles from different cancer entities into a defined set of neutrophil subclusters with distinct phenotypic properties, to step beyond the traditional binary N1/2 classification. Finally, we discuss potential ways to exploit these neutrophil states in the setting of anticancer therapy.

Glucocorticoid treatment influences prostate cancer cell growth and the tumor microenvironment via altered glucocorticoid receptor signaling in prostate fibroblasts.

Despite significant therapeutic advances in recent years, treatment of metastatic prostate cancer (PCa) remains palliative, owing to the inevitable occurrence of drug resistance. There is increasing evidence that epithelial glucocorticoid receptor (GR) signaling and changes in the tumor-microenvironment (TME) play important roles in this process. Since glucocorticoids (GCs) are used as concomitant medications in the course of PCa treatment, it is essential to investigate the impact of GCs on stromal GR signaling in the TME. Therefore, general GR mRNA and protein expression was assessed in radical prostatectomy specimens and metastatic lesions. Elevated stromal GR signaling after GC treatment resulted in altered GR-target gene, soluble protein expression, and in a morphology change of immortalized and primary isolated cancer-associated fibroblasts (CAFs). Subsequently, these changes affected proliferation, colony formation, and 3D-spheroid growth of multiple epithelial PCa cell models. Altered expression of extra-cellular matrix (ECM) and adhesion-related proteins led to an ECM remodeling. Notably, androgen receptor pathway inhibitor treatments did not affect CAF viability. Our findings demonstrate that GC-mediated elevated GR signaling has a major impact on the CAF secretome and the ECM architecture. GC-treated fibroblasts significantly influence epithelial tumor cell growth and must be considered in future therapeutic strategies.

Whole stromal fibroblast signature is linked to specific chemokine and immune infiltration patterns and to improved survival in NSCLC.

Cancer associated fibroblasts (CAF) are known to orchestrate multiple components of the tumor microenvironment, whereas the influence of the whole stromal-fibroblast compartment is less understood. Here, an extended stromal fibroblast signature was investigated to define its impact on immune cell infiltration. The lung cancer adenocarcinoma (LUAD) data set of the cancer genome atlas (TCGA) was used to test whole stroma signatures and cancer-associated fibroblast signatures for their impact on prognosis. 3D cell cultures of the NSCLC cancer cell line A549 together with the fibroblast cell line SV80 were used in combination with infiltrating peripheral blood mononuclear cells (PBMC) for in-vitro investigations. Immune cell infiltration was assessed via flow cytometry, chemokines were analyzed by immunoassays and RNA microarrays. Results were confirmed in specimens from NSCLC patients by flow cytometry or immunohistochemistry as well as in the TCGA data set. The TCGA analyses correlated the whole stromal-fibroblast signature with an improved outcome, whereas no effect was found for the CAF signatures. In 3D microtumors, the presence of fibroblasts induced infiltration of B cells and CD69+CD4+ T cells, which was linked to an increased expression of CCL13 and CXCL16. The stroma/lymphocyte interaction was confirmed in NSCLC patients, as stroma-rich tumors displayed an elevated B cell count and survival in the local cohort and the TCGA data set. A whole stromal fibroblast signature was associated with an improved clinical outcome in lung adenocarcinoma and in vitro and in vivo experiments suggest that this signature increases B and T cell recruitment via induction of chemokines.

The LARP1 homolog Slr1p controls the stability and expression of proto-5'TOP mRNAs in fission yeast.

Messenger RNAs (mRNAs) in higher eukaryotes that encode proteins important for the assembly of the translational apparatus (e.g., ribosomal proteins) often harbor a pyrimidine-rich motif at the extreme 5' end known as a 5' terminal oligopyrimidine (5'TOP) sequence. Members of the La-related protein 1 (LARP1) family control 5'TOP expression through a conserved DM15 motif, but the mechanism is not well understood. 5'TOP motifs have not been described in many lower organisms, and fission yeast harbors a LARP1 homolog that also lacks a DM15 motif. In this work, we show that the fission yeast LARP1 homolog, Slr1p, controls the translation and stability of mRNAs encoding proteins analogous to 5'TOP mRNAs in higher eukaryotes, which we thus refer to as proto-5'TOPs. Our data suggest that the LARP1 DM15 motif and the mRNA 5'TOP motif may be features that were scaffolded over a more fundamental mechanism of LARP1-associated control of gene expression.

Pembrolizumab in mCRPC - Combination therapies as breakthrough to success?

PURPOSE OF REVIEW: Immune checkpoint inhibitors (ICIs) have shown promising antitumor activity in various malignant diseases. This narrative review provides an update on ongoing clinical studies investigating the only FDA-approved ICI programmed death receptor 1 (PD-1) inhibitor pembrolizumab in mono- and combination therapy in patients with metastatic castration-resistant prostate cancer (mCRPC). RECENT FINDINGS: Although most clinical trials investigating pembrolizumab as mono- or combinational therapy did not meet their primary endpoints, there exist subgroups of patients that demonstrate impressive responses rates justifying further investigation of ICI in prostate cancer. Beside combination of pembrolizumab with approved mCRPC agents, innovative approaches, like combining pembrolizumab with radioligands, deoxyribonucleic acid vaccines or innovative immunotherapeutic agents (i.e., ONC-392, AMG160, BXCL701) are ongoing exerting promising preliminary findings. SUMMARY: ICI monotherapy seems to be effective in a small biomarker-preselected population, however, there is evidence that especially novel ICI combination approaches can improve patient survival, which could ultimately refocus and revolutionize the treatment of mCRPC.

Tissue iron distribution in patients with anemia of inflammation: Results of a pilot study.

Anemia of inflammation (AI) is frequently present in subjects with inflammatory disorders, primarily caused by inflammation-driven iron retention in macrophages. So far, only limited data on qualitative and quantitative estimates of tissue iron retention in AI patients exist. We performed a prospective cohort study analyzing splenic, hepatic, pancreatic, and cardiac iron content with MRI-based R2*-relaxometry in AI patients, including subjects with concomitant true iron deficiency (AI+IDA) hospitalized between 05/2020-01/2022. Control groups were individuals without inflammation. Spleen R2* values in AI patients with ferritin ≤200 µg/L (AI+IDA) were comparable with those found in controls. In AI patients with ferritin >200 µg/L, spleen (47.6 s-1 vs. 19.3 s-1 , p < .001) and pancreatic R2* values (32.5 s-1 vs. 24.9 s-1 , p = .011) were significantly higher compared with controls, while liver and heart R2*-values did not differ. Higher spleen R2* values were associated with higher ferritin, hepcidin, CRP, and IL-6 concentrations. Spleen R2* values normalized in AI patients after recovery (23.6 s-1 vs. 47.6 s-1 , p = .008), while no changes were found in patients with baseline AI+IDA. This is the first study investigating tissue iron distribution in patients with inflammatory anemia and AI with concomitant true iron deficiency. The results support the findings in animal models demonstrating iron retention in macrophages, which are primarily accumulating in the spleen under inflammatory conditions. MRI-related iron measurement may help to better characterize actual iron needs and to define better biomarker thresholds in the diagnosis of true ID in patients with AI. It may qualify as a useful diagnostic method to estimate the need for iron supplementation and to guide therapy.

Compartmentalization of the host microbiome: how tumor microbiota shapes checkpoint immunotherapy outcome and offers therapeutic prospects.

The host microbiome is polymorphic, compartmentalized, and composed of distinctive tissue microbiomes. While research in the field of cancer immunotherapy has provided an improved understanding of the interaction with the gastrointestinal microbiome, the significance of the tumor-associated microbiome has only recently been grasped. This article provides a state-of-the-art review about the tumor-associated microbiome and sheds light on how local tumor microbiota shapes anticancer immunity and influences checkpoint immunotherapy outcome. The direct route of interaction between cancer cells, immune cells, and microbiota in the tumor microenvironment is emphasized and advocates a focus on the tumor-associated microbiome in addition to the spatially separated gut compartment. Since the mechanisms underlying checkpoint immunotherapy modulation by tumor-associated microbiota remain largely elusive, future research should dissect the pathways involved and outline strategies to therapeutically modulate microbes and their products within the tumor microenvironment. A more detailed knowledge about the mechanisms governing the composition and functional quality of the tumor microbiome will improve cancer immunotherapy and advance precision medicine for solid tumors.

High-resolution single-cell atlas reveals diversity and plasticity of tissue-resident neutrophils in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is characterized by molecular heterogeneity with diverse immune cell infiltration patterns, which has been linked to therapy sensitivity and resistance. However, full understanding of how immune cell phenotypes vary across different patient subgroups is lacking. Here, we dissect the NSCLC tumor microenvironment at high resolution by integrating 1,283,972 single cells from 556 samples and 318 patients across 29 datasets, including our dataset capturing cells with low mRNA content. We stratify patients into immune-deserted, B cell, T cell, and myeloid cell subtypes. Using bulk samples with genomic and clinical information, we identify cellular components associated with tumor histology and genotypes. We then focus on the analysis of tissue-resident neutrophils (TRNs) and uncover distinct subpopulations that acquire new functional properties in the tissue microenvironment, providing evidence for the plasticity of TRNs. Finally, we show that a TRN-derived gene signature is associated with anti-programmed cell death ligand 1 (PD-L1) treatment failure.

Multi-omic Characterization of Pancreatic Ductal Adenocarcinoma Relates CXCR4 mRNA Expression Levels to Potential Clinical Targets.

PURPOSE: Chemokines are essential for immune cell trafficking and are considered to have a major impact on the composition of the tumor microenvironment. CX-chemokine receptor 4 (CXCR4) is associated with poor differentiation, metastasis, and prognosis in pancreatic ductal adenocarcinoma (PDAC). This study provides a comprehensive molecular portrait of PDAC according to CXCR4 mRNA expression levels. EXPERIMENTAL DESIGN: The Cancer Genome Atlas database was used to explore molecular and immunologic features associated with CXCR4 mRNA expression in PDAC. A large real-word dataset (n = 3,647) served for validation and further exploratory analyses. Single-cell RNA analyses on a publicly available dataset and in-house multiplex immunofluorescence (mIF) experiments were performed to elaborate cellular localization of CXCR4. RESULTS: High CXCR4 mRNA expression (CXCR4high) was associated with increased infiltration of regulatory T cells, CD8+ T cells, and macrophages, and upregulation of several immune-related genes, including immune checkpoint transcripts (e.g., TIGIT, CD274, PDCD1). Analysis of the validation cohort confirmed the CXCR4-dependent immunologic TME composition in PDAC irrespective of microsatellite instability-high/mismatch repair-deficient or tumor mutational burden. Single-cell RNA analysis and mIF revealed that CXCR4 was mainly expressed by macrophages and T-cell subsets. Clinical relevance of our finding is supported by an improved survival of CXCR4high PDAC. CONCLUSIONS: High intratumoral CXCR4 mRNA expression is linked to a T cell- and macrophage-rich PDAC phenotype with high expression of inhibitory immune checkpoints. Thus, our findings might serve as a rationale to investigate CXCR4 as a predictive biomarker in patients with PDAC undergoing immune checkpoint inhibition.

Comprehensive characterization of the prostate tumor microenvironment identifies CXCR4/CXCL12 crosstalk as a novel antiangiogenic therapeutic target in prostate cancer.

BACKGROUND: Crosstalk between neoplastic and stromal cells fosters prostate cancer (PCa) progression and dissemination. Insight in cell-to-cell communication networks provides new therapeutic avenues to mold processes that contribute to PCa tumor microenvironment (TME) alterations. Here we performed a detailed characterization of PCa tumor endothelial cells (TEC) to delineate intercellular crosstalk between TEC and the PCa TME. METHODS: TEC isolated from 67 fresh radical prostatectomy (RP) specimens underwent multi-omic ex vivo characterization as well as orthogonal validation of both TEC functions and key markers by immunohistochemistry (IHC) and immunofluorescence (IF). To identify cell-cell interaction targets in TEC, we performed single-cell RNA sequencing (scRNA-seq) in four PCa patients who underwent a RP to catalogue cellular TME composition. Targets were cross-validated using IHC, publicly available datasets, cell culture expriments as well as a PCa xenograft mouse model. RESULTS: Compared to adjacent normal endothelial cells (NEC) bulk RNA-seq analysis revealed upregulation of genes associated with tumor vasculature, collagen modification and extracellular matrix remodeling in TEC. PTGIR, PLAC9, CXCL12 and VDR were identified as TEC markers and confirmed by IF and IHC in an independent patient cohort. By scRNA-seq we identified 27 cell (sub)types, including endothelial cells (EC) with arterial, venous and immature signatures, as well as angiogenic tip EC. A focused molecular analysis revealed that arterial TEC displayed highest CXCL12 mRNA expression levels when compared to all other TME cell (sub)populations and showed a negative prognostic role. Receptor-ligand interaction analysis predicted interactions between arterial TEC derived CXCL12 and its cognate receptor CXCR4 on angiogenic tip EC. CXCL12 was in vitro and in vivo validated as actionable TEC target by highlighting the vessel number- and density- reducing activity of the CXCR4-inhibitor AMD3100 in murine PCa as well as by inhibition of TEC proliferation and migration in vitro. CONCLUSIONS: Overall, our comprehensive analysis identified novel PCa TEC targets and highlights CXCR4/CXCL12 interaction as a potential novel target to interfere with tumor angiogenesis in PCa.

Hairy Cell Leukemia Patients Have a Normal Life Expectancy-A 35-Year Single-Center Experience and Comparison with the General Population.

Classic hairy cell leukemia (HCL) is an uncommon hematologic malignancy characterized by an excellent prognosis since purine analogues (PA), such as cladribine (2-CdA), have been introduced in the 1990s. However, most data on long-term outcomes is gathered from patients treated with PA first-line or include limited information on previous treatment outcomes, i.e., Interferon-α (IFN-α). Survival curves from previous series did not reach a plateau, indicating that nearly all patients ultimately relapse. Yet, overall survival (OS) data were rarely corrected for life expectancy of the general population. We here report 83 consecutive HCL patients treated between 1983 and 2017 at the University Center in Innsbruck, Austria. Median follow-up was 170 months (1-498). IFN-α, the first-line treatment of choice before 1990, was administered to 24 patients, achieving an overall response rate (ORR) of 86% and an unconfirmed complete remission (CRu) in 23%. All these patients relapsed after a median progression-free survival (PFS) of 30 months (3-80), but either remained drug-sensitive upon re-exposure to IFN-α or were successfully salvaged with PA. All 42 patients exposed to first-line 2-CdA responded (ORR of 100%). Sixteen patients received two to four successive courses of PA with a continuous decrease in the response quality (CRu rate 85.7% 1st-line vs. 41.5% 3rd-line treatment). Median PFS was not reached in both treatment-naïve patients and those retreated at first relapse. Although pretreatment with IFN-α was associated with a shortened median PFS of 81 months (43-118) after PA therapy, this tendency of inferior PFS did not result in inferior OS. OS of all 83 patients was excellent and equivalent to that of age-, sex-, and diagnostic period-matched controls from the Tyrolean general population (standardized mortality ratio 0.8), regardless of their age at diagnosis or whether they were diagnosed until or after the year 2000. These results confirm that HCL patients may look forward to a normal lifespan when treated with PA irrespective of their pretreatment history.