Moving Beyond Morphology: Toward a Morpho-Molecular Classification of Pleural Mesothelioma

Advances in the Treatment of Malignant Pleural Mesothelioma

Pleural mesothelioma is a rare but aggressive form of cancer. Local recurrence represents the majority of treatment failures and overall survival (OS) outcomes remain dismal. Adding locoregional treatment with radiotherapy after surgical resection has been considered but its role remains uncertain. The purpose of this study was to evaluate the outcomes of adjuvant radiation therapy (RT) for patients with malignant pleural mesothelioma. The National Cancer Data Base (NCDB) was queried (2004-2013) for patients with malignant mesothelioma. Patients were divided into three groups: observation, surgery alone, and surgery followed by adjuvant RT. Statistics included Fisher's exact or Chi square tests to analyze categorical proportions between groups, Kaplan-Meier analysis to evaluate OS, and Cox proportional hazards modeling to determine variables associated with OS. Propensity matching was performed to make comparisons between homogenous groups. Overall, the surgery plus radiotherapy group had a higher median survival (21.4months) compared with surgery alone (16.59months) [p < 0.001]. RT was more likely to be delivered after extrapleural pneumonectomy than with lung-sparing surgical approaches. On multivariable analysis, receipt of surgery plus radiotherapy, chemotherapy administration, and higher socioeconomic status were associated with improved OS (p < 0.0001). After propensity matching, receipt of surgery plus radiotherapy and chemotherapy administration were still associated with improved OS (p < 0.05). In the treatment of malignant pleural mesothelioma, adjuvant radiotherapy after surgical intervention was associated with improved OS. This study is the largest study of adjuvant radiotherapy to date, and our findings highlight the need for additional prospective data.

Pleural mesothelioma (PM) is a rare and aggressive cancer arising from pleural mesothelial cells with a strong association to asbestos exposure. Among the diagnostic strategies available are noninvasive techniques including thoracic ultrasound (TUS), computed tomography (CT) scans, positron emission tomography (PET-CT), and invasive procedures such as thoracoscopy and pleural biopsy. Accurate identification of the histological subtype is critical for tailoring treatment strategies. The standard treatment for unresectable PM has traditionally been chemotherapy, particularly platinum and pemetrexed. However, recent advances in translational clinical research, including immune checkpoint inhibitors (ICIs), are changing the therapeutic landscape, offering new opportunities for personalized treatment. The recent FDA approval of nivolumab and ipilimumab combination therapy as a first-line treatment has significantly improved outcomes, especially for nonepithelioid subtypes. Ongoing studies are exploring additional immune-targeted therapies such as VISTA, LAG-3, and dendritic cell-based therapies. Early detection, refined biomarker identification, and a deeper understanding of the tumor microenvironment remain essential to improving PM prognosis and patient survival. This review provides a comprehensive exploration of the epidemiology, etiology, clinical manifestations, diagnostic approaches (including immunohistochemical and molecular markers), staging, and current treatment strategies for PM.

Current risk stratification and treatment decision-making for bladder cancer informed by histopathology as well as molecular diagnostics face limitations. This review summarizes recent advancements in single-cell and spatial omics methodologies for understanding bladder cancer biology and their potential impact on development of novel therapeutic strategies. Single-cell RNA sequencing and spatial omics techniques offer unprecedented insights into various aspects of tumor microenvironment (TME), bladder cancer heterogeneity, cancer stemness, and cellular plasticity. Studies have identified multiple malignant cell subpopulations within tumors, revealing diverse transcriptional states and clonal evolution. Additionally, intratumor heterogeneity has been linked to tumor progression and therapeutic response. Immune cell composition analysis has revealed immunosuppressive features in the TME, impacting treatment response. Furthermore, studies have elucidated the role of cancer-associated fibroblasts and endothelial cells in shaping the tumor immune landscape and response to therapy. Single-cell and spatial omics technologies have revolutionized our understanding of bladder cancer biology, uncovering previously unseen complexities. These methodologies provide valuable insights into tumor heterogeneity and microenvironmental interactions, with implications for therapeutic development. However, challenges remain in translating research findings into clinical practice and implementing personalized treatment strategies. Continued interdisciplinary collaboration and innovation are essential for overcoming these challenges and leveraging the full potential of single-cell and spatial omics in improving bladder cancer diagnosis and treatment.

2551 Background: A multimodal approach based on debulking surgery is beneficial in the treatment of pleural mesothelioma (PM). In addition, hyperthermic intra-thoracic chemotherapy significantly improves patient survival. Chemotherapeutic agents such as gemcitabin (Gem) have proven their efficacy in PM. The aim of this study was to assess the feasibility, tolerability and pharmacokinetics (PK) of gemcitabine (Gem)-cisplatine (Pt) both administered using this new intra-thoracic hyperthermic schedule. Methods: A phase II study was conducted in patients with T<3, N<2, M0 epithelioid mesothelioma. After neoadjuvant chemotherapy (Gem-Pt), extra-pleural pneumonectomy with diaphragmatic and pericardic reconstruction was performed, followed by a 1-hour intra-thoracic hyperthermic infusion of Pt (100 mg/m²) + Gem (1250 mg/m²). Pleural and blood samples were analyzed for Gem and its active metabolite difluorodeoxyuridine (dFdU, HPLC assay) and for total and free Pt concentrations (atomic absorption spectrophotometry). Five patients were analyzed for PK. Results: No major chemotherapy-related side effect was reported. Main PK data are the following: the maximum dFdU concentration was reached between 1.25 and 1.5 h in pleura, and between 1.25 and 4 h in plasma. For free Pt, maximum concentrations were observed between 0.25 and 1 h in pleura and between 1 and 1.25 h in plasma. Large inter-patient variability was observed for both dFdU and free Pt PK. Importantly, the local exposure to dFdU was 2.5 to 9-fold that of plasma exposure. For free Pt, pleural exposure was 8.7 to 30-fold greater than plasma exposure. Conclusions: These data indicate a markedly higher pleural exposure as compared to systemic exposure for dFdU and Pt. Along with satisfactory tolerance, these results support the potential benefit offered by this new intra-thoracic hyperthermic schedule in the management of PM. Patient AUC0–48h dFdU (μM.h) AUC0–48h free Pt (mg/l.h) Plasma Pleura Pleura/plasma Plasma Pleura Pleura/plasma 1 913 2,350 2.57 6.96 60.65 8.71 2 304 1,396 4.59 5.44 not done - 3 379 953 2.51 1.87 56.68 30.31 4 537 4,856 9.04 4.26 100.87 23.68 5 386 1,453 3.76 1.82 29.72 16.29 No significant financial relationships to disclose.

TPS8601 Background: Pleural mesothelioma (PM) is a highly aggressive cancer of the pleura, predominantly caused by prior asbestos exposure. Currently, there is no approved standard therapy for the treatment of early-stage PM. In most cases a multimodal therapy is recommended consisting of locoregional treatment by surgical cytoreduction via extended pleurectomy/decortication (eP/D), which, if feasible, can be combined with hyperthermic intrathoracic chemoperfusion (HITOC), together with inductive or adjuvant chemotherapy. Considering the immunogenic effects of chemotherapy on the tumor microenvironment, synergistic effects are expected when such a treatment is combined with immune checkpoint inhibitor therapy. In addition, interactions between immune infiltrates and mesothelioma cells play a role in the advent of PM, also implying a beneficial role for immunotherapy in this entity. This is also supported by recent clinical data that demonstrated beneficial effects of immune checkpoint inhibitors in patients with advanced PM. The NICITA trial is an investigator-initiated trial, investigating the combination of adjuvant chemotherapy with immune checkpoint inhibitor compared to chemotherapy alone in radically resected patients with early stage PM. Methods: The NICITA trial is a randomized, open-label, phase II clinical trial that is conducted in 14 centers across Germany. Eligible patients have been diagnosed with PM in tumor stages I-III (UICC 8th edition) and epithelioid subtype, and must have undergone cytoreductive surgery by eP/D with or without HITOC. Patients will be randomized 1:1 to receive either a combination of 4 cycles of pemetrexed/platinum-based adjuvant chemotherapy and nivolumab (480 mg q4w) followed by nivolumab maintenance therapy (12 cycles, 480 mg q4w) or 4 cycles of adjuvant chemotherapy only. Stratification will take place according to previous HITOC treatment (yes vs. no), ECOG status (0,1 vs. 2), and achievement of macroscopic complete resection (yes vs. no). The primary endpoint of this trial is time-to-next-treatment. Secondary endpoints include additional measures of efficacy (progression-free survival, overall survival, measures of treatment-beyond-progression) and quality of life, as well as the assessment of safety. Furthermore, a comprehensive longitudinal collection of biomarker samples, including tumor tissue, blood, and stool samples, for an accompanying translational research project is implemented in this clinical trial. Sample size justification: the recruitment of 46 patients to each arm with a low drop-out rate of 13% appears feasible resulting in 40 patients to be analyzed per arm. This sample size will permit a descriptive comparison and adequately describe the tested treatment options as deduced from the precision of the median TNT confidence interval estimate. As of February 2nd 2023, 85 of planned 92 patients have been enrolled into the NICITA trial. Clinical trial information: NCT04177953 .

Pleural mesothelioma (PM) is a rare, aggressive malignancy of the pleura. The PM diagnosis is challenging, both in terms of imaging, sampling of representative biopsies and pathological examination, and the treatment options are limited. The main aim of this article is to summarize the most recent advances in the diagnosis and management of PM as presented in the Danish clinical guidelines for PM. An expert task force consisting of a lung physician, a pathologist, an oncologist, and a thoracic surgeon conducted an ad hoc literature search during November 2023-July 2024, reviewed the evidence and developed the current clinical guidelines after reaching consensus. The article appraises the radiological modalities and biopsy techniques that are employed in PM diagnostics, provides a comprehensive overview of the evidence behind the PM histopathological and cytological diagnosis, and asserts the state of the art and novel developments of oncological, multimodal, and palliative PM treatment and follow-up that are portrayed in the Danish clinical guidelines.

In order to explore valproate's spectrum of efficacy in rapid-cycling bipolar disorder, 55 patients underwent a prospective, open, 7.8-month trial designed to assess the drug's acute and prophylactic properties. Twenty patients received monotherapy, and 35 received combination therapy. Moderate to marked acute antidepressant responses were seen in 47% of the patients, prophylactic antidepressant responses in 76%, acute antimanic responses in 91%, prophylactic antimanic responses in 94%, acute responses in mixed states in 85%, and prophylactic responses in mixed states in 93%. Consistent with other anticonvulsant literature, these data suggest that valproate has marked antimanic and mixed state efficacy, but minimal to moderate antidepressant properties.

This month's issue of the Archives of Pathology & Laboratory Medicine includes a unique special section on "Molecular Signatures of Lung and Pleural Tumors" that represents the proceedings of a special Joint Symposium of the European Working Groups for Molecular Pathology and Pulmonary Pathology at the 21st European Congress of Pathology in Istanbul, Turkey, in September 2007. This symposium and the subsequent special section were organized by Dr Helmut Popper of the Institute of Pathology at the Medical University of Graz, Graz, Austria, president elect of the Austrian Society of Pathologists and immediate past president of the European Working Group for Pulmonary Pathology. In this brief review, we have provided some definitions of terms and concepts used in the proceedings of the "Molecular Signatures of Lung and Pleural Tumors" special section for those readers who are not already familiar with molecular pathology.Molecular pathology may have once been a specialized component of the research laboratory or the clinical laboratory, but today molecular diagnostic and prognostic techniques are in common use within the anatomic pathology laboratory, especially in the realm of infectious organism diagnosis and cancer diagnosis. Molecular testing continues to expand as more easily obtainable archival paraffin-embedded tissue replaces fresh and frozen tissues as the source of DNA and RNA needed for molecular analysis, and as newer technologies allow for more streamlined methods of testing. This review addresses the increased application of molecular testing in lung pathology, specifically how the current state of molecular pathology may be applied to practical, everyday lung pathology diagnosis.Genes, made up of nucleic acids, contain the information necessary for the construction of proteins from amino acids within a cell. Genes code for proteins required for metabolic reactions and cellular structure. DNA makes up genes, and RNA transcribes the genetic code held within the DNA into proteins. The genetic code within the genes is composed of nucleic acids, for which nucleotides are the building blocks. Nucleotides, made up of a sugar-phosphate backbone with a nitrogenous base, are either purines—adenine (A) and guanine (G) in DNA and RNA— or pyrimidines—thymine (T) and cytosine (C) in DNA (uracil [U] replaces T in RNA). The nucleotides that make up the genes are arranged in a double-stranded right-handed helix. Nucleotides in DNA are arranged sequentially so that a gene will code for a matching protein. Within a double helix pattern, A, a purine, always binds with T, a pyrimidine, and G always binds with C, giving a nucleotide sequence for which 1 strand is a "mirror image" of the other strand.1–6There are 46 chromosomes (23 pairs) in a human diploid cell, on which all genes are located. Chromosomes are paired, and as such a gene is found on a locus on each of the 2 paired chromosomes, giving 2 copies, or alleles, of genes. Gametes are haploid rather than diploid and therefore contain only 1 allele for each gene. Diploid status is reestablished when the nuclear material from an egg and sperm combine during fertilization.1–6Transcription, the synthesis of messenger RNA (mRNA) from a DNA strand, is a key step in the formation of protein coded by DNA. During transcription, enzymes called topoisomerases break a DNA strand and allow the DNA double-helix to uncoil, giving 2 DNA strands, 1 of which is the template for mRNA, called the DNA template. Base pairs are matched with the DNA template to produce a mirror image of the DNA template, except with the substitution of U for T, forming a strand of mRNA. A series of 3 base pairs in a gene, called a codon, code for a specific amino acid, so that a series of codons code for a particular sequence of amino acids resulting in the synthesis of a specific protein. Translation, the assembly of the protein molecule from the mRNA template, occurs with the addition of amino acids in a particular sequence based on the specificity of the mRNA. Transfer RNA assists in translation.1–6After translation, modifications to the newly formed protein occur in order for it to function, to move within the cell, or to fold properly. Methylation, acetylation, phosphorylation, glycosylation, posttranslational cleavage, and the addition of lipid groups are examples of posttranslational modifications. End regions of chromosomes are made up of telomeres, hundreds of repeats of the nucleotide sequence TTAGGG. Some of these telomere sequences are lost each time a cell divides, until they are lost and the cell can no longer divide. This process is called senescence. A polymerase called telomerase is able to replace the DNA sequences at the end regions, allowing for continuing cell division—a significant feature in some cancers.1–6The polypeptide chain formed by the specific amino acid sequence causes the newly formed protein to fold into a tertiary arrangement giving it a 3-dimensional structure. Often, the newly formed protein is inert until made functional by a posttranslational modification such as phosphorylation or proteolytic cleavage. Phosphorylation, the addition to the protein of a phosphate group catalyzed by enzymes called kinases, may cause, for example, translocation of the protein from the cytosol into the nucleus. Dephosphorylation is the removal of a phosphate group catalyzed by enzymes called phosphatases. Phosphorylation and dephosphorylation of proteins are often important in the activation and deactivation of cell cycle proteins, signaling pathway proteins, and transcription factor proteins.1–6Protein degradation is necessary to remove damaged proteins and limit signaling proteins such as those involved in cell survival and cell death. This degradation often needs to proceed quickly. Reversible cross-linkage to a polypeptide, termed ubiquitin, leads to the rapid degradation of proteins and is called ubiquinylation or polyubiquinylation.7The control of gene expression is for the most part controlled by regulation of transcription initiation. Proteins called transcription factors, also termed transactivators or trans-acting factors, bind to DNA and regulate RNA polymerase activity, affecting gene expression either by inducing or activating the gene or by inhibiting the gene by reducing transcription levels.8–13 Transcription factors are necessary for RNA polymerase to initiate transcription, and transcription factors called transcriptional activators stimulate transcription of an RNA molecule from its DNA template.1415A variety of techniques exist for molecular pathology diagnosis, including nucleic acid extraction, Southern blotting, restriction fragment length polymorphism, sequencing, liquid bead microarrays, mass spectrometry, and comparative genomic hybridization, among others. Nucleic acid extraction historically has used organic techniques using chloroform and phenol; however, automated nucleic acid extraction exists today and is frequently used to purify nucleic acids for their use with other molecular methods. For practical laboratory-based molecular diagnosis of lung disease, polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) are 2 of the most important techniques commonly used.In situ hybridization uses DNA or RNA probes to evaluate intact cells for genetic changes. Probes visualized with a chromogen that produces a colored chemical at the reaction site is called chromogenic in situ hybridization and probes using fluorescent labels are called FISH. Evaluation of genetic alterations within intact cells allows for the detection of genetic alterations occurring in a specific group of cells or within a small number of examined cells and is a major benefit of the use of in situ hybridization in anatomic pathology. It is commonly used with cytologic and surgical specimens to detect tumor cells and certain microorganisms, and with surgical specimens of tumors for its prognostic utility and to determine treatment response.16–22 Chromogenic in situ hybridization uses a probe that can be seen as a chromogenic reaction under light microscopy, whereas FISH is available as probe sets and multiprobe FISH cocktails. Peripheral blood, urine, sputum, endoscopic brushings and washings, and paraffin-embedded, formalin-fixed tissue are all suitable for FISH. However, fixation of tissue with formalin for longer than 48 hours may yield poorer FISH results.23DNA and RNA probes hybridize to a specific target sequence that is of interest, for example, genes implicated in a specific type of cancer or an inherited disease, or to a certain microorganism.16–22 Probes are made using DNA fragments cloned from yeast or bacterial artificial chromosomes and, with FISH, are directly or indirectly fluorophore labeled, most often using Texas Red, fluorescing red, and fluorescein isothiocyanate, fluorescing green.24 Directly labeled probes have a fluorophore-labeled nucleotide inserted into the probe, so binding of the probe to its target in 1 hybridization step is all that is required to visualize the probe. Indirectly labeled probes have a reporter molecule such as biotin or digoxigenin attached covalently, requiring the additional step of the application of a fluorophore-labeled avidin or fluorophore-labeled antidigoxigenin. The additional step required with indirectly labeled probes is a disadvantage; however, indirectly labeled probes generally allow for stronger signals because of greater signal amplification.24 There are 4 general types of probes: chromosome enumeration probes, locus-specific indicator probes, telomeric probes, and chromosome paints. Chromosome enumeration probes hybridize to repetitive DNA sequences located near chromosome centromeres, and because the loss of a centromere is generally indicative of the loss of an entire chromosome, they are used to enumerate the number of copies of a certain chromosome within a cell.2526 Locus-specific probes are probes to unique sequences and are most frequently used to determine whether specific genes are amplified, translocated, or deleted. Telomeric probes hybridize to unique DNA sequences located very close to telomeres. The probes do not hybridize to telomeric sequences. Chromosomal paints are a mix of probes that probe to the entire length of 1 or more chromosomes.A FISH specimen must undergo prehybridization to allow a probe to efficiently hybridize to cellular DNA targeted by the probe while protecting the cell from morphologic disruption. Following prehybridization, the probe and cellular DNA are denatured so that the probe can hybridize to the cellular DNA it is targeting. Hybridization usually takes between 4 and 12 hours. A type of DNA called Cot DNA is added during hybridization to hybridize highly repetitive DNA sequences that are located within the genome so that the probe DNA does not nonspecifically bind these repetitive sequences and yield a multitude of nonspecific signals rather than the appropriately specific signal.24252728 The nonbound probe is then removed by washing, a nuclear counterstain that weakly fluoresces is added so that the nucleus can be identified, and an antifade is added to retard fluorophore photobleaching. The signal produced by the FISH fluorophore is then examined with fluorescence microscopy.Since its introduction in 1985,29 PCR has been refined to be an efficient and sensitive method of studying the molecular pathology of primary and metastatic neoplasms, inflammatory mechanisms, and infectious diseases.30–37 Today, automated instruments designed for the laboratory tabletop are available. Polymerase chain reaction amplifies DNA via a repeated 3-step process of denaturation, annealing, and extension. Double-stranded DNA that is the target is denatured at high temperature to yield 2 intact single strands of complementary DNA. Then at a lower temperature, specially designed single-stranded DNA primers anneal or bind to a specific targeted area of the single-stranded DNA. Because there are very large numbers of DNA primers relative to the full-length complementary DNA strand, the target DNA anneals with the primer DNA much more frequently than with the complementary DNA when cooling occurs. In the third step, extension, Taq polymerase identifies the now partially double-stranded DNA and extends the primers by polymerization, to yield, at the end of the first cycle, 2 double-stranded copies of a portion of the target DNA generated from 1 copy. Because the DNA primers only recognize the DNA for which they have been specifically designed, only that specific segment of DNA, making up a small part of the entire DNA present in the original DNA, is preferentially amplified. Subsequent cycles produce numerous shorter double-stranded DNA PCR products, so that more than a billion copies of the original double-stranded DNA are produced after 30 cycles, and more than a trillion are produced after 40 cycles. Following amplification, post-PCR analysis of the markedly increased amount of targeted DNA sequence product can be performed. The target sequence can be shown to be present in a specimen by the use of amplicons run on polyacrylamide or agarose electrophoresis, or via Southern blotting with probe hybridization, comparing the lengths of the targeted DNA sequence with DNA "ladder" markers. DNA sequencing can be performed on the amplicons, or studies to identify mutations may also be performed.3839 Because PCR enormously amplifies DNA, great caution must be used in performing PCR to avoid cross-contamination of a specimen with even very small amounts of DNA.Real-time PCR is becoming a more and more popular method of molecular pathology research and diagnosis that eliminates the need for post-PCR analysis and allows for relatively quick detection of DNA targets, including specific mutations.40–44The best way to examine specific genes present in a certain cell type, such as in tumor cells, is to examine those cells' mRNA. As RNA is not stable enough to work with easily in a laboratory, reverse transcription can be used to convert mRNA into its complementary DNA. With reverse transcription, mRNA is the template used for the production of a strand of DNA, opposite or reverse of typical cellular transcription. The complementary DNA can be used as the template for PCR in a process called reverse transcriptase–PCR (RT-PCR). Reverse transcriptase–PCR can be used to examine genes that are expressed, overexpressed, underexpressed, or not expressed in a specific cell type by the isolation of specific mRNA.44–47 Altered gene expression is a characteristic of malignant transformation, and those alterations allow for the identification of the presence of cancer cells via the detection of mRNA transcripts specific to those tumor cells. Tumor markers have been identified that are specific to solid organ cancers, and RT-PCR is highly sensitive in detecting differentially expressed tumor-related mRNAs. Some studies have indicated that RT-PCR can detect as few as 1 cancer cell in a million normal cells.4448 Real-time quantitative RT-PCR has become popular for detecting and quantifying RNA targets in a variety of cancers. It requires no post-PCR analysis and is efficient and automated. Several studies have used real-time quantitative RT-PCR to evaluate lymph nodes for micrometastases, including for non–small cell lung carcinoma (NSCLC), and to examine peripheral blood for potential dissemination of lung cancer cells during lobectomy.49–52Normal human DNA contains 2 alleles for every genetic locus, the majority of which are identical, or homozygous, and the loss of 1 allele results in no pathologic change. Some genetic loci have 2 differing copies of alleles and are heterozygous. The majority of these heterozygous alleles allow for normal variance and do not result in pathologic changes; however, some of these heterozygous loci have the potential to cause pathologic genetic variations, with resultant disease. If there is a loss of the normal, or "wild-type," allele at a locus, with resultant "loss of heterozygosity," the remaining aberrant allele can cause cellular damage. At homozygous loci, the loss of an allele can occur and be followed by gene silencing or point mutation, causing loss of tumor suppressor genes.5354 Detection of loss of heterozygosity using older methods such as Southern blot analysis and restriction fragment length polymorphism analysis are low-throughput, tedious, and inefficient; however, newer, high-throughput single nucleotide polymorphism arrays have allowed for more efficient examination of loss of heterozygosity.55–57Cancers, including lung cancers, commonly exhibit loss of heterozygosity, causing the inactivation or silencing of genes critical for growth regulation and homeostasis. Cigarette smoking has been associated with loss of heterogeneity of sites on chromosome 3, and the association is greater in patients who began smoking at a young age.58–61 More than 90% of small cell carcinomas and more than 70% of NSCLCs contain loss of heterozygosity.576263 Among NSCLCs, squamous cell carcinomas exhibit loss of heterozygosity in more than 90% of cases, compared with adenocarcinomas, showing loss of heterozygosity in approximately 70% of cases. In NSCLC, loss of heterozygosity generally involves genetic foci on chromosomes 1p, 3p, 8p, 9p, 13q, 17p, 19p, Xp, and Xq. In small cell carcinomas, loss of heterozygosity generally involves chromosomes 3p, 4q, 5q, 4q, 10q, 13q, 15q, and 17p.5762–66 Losses found in both small cell carcinomas and NSCLCs, involving chromosomes 3p, 13q, and 17p, are probably related to inactivation of critical tumor suppressor genes including retinoblastoma, p53, and fragile histidine triad (FHIT).576263 Loss of heterozygosity in premalignant conditions and malignant diseases of the lung represents both early- and late-stage changes in the progression of disease; however, the continuum of losses makes it hard to evaluate the specific contribution of each loss. Loss of heterozygosity has also been identified in some benign lung diseases, including asthma and chronic obstructive pulmonary disease, probably reflecting the genetic predisposition identified in these diseases.67–70 Loss of heterozygosity has also been identified in cases of usual interstitial pneumonia (idiopathic pulmonary fibrosis) and suggests premalignant potential in those cases.71Extracellular messenger molecules such as hormones, inflammatory cytokines, and growth factors, called ligands, bind to specific cell surface receptors and activate messengers within the cytosol leading eventually to activation of nuclear transcription factors that, due to the extracellular message, direct the transcription of a specific gene product, such as the transcription of a protein involved in cell growth. This cascade of events is termed signal transduction, and the series of steps within the cascade is termed signal transduction pathway or signaling pathway. Growth factor receptors are a common cell surface receptor, on which polypeptide growth factors such as epidermal growth factor are ligands attaching to those receptor protein-tyrosine kinases, activating the receptor and causing it to bind with intracellular proteins, which in turn continue the signaling pathway. Epidermal growth factor receptor is a member of the type I growth factor receptor tyrosine kinase family. Epidermal growth factor receptor has other ligands that bind to it other than epidermal growth factor, including transforming growth factor α, and these ligands, receptors, and signaling pathways play a central role in many lung cancers as well as some nonneoplastic pulmonary diseases.72–77The extracellular ligands' "messages" are transmitted via a signaling pathway. Cell differentiation and proliferation, cell survival, and cell death and apoptosis are regulated by signaling pathways, the majority of which "cross-talk" with other signaling pathways in a complex manner. Several important signaling pathways have been well studied. For example, the Wnt/B/catenin pathway, termed the canonical Wnt signaling pathway, involves Wnt binding to Frizzled cell surface receptors, which in turn activate Disheveled, causing the inhibition of protein kinase glycogen synthase kinase 3, which in turn releases dephosphorylated β-catenin from the adenomatous polyposis coli–axin complex. β-Catenin associates with T-cell factor/lymphoid enhancer–binding factor transcription factors causing the induction of Myc.78–84 Other important signaling pathways include the JAK/STAT pathway, involving signal transducers and activators of transcription (STAT) proteins and Janus kinase (JAK) nonreceptor protein tyrosine kinases; the Ras/Raf-1/MAPK pathway, a significant pathway in carcinogenesis, including epithelial cell proliferation; the nuclear factor-κB transcription factor and nuclear factor-κB signaling pathways that regulate immune system proteins, cell survival and proliferation proteins, and apoptosis proteins; and the PI3K/Akt/ mTOR pathway important in regulating cell survival; among many others.85–93Errors in replication, extracellular influences such as UV light, radiation, and chemicals, and endogenous influences such as oxygen radicals routinely cause DNA damage, generally depurination, and with the of a chemical group to DNA, the of which is termed an Several DNA pathways exist to the damaged DNA and replace it with newly DNA based on its complementary DNA DNA pathways are important in an to lung cancer and to lung cancer The base pathway small foci of DNA including or single or fragments and The nucleotide pathway DNA involving both The be because no for the DNA segment is available. can cause DNA such as chemical and The DNA pathway, also termed the break pathway, involves a cascade of events and to double-stranded The direct pathway DNA by in cell cycle is a series of very regulated events cell proliferation, including into DNA replication, replication, into cell cell and cell The cell cycle is into at for DNA synthesis or of and with nuclear and cellular which control of DNA and cell in to and with proteins called that regulate the progression of the series of steps in the cell cycle by activating and proteins by phosphorylation, including proteins that as on cell cycle progression and cell The cell cycle is controlled by many pathways and and and it may be appropriately or in inflammatory Cell cycle regulation loss is a very important step in cell proliferation during cycle damaged DNA to be on to cells by the cell cycle at specific It allows damaged DNA to be the is to cells into apoptosis cell The primary in the cell cycle is the restriction point in to the cell cycle occurs. Other include an and a A complex of proteins, the complex and the detect DNA at these DNA is the DNA is and the cell cycle factor signaling the cell cycle and the the however, once the cell the cell restriction it no longer requires growth factor and the cell is to the cell The gene product, progression past the restriction point of the cell cycle and the expression of genes involved in DNA of the cell cycle also on activation of of the of p53, the gene product, is to the DNA the of the cell cycle at in to DNA or to the induction of apoptosis when is As has been to as the of the and have critical in the of the cell cycle, and of and are the most common associated with the cell cycle of however, due to the large number of and pathways, many can produce direct or loss. The gene for 2 protein products, a kinase and of either of these can produce of the or of and as well as other genes and their or of and p53, can produce loss of cell cycle control the direct loss of and and prognostic techniques using molecular are used in anatomic pathology genes for proliferation, and the used for of molecular and factors involved in genetic will the current and molecular testing and their to the diagnosis and of lung

Spatial transcriptomics has emerged as a transformative technology in biomedical research, offering unprecedented insights into gene and protein expression within their native tissue context. Unlike conventional bulk or single-cell sequencing approaches, spatial omics has the advantage of preserving the spatial structure of tissues, allowing researchers to directly map molecular information onto histological structures. This review provides an overview of the current state of spatial omics technologies, highlighting their application in cancer research. Spatial omics has enabled detailed characterization of the tumor microenvironment (TME), revealing spatial heterogeneity, immune cell infiltration patterns, and complex mechanisms of tumor progression and therapy resistance across various cancer types. The review covers future directions, including artificial intelligence–driven analytics, improved standardization, and cost reduction to accelerate clinical translation. Ultimately, spatial omics is poised to play a central role in precision oncology, enabling a deeper understanding of tumor biology and informing more effective individualized treatment strategies.

The authors investigated the pharmacological treatment of delusional depression by assigning patients on a random double-blind basis to amitriptyline alone, perphenazine alone, or a combination of the two. Fourteen (78%) of the 18 patients assigned to amitriptyline plus perphenazine were responders, compared with seven (41%) of 17 patients treated with amitriptyline alone and three (19%) of the 16 patients treated with perphenazine alone. The combination of amitriptyline and perphenazine was clearly superior (p less than .01).

Background: Pleural mesothelioma (PM) is an aggressive malignancy with a poor prognosis. The clinical efficacy of standard therapy with immune checkpoint inhibitors (ICI) is limited and heterogeneous across PM subtypes. Tumor-intrinsic characteristics (i.e., inflammatory phenotype, molecular features, DNA methylation) may influence immune responsiveness, but predictive biomarkers of ICI therapy efficacy in PM are still lacking. Methods: NIBIT-EPI-MESO is a retrospective, multicenter study, sponsored by the NIBIT Foundation, evaluating biological correlates of clinical outcomes in PM patients (pts) treated with ICI (i.e., anti-CTLA-4 plus anti-PD-1, anti-CTLA-4 plus anti-PD-L1, or anti-CTLA-4 monotherapy). Pre-ICI therapy FFPE tumor samples were analyzed by RRBS methylation (n=83 pts) and RNA-seq (n=82 pts), with methylation subtypes defined by consensus clustering of the top 1% most variable CpGs. Tumor microenvironment (TME) was characterized by multiplex immunofluorescence analysis of CD4, CD8, CD20, CD68, CD163 (n=35 pts). Integrated multi-omics analyses were used to associate tumor biology with clinical outcome of PM pts. Results. Unsupervised methylation profiling identified four PM subsets with increasingly global DNA methylation levels: demethylated (DEM), LOW, intermediate (INT), and CpG island methylator phenotype (CIMP). Methylation subtypes were significantly associated with response to ICI, with LOW/DEM enriched among responder (R) pts and INT/CIMP in non-R pts (p= 0.002); no association of response to ICI was found with PM histotype (p=0.33). The LOW subset exhibited the longest median overall survival (mOS) and the highest 3-year OS rate, expressed genes involved in pathways associated with innate and adaptive immune responses, and showed an “inflamed” TME (i.e., CD8+ T cells, CD20+ B cells). Conversely, the CIMP subtype had the shortest mOS and OS rate, was characterized by genes enriched in developmental, morphogenetic and cell cycle-related processes, along with a “desert” TME. Functional characterization of the identified methylation classes of PM was validated in the MESOMICS dataset. Accordingly, a PM methylation subtype classifier was developed to predict response to ICI therapy. Conclusions: Tumor DNA methylation defines biologically and clinically distinct immune phenotypes in PM and robustly predicts clinical response and long-term survival in ICI-treated PM patients, regardless of tumor histology. Citation Format: Luana Calabrò, Francesca Pia Caruso, Alessia Covre, Teresa Maria Rosaria Noviello, Maria Fortunata Lofiego, Rossella Tufano, Luigi Ferraro, Piera Grisolia, Antonio De Falco, Vincenzo Lagano, Francesco Sgambelluri, Giovanna Sabella, Giulia Rossi, Giulia Gibilisco, Francesco Marzani, Emma Bello, Elena Simonetti, Vincenzo D'Alonzo, Michele Caraglia, Sandra Coral, Antonina De Angelis, Luigi Cerbone, Sara Delfanti, Federica Grosso, Anna Maria Di Giacomo, Massimo Milione, Roberta Mortarini, Andrea Anichini, Michele Ceccarelli, Michele Maio. Tumor methylation subtypes predict the clinical outcome to immunotherapy in pleural mesothelioma patients from the NIBIT-EPI-MESO study [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr LB-C009.

Cell annotation is a crucial methodological component to interpreting single cell and spatial omics data. These approaches were developed for single cell analysis but are often biased, manually curated and yet unproven in spatial omics. Here we apply a stemness model for assessing oncogenic states to single cell and spatial omic cancer datasets. This one-class logistic regression machine learning algorithm is used to extract transcriptomic features from non-transformed stem cells to identify dedifferentiated cell states in tumors. We found this method identifies single cell states in metastatic tumor cell populations without the requirement of cell annotation. This machine learning model identified stem-like cell populations not identified in single cell or spatial transcriptomic analysis using existing methods. For the first time, we demonstrate the application of a ML tool across five emerging spatial transcriptomic and proteomic technologies to identify oncogenic stem-like cell types in the tumor microenvironment.

Recent advances in self-supervised foundation models for computational cancer histopathology have shown great promise for predicting clinical phenotypes from routine hematoxylin and eosin (H&amp;E)-stained images. However, application of these models in the context of discovery, for example for identification of novel biomarkers or drug targets, remains challenging as this typically requires linking whole slide imaging (WSI) data to molecular data, which has historically been scarce. Recently, spatial omics (SO) has emerged as a powerful technology for high-throughput profiling of RNAs and proteins directly within cancer tissues. This presents new and exciting opportunities to improve histopathology models for making molecular predictions about the tumor microenvironment. In this work, we develop machine learning models that can generate transcriptomic profiles at two different spatial scales: (1) cellular-level resolution, i.e. SO, and (2) slide-level resolution, i.e. bulk RNA-sequencing. We present a preprocessing and benchmarking framework to enable rigorous comparison of models on an in-house SO dataset, as well as a public bulk RNA-seq dataset (TCGA-COAD/READ), of colorectal cancer. We fine-tune recently published digital pathology foundation models for these tasks, and find that they outperform both general pathology-naïve image foundation models, as well as specialized digital pathology deep learning models. We also find that existing models cannot be simultaneously trained using supervision from multiple spatial resolutions. To address this, we propose a graph neural network (GNN) modeling approach that can model both local interactions between cells within tumor niches, as well as global interactions across tumor regions. We show that our GNN model outperforms existing models for both cell-level and slide-level tasks. This work presents a novel multi-scale framework for predicting transcriptome profiles from WSIs. Citation Format: Tianyi Zhang, Malay Singh, Dwivedi Naman, Timothy Antoni, Ju Can, Firas Elbayoumi, Iain Bee Huat Tan, Tiantian He, Hwee Kuan Lee, Shyam Prabhakar, Grace Hui Ting Yeo. Multi-Scale Modeling for Prediction of Spatial Single-Cell and Bulk Transcriptomic Profiles from Whole-Slide Images of Colorectal Cancer [abstract]. In: Proceedings of Frontiers in Cancer Science 2024; 2024 Nov 13-15; Singapore. Philadelphia (PA): AACR; Cancer Res 2025;85(15_Suppl):Abstract nr P24.

The tumor microenvironment (TME) actively contribute to tumor development and treatment response. The interplay between tumor cells, immune cells, fibroblasts and blood vessels contribute to immune escape and drug resistance. Prior to treatment, higher tumor infiltrating lymphocytes correlate with better survival, while greater stromal content is linked to poor survival. Studying the composition and dynamics of the TME is essential for improving patient stratification, however a scalable tool for addressing this question is still lacking. Spatially resolved omics technologies allow for charting tissue architecture at the individual cell level, though large-scale studies remain challenging due to high expenses. In contrast, hematoxylin and eosin (H&amp;E) slides are a cost-effective modality that provide rich morphological information for studying spatial biology. However, their use relies on pathologist interpretation. A key area of research in digital pathology has been automating cell (type) identification, predicting nuclei location and cell type in H&amp;E slides. Existing deep learning models are limited by the quantity and diversity of training data, which requires pathologists to carefully annotate the location and identity of large volume of cells. To date, the largest dataset comprises approximately 200,000 cells, annotated with four cell types across 19 cancer types. We propose a novel approach of automated “molecular annotation”, where cell types and location on H&amp;E slides are annotated with the aid of spatial proteomics, in place of pathologist annotation. Specifically, samples were profiled with both modalities. From the spatial omics modality, pixels were first segmented into cells, followed by cell clustering and cluster annotation based on molecular features of the cells. The location and identity of all cells on the tissue were then identified. These information were subsequently transferred to the H&amp;E image by alignment at single-cell resolution, forming a dataset annotated with molecular ground truth. With a spatial omics dataset of two spatial proteomics slides from colorectal cancer patients, we obtained 160,000 annotated cells including 50,000 immune cells, 25,000 tumor cells, 12,600 stroma, 7,900 endothelial cells, among others. The size of this dataset is close to the largest annotated dataset publicly available to date. We then use this dataset to benchmark existing state-of-the-art deep-learning based cell type predictions models, as well as to fine-tune existing models for predicting cells in the colorectal tumor microenvironment. This proof-of-concept study aims to demonstrate the feasibility of molecular annotation approach. By including more spatial omics data, this approach can boost the performance of existing pre-trained models and enhance generalizability to specific tumor types. It opens up the opportunity to harness millions of cells for deep learning models to predict cell types on H&amp;E slides, make AI models a cost-effective option for studying the TME. Citation Format: Siao-Han Wong, Benedikt Brors, Sonja Loges. Empowering AI-driven prediction of the tumor microenvironment from histopathology images via molecular annotation [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Artificial Intelligence and Machine Learning; 2025 Jul 10-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(13_Suppl):Abstract nr B053.