Caracterización de la estadificación molecular en carcinoma de colon. Correlación clínico-histológica

[spa] ANTECEDENTES: El tratamiento del carcinoma colorectal (CCR) en estadios I-II es quirúrgico, aunque un 25% de pacientes pueden recidivar. El estadio ganglionar es un factor pronóstico independiente y determina el tratamiento quimioterápico. El estadiaje ganglionar histológico (pN) se realiza con tinción de hematoxilina-eosina (HE), método poco sensible para detectar pacientes con riesgo de recidiva. Las técnicas moleculares detectan células tumorales en el 25-50% de los ganglios linfáticos (GLs) de CCR que se han diagnosticado como negativos por HE, las cuales se asocian en el CCR precoz a mayor riesgo de recidiva y peor supervivencia. Sin embargo, el diagnóstico molecular es complejo y costoso, impidiendo su uso en la práctica clínica. OBJETIVOS, ESTUDIOS REALIZADOS Y RESULTADOS OBTENIDOS: El primer estudio que compone la tesis doctoral es multicéntrico y prospectivo. Tiene como objetivo determinar la relación entre la carga tumoral molecular en GLs y los factores de riesgo convencionales en pacientes con cáncer de colon en estadios I-II. Se obtuvieron 1940 GLs de 149 pacientes con cáncer de colon con estadio histológico pN0. Se cuantificó la cantidad de ARNm mensajero (ARNm) de citoqueratina 19 (CK19) en los GLs mediante la técnica Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) denominada One-Step Nucleic Acid Amplification. Se definió la carga tumoral total (CTT) de cada paciente como la suma de todas las copias de ARNm de CK19/μL de cada GL positivo por colectomía. Se obtuvo una mediana de 15 GLs por caso (RIC 12;20). La positividad molecular se correlacionó con la presencia de áreas de alto grado (p <0,01), histología mucinosa/anillo de sello (p = 0,017), sexo masculino (p = 0,02), número GLs aislados (p = 0,012) y el peso total de GLs por caso (p < 0,01). La CTT se relacionó con el estadio pT (p = 0,01) y tamaño tumoral (p <0,01) en los tumores de bajo grado. El estudio de regresión logística multivariante mostró una correlación independiente de positividad molecular con el género, grado tumoral y número de GLs en fresco [AUC = 0,71 (IC del 95% = 0,62 a 0,79)]. El objetivo del segundo estudio es evaluar el impacto del tatuaje endoscópico prequirúrgico en la detección molecular de carga tumoral en GLs de neoplasias de colon en estadios iniciales. Métodos: Se trata de un estudio de cohorte prospectivo basado en una población de cribado de CCR en un hospital universitario terciario. Se evaluaron los GLs de colectomías con y sin tatuaje endoscópico preoperatorio mediante dos métodos, HE y RT-LAMP. Se comparó la cantidad de la carga tumoral y los valores de GLs obtenidos entre especímenes tatuados y no tatuados. Resultados: Se evaluaron mediante HE y RT-LAMP 936 GLs obtenidos de 71 colectomías que contenían carcinomas precoces y adenomas endoscópicamente irresecables (8 pT0, 17 pTis, 27 pT1, 19 pT2); 47 de 71 casos (66,2%) estaban tatuados. La positividad molecular en GLs se correlacionó con la presencia de tatuaje ganglionar [p <0,001; odds ratio 3.1 (95% IC 1.7 a 5.5)]. Se obtuvo un número significativamente mayor de GLs en especímenes tatuados en comparación con los no tatuados (mediana 17 GLs vs. 14,5 GLs; p = 0,019). CONCLUSIONES: Los resultados de los estudios de la presente tesis doctoral muestran que la detección de ARNm de CK19 en ganglios linfáticos se correlaciona con factores de alto riesgo clásicos en pacientes con cáncer de colon en estadio I-II. La CTT es una medida cuantitativa y objetiva que puede contribuir a una mejor estadificación de pacientes con cáncer de colon precoz. Asimismo, el tatuaje endoscópico permite la detección de los ganglios linfáticos más proclives a albergar células tumorales y aumenta el número de ganglios linfáticos aislados.[emg] Stage I–II colorectal cancer patients are surgically treated although up to 25 % will recur from disease. Molecular tumour detection in LN of early-stage patients is associated with an increased risk of disease recurrence and poor survival. The first study is a prospective multicentre study aimed to determine the relationship between LN molecular tumour burden and conventional high-risk factors in stage I–II colon cancer patients. A total of 1940 LN from 149 pN0 colon cancer patients were analysed for the amount of tumour CK19 mRNA with the quantitative OSNA molecular assay. Patient’s total tumour load (TTL) resulted from the sum of all CK19 mRNA tumour copies/μL of each positive LN from the colectomy specimen. A median of 15 LN were procured per case. Molecular positivity correlated with high-grade, mucinous/signet ring type, male gender, number of collected LN and total LN weight per case (p≤0.02). The TTL was related to pT stage and tumour size in low-grade tumours (p≤0.01). Multivariate logistic regression showed independent correlation of molecular positivity with gender, tumour grade and number of fresh LN (AUC=0.71). The second study aimed to evaluate the impact of presurgical endoscopic tattooing in early colon neoplasms (i.e. amount of tumour burden in LNs and LN yields). A prospective cohort study from a CRC screening-based population was performed. LNs from colectomies with and without preoperative endoscopic tattooing were assessed by HE and OSNA. HE and OSNA analyses of 936 LNs were performed from 71 colectomies containing early carcinomas and endoscopically unresectable adenomas; 47/71 cases were tattooed. Molecular positivity correlated with the presence of tattoo in LN (p<0.001; OR 3.1). A significantly higher number of LNs were obtained in tattooed specimens (median 17 LN vs. 14.5 LN; p=0.019). In conclusion, lymph node CK19 mRNA detection correlates with classical high-risk factors in stage I–II colon cancer patients. TTL is a quantitative and objective measure that may help to better stage early colon cancer patients. Additionally, endoscopic tattooing enables the analysis of those LNs most prone to harbour tumour cells and improves the number of LN harvested

Sporadic Creutzfeldt-Jakob disease VM1: phenotypic and molecular characterization of a novel subtype of human prion disease

Creutzfeldt-Jakob disease; Prion disease; Prion strainsMalaltia de Creutzfeldt-Jakob; Malalties priòniques; Soques de prionsEnfermedad de Creutzfeldt-Jakob; Enfermedad priónica; Cepas de prionesThe methionine (M)-valine (V) polymorphic codon 129 of the prion protein gene (PRNP) plays a central role in both susceptibility and phenotypic expression of sporadic Creutzfeldt-Jakob diseases (sCJD). Experimental transmissions of sCJD in humanized transgenic mice led to the isolation of five prion strains, named M1, M2C, M2T, V2, and V1, based on two major conformations of the pathological prion protein (PrPSc, type 1 and type 2), and the codon 129 genotype determining susceptibility and propagation efficiency. While the most frequent sCJD strains have been described in codon 129 homozygosis (MM1, MM2C, VV2) and heterozygosis (MV1, MV2K, and MV2C), the V1 strain has only been found in patients carrying VV. We identified six sCJD cases, 4 in Catalonia and 2 in Italy, carrying MV at PRNP codon 129 in combination with PrPSc type 1 and a new clinical and neuropathological profile reminiscent of the VV1 sCJD subtype rather than typical MM1/MV1. All patients had a relatively long duration (mean of 20.5 vs. 3.5 months of MM1/MV1 patients) and lacked electroencephalographic periodic sharp-wave complexes at diagnosis. Distinctive histopathological features included the spongiform change with vacuoles of larger size than those seen in sCJD MM1/MV1, the lesion profile with prominent cortical and striatal involvement, and the pattern of PrPSc deposition characterized by a dissociation between florid spongiform change and mild synaptic deposits associated with coarse, patch-like deposits in the cerebellar molecular layer. Western blot analysis of brain homogenates revealed a PrPSc type 1 profile with physicochemical properties reminiscent of the type 1 protein linked to the VV1 sCJD subtype. In summary, we have identified a new subtype of sCJD with distinctive clinicopathological features significantly overlapping with those of the VV1 subtype, possibly representing the missing evidence of V1 sCJD strain propagation in the 129MV host genotype

Cognitive decline in amyotrophic lateral sclerosis: Neuropathological substrate and genetic determinants

Proteïna TDP-43; Esclerosi lateral amiotròfica; Demència frontotemporalProteína TDP-43; Esclerosis lateral amiotrófica; Demencia frontotemporalTDP-43 protein; Amyotrophic lateral sclerosis; Frontotemporal dementiaCognitive impairment and behavioral changes in amyotrophic lateral sclerosis (ALS) are now recognized as part of the disease. Whether it is solely related to the extent of TDP-43 pathology is currently unclear. We aim to evaluate the influence of age, genetics, neuropathological features, and concomitant pathologies on cognitive impairment in ALS patients. We analyzed a postmortem series of 104 ALS patients and retrospectively reviewed clinical and neuropathological data. We assessed the burden and extent of concomitant pathologies, the role of APOE ε4 and mutations, and correlated these findings with cognitive status. We performed a logistic regression model to identify which pathologies are related to cognitive impairment. Cognitive decline was recorded in 38.5% of the subjects. Neuropathological features of frontotemporal lobar degeneration (FTLD) were found in 32.7%, explaining most, but not all, cases with cognitive impairment. Extent of TDP-43 pathology and the presence of hippocampal sclerosis were associated with cognitive impairment. Mutation carriers presented a higher burden of TDP-43 pathology and FTLD more frequently than sporadic cases. Most cases (89.4%) presented some degree of concomitant pathologies. The presence of concomitant pathologies was associated with older age at death. FTLD, but also Alzheimer’s disease, were the predominant underlying pathologies explaining the cognitive impairment in ALS patients. In sum, FTLD explained the presence of cognitive decline in most but not all ALS cases, while other non-FTLD related findings can influence the cognitive status, particularly in older age groups.SBE is a recipient of the Rio-Hortega post-residency grant from the Instituto de Salud Carlos III, Spain. This study was partially funded by Fundació Marató de TV3 (grant no. 20141610 to EG and no. 20143710 to RRG) and Fondo Europeo de Desarrollo Regional (FEDER) (PI15/01618 to RRG). AA is funded by Departament de Salut de la Generalitat de Catalunya, Pla estratègic de recerca i innovació en salut (PERIS) 2016–2020 (SLT002/16/00329). JG is recipient of the Instituto de Salud Carlos III-FEDER grants (PI16/01673 and PI19/00593

Preclinical studies with glioblastoma brain organoid co-cultures show efficient 5-ALA photodynamic therapy

Abstract: Background: The high recurrence of glioblastoma (GB) that occurs adjacent to the resection cavity within two years of diagnosis urges an improvement of therapies oriented to GB local control. Photodynamic therapy (PDT) has been proposed to cleanse infiltrating tumor cells from parenchyma to ameliorate short long-term progression-free survival. We examined 5-aminolevulinic acid (5-ALA)- mediated PDT effects as therapeutical treatment and determined optimal conditions for PDT efficacy without causing phototoxic injury to the normal brain tissue. Methods: We used a platform of Glioma Initiation Cells (GICs) infiltrating cerebral organoids with two different glioblastoma cells, GIC7 and PG88. We measured GICs-5-ALA uptake and PDT/5-ALA activity in dose-response curves and the efficacy of the treatment by measuring proliferative activity and apoptosis. Results: 5-ALA (50 and 100  g/mL) was applied, and the release of protoporphyrin IX (PpIX) fluorescence measures demonstrated that the emission of PpIX increases progressively until its stabilization at 24 h. Moreover, decreased proliferation and increased apoptosis corroborated the effect of 5-ALA/PDT on cancer cells without altering normal cells. Conclusions: We provide evidence about the effectiveness of PDT to treat high proliferative GB cells in a complex in vitro system, which combines normal and cancer cells and is a useful tool to standardize new strategic therapies

Molecularly determined total tumour load in lymph nodes of stage I–II colon cancer patients correlates with high-risk factors. A multicentre prospective study

Stage I–II (pN0) colorectal cancer patients are surgically treated although up to 25 % will eventually die from disease recurrence. Lymph node (LN) status is an independent prognostic factor in colorectal cancer (CRC), and molecular tumour detection in LN of early-stage CRC patients is associated with an increased risk of disease recurrence and poor survival. This prospective multicentre study aimed to determine the relationship between LN molecular tumour burden and conventional high-risk factors in stage I–II colon cancer patients. A total of 1940 LN from 149 pathologically assessed pN0 colon cancer patients were analysed for the amount of tumour cytokeratin 19 (CK19) messenger RNA (mRNA) with the quantitative reverse transcription loop-mediated isothermal amplification molecular assay One-Step Nucleic Acid Amplification. Patient’s total tumour load (TTL) resulted from the sum of all CK19 mRNA tumour copies/μL of each positive LN from the colectomy specimen. A median of 15 LN were procured per case (IQR 12;20). Molecular positivity correlated with high-grade (p < 0.01), mucinous/signet ring type (p = 0.017), male gender (p = 0.02), number of collected LN (p = 0.012) and total LN weight per case (p < 0.01). The TTL was related to pT stage (p = 0.01) and tumour size (p < 0.01) in low-grade tumours. Multivariate logistic regression showed independent correlation of molecular positivity with gender, tumour grade and number of fresh LN [AUC = 0.71 (95 % CI = 0.62–0.79)]. Our results show that lymph node CK19 mRNA detection correlates with classical high-risk factors in stage I–II colon cancer patients. Total tumour load is a quantitative and objective measure that may help to better stage early colon cancer patients.Work supported by the Banc de Tumors-Biobanc Hospital Clinic-IDIBAPS and Xarxa de Bancs de Tumors de Catalunya (XBTC), and by grants from the Fundación Científica de la Asociación Española Contra el Cáncer (GCB13131592CAST), Ministerio de Economía y Competitividad (SAF2014–54,453-R), Agència de Gestió d’Ajuts Universitaris i de Recerca (2014SGR135), and by Sysmex Coorp Spain (Sant Just Desvern, Spain). CIBERehd is funded by the Instituto de Salud Carlos II

Budget Impact Analysis of Molecular Lymph Node Staging Versus Conventional Histopathology Staging in Colorectal Carcinoma

Background: The presence of lymph node (LN) metastasis is a critical prognostic factor in colorectal cancer (CRC) patients and is also an indicator for adjuvant chemotherapy. The gold standard (GS) technique for LN diagnosis and staging is based on the analysis of haematoxylin and eosin (H&E)-stained slides, but its sensitivity is low. As a result, patients may not be properly diagnosed and some may have local recurrence or distant metastases after curative-intent surgery. Many of these diagnostic and treatment problems could be avoided if the one-step nucleic acid amplification assay (OSNA) was used rather than the GS technique. OSNA is a fast, automated, standardised, highly sensitive, quantitative technique for detecting LN metastases. Objectives: The aim of this study was to assess the budget impact of introducing OSNA LN analysis in early-stage CRC patients in the Spanish National Health System (NHS). Methods: A budget impact analysis comparing two scenarios (GS vs. OSNA) was developed within the Spanish NHS framework over a 3-year time frame (2017-2019). The patient population consisted of newly diagnosed CRC patients undergoing surgical treatment, and the following costs were included: initial surgery, pathological diagnosis, staging, follow-up expenses, systemic treatment and surgery after recurrence. One- and two-way sensitivity analyses were performed. Results: Using OSNA instead of the GS would have saved 1,509,182, 6,854,501 and 10,814,082 during the first, second and third years of the analysis, respectively, because patients incur additional costs in later years, leading to savings of more than 19 million for the NHS over the 3-year time horizon. Conclusions: Introducing OSNA in CRC LN analysis may represent not only an economic benefit for the NHS but also a clinical benefit for CRC patients since a more accurate staging could be performed, thus avoiding unnecessary treatments

Cognitive decline in amyotrophic lateral sclerosis: Neuropathological substrate and genetic determinants

Cognitive impairment and behavioral changes in amyotrophic lateral sclerosis (ALS) are now recognized as part of the disease. Whether it is solely related to the extent of TDP-43 pathology is currently unclear. We aim to evaluate the influence of age, genetics, neuropathological features, and concomitant pathologies on cognitive impairment in ALS patients. We analyzed a postmortem series of 104 ALS patients and retrospectively reviewed clinical and neuropathological data. We assessed the burden and extent of concomitant pathologies, the role of APOE ε4 and mutations, and correlated these findings with cognitive status. We performed a logistic regression model to identify which pathologies are related to cognitive impairment. Cognitive decline was recorded in 38.5% of the subjects. Neuropathological features of frontotemporal lobar degeneration (FTLD) were found in 32.7%, explaining most, but not all, cases with cognitive impairment. Extent of TDP-43 pathology and the presence of hippocampal sclerosis were associated with cognitive impairment. Mutation carriers presented a higher burden of TDP-43 pathology and FTLD more frequently than sporadic cases. Most cases (89.4%) presented some degree of concomitant pathologies. The presence of concomitant pathologies was associated with older age at death. FTLD, but also Alzheimer's disease, were the predominant underlying pathologies explaining the cognitive impairment in ALS patients. In sum, FTLD explained the presence of cognitive decline in most but not all ALS cases, while other non-FTLD related findings can influence the cognitive status, particularly in older age groups

Homozygous R136S mutation in PRNP gene causes inherited early onset prion disease

Altres ajuts: Fundació la Marató de TV3/201821-31Background: More than 40 pathogenic heterozygous PRNP mutations causing inherited prion diseases have been identified to date. Recessive inherited prion disease has not been described to date. Methods: We describe the clinical and neuropathological data of inherited early-onset prion disease caused by the rare PRNP homozygous mutation R136S. In vitro PrP propagation studies were performed using recombinant-adapted protein misfolding cyclic amplification technique. Brain material from two R136S homozygous patients was intracranially inoculated in TgMet129 and TgVal129 transgenic mice to assess the transmissibility of this rare inherited form of prion disease. Results: The index case presented symptoms of early-onset dementia beginning at the age of 49 and died at the age of 53. Neuropathological evaluation of the proband revealed abundant multicentric PrP plaques and Western blotting revealed a ~ 8 kDa protease-resistant, unglycosylated PrP fragment, consistent with a Gerstmann-Sträussler-Scheinker phenotype. Her youngest sibling suffered from progressive cognitive decline, motor impairment, and myoclonus with onset in her late 30s and died at the age of 48. Genetic analysis revealed the presence of the R136S mutation in homozygosis in the two affected subjects linked to homozygous methionine at codon 129. One sibling carrying the heterozygous R136S mutation, linked to homozygous methionine at codon 129, is still asymptomatic at the age of 74. The inoculation of human brain homogenates from our index case and an independent case from a Portuguese family with the same mutation in transgenic mice expressing human PrP and in vitro propagation of PrP studies failed to show disease transmissibility. Conclusion: In conclusion, biallelic R136S substitution is a rare variant that produces inherited early-onset human prion disease with a Gerstmann-Sträussler-Scheinker neuropathological and molecular signature. Even if the R136S variant is predicted to be "probably damaging", heterozygous carriers are protected, at least from an early onset providing evidence for a potentially recessive pattern of inheritance in human prion diseases

Gal-1 Expression Analysis in the GLIOCAT Multicenter Study: Role as a Prognostic Factor and an Immune-Suppressive Biomarker

Glioblastoma (GBM) is the most frequent primary malignant brain tumor and has a dismal prognosis. Unfortunately, despite the recent revolution of immune checkpoint inhibitors in many solid tumors, these have not shown a benefit in overall survival in GBM patients. Therefore, new potential treatment targets as well as diagnostic, prognostic, and/or predictive biomarkers are needed to improve outcomes in this population. The beta-galactoside binding protein Galectin-1 (Gal-1) is a protein with a wide range of pro-tumor functions such as proliferation, invasion, angiogenesis, and immune suppression. Here, we evaluated Gal-1 expression by immunohistochemistry in a homogenously treated cohort of GBM (the GLIOCAT project) and correlated its expression with clinical and molecular data. We observed that Gal-1 is a negative prognostic factor in GBM. Interestingly, we observed higher levels of Gal-1 expression in the mesenchymal/classical subtypes compared to the less aggressive proneural subtype. We also observed a Gal-1 expression correlation with immune suppressive signatures of CD4 T-cells and macrophages, as well as with several GBM established biomarkers, including SHC1, PD-L1, PAX2, MEOX2, YKL-40, TCIRG1, YWHAG, OLIG2, SOX2, Ki-67, and SOX11. Moreover, Gal-1 levels were significantly lower in grade 4 IDH-1 mutant astrocytomas, which have a better prognosis. Our results confirm the role of Gal-1 as a prognostic factor and also suggest its value as an immune-suppressive biomarker in GBM

Post-mortem neuropathologic examination of a 5-case series of CAR T-cell treated patients

Introduction: Chimeric antigen receptor (CAR) T-cell therapy is a promising immunotherapy for the treatment of refractory hematopoietic malignancies. Adverse events are common, and neurotoxicity is one of the most important. However, the physiopathology is unknown and neuropathologic information is scarce.Materials and methods: Post-mortem examination of 6 brains from patients that underwent CAR T-cell therapy from 2017 to 2022. In all cases, polymerase chain reaction (PCR) in paraffin blocks for the detection of CAR T cells was performed.Results: Two patients died of hematologic progression, while the others died of cytokine release syndrome, lung infection, encephalomyelitis, and acute liver failure. Two out of 6 presented neurological symptoms, one with extracranial malignancy progression and the other with encephalomyelitis. The neuropathology of the latter showed severe perivascular and interstitial lymphocytic infiltration, predominantly CD8+, together with a diffuse interstitial histiocytic infiltration, affecting mainly the spinal cord, midbrain, and hippocampus, and a diffuse gliosis of basal ganglia, hippocampus, and brainstem. Microbiological studies were negative for neurotropic viruses, and PCR failed to detect CAR T -cells. Another case without detectable neurological signs showed cortical and subcortical gliosis due to acute hypoxic-ischemic damage. The remaining 4 cases only showed a mild patchy gliosis and microglial activation, and CAR T cells were detected by PCR only in one of them.Conclusions: In this series of patients that died after CAR T-cell therapy, we predominantly found non-specific or minimal neuropathological changes. CAR T-cell related toxicity may not be the only cause of neurological symptoms, and the autopsy could detect additional pathological findings