Differentially Expressed Genes Involved in Primary Resistance to Immunotherapy in Patients with Advanced-Stage Pulmonary Cancer.

In the last few years, nivolumab has become the standard of care for advanced-stage lung cancer patients. Unfortunately, up to 60% of patients do not respond to this treatment. In our study, we identified variations in gene expression related to primary resistance to immunotherapy. Bronchoscopy biopsies were obtained from advanced non-small cell lung cancer (NSCLC) patients previously characterized as responders or non-responders after nivolumab treatment. Ten tumor biopsies (from three responders and seven non-responders) were analyzed by the differential expression of 760 genes using the NanoString nCounter platform. These genes are known to be involved in the response to anti-PD1/PD-L1 therapy. All the patients were treated with nivolumab. Examining the dysregulated expression of 24 genes made it possible to predict the response to nivolumab treatment. Supervised analysis of the gene expression profile (GEP) revealed that responder patients had significantly higher levels of expression of CXCL11, NT5E, KLRK1, CD3G, GZMA, IDO1, LCK, CXCL9, GNLY, ITGAL, HLA-DRB1, CXCR6, IFNG, CD8A, ITK, B2M, HLA-B, and HLA-A than did non-responder patients. In contrast, PNOC, CD19, TP73, ARG1, FCRL2, and PTGER1 genes had significantly lower expression levels than non-responder patients. These findings were validated as predictive biomarkers in an independent series of 201 patients treated with nivolumab (22 hepatocellular carcinomas, 14 non-squamous cell lung carcinomas, 5 head and neck squamous cell carcinomas, 1 ureter/renal pelvis carcinoma, 120 melanomas, 4 bladder carcinomas, 31 renal cell carcinomas, and 4 squamous cell lung carcinomas). ROC curve analysis showed that the expression levels of ITK, NT5E, ITGAL, and CD8A were the best predictors of response to nivolumab. Further, 13/24 genes showed an adverse impact on overall survival (OS) in an independent, large series of patients with NSCLC (2166 cases). In summary, we found a strong association between the global GEP of advanced NSCLC and the response to nivolumab. The classification of NSCLC patients based on GEP enabled us to identify those patients who genuinely benefited from treatment with immune checkpoint inhibitors (ICIs). We also demonstrated that abnormal expression of most of the markers comprising the genomic signature has an adverse influence on OS, making them significant markers for therapeutic decision-making. Additional prospective studies in larger series of patients are required to confirm the clinical utility of these biomarkers.

Primary Cutaneous Marginal Zone Lymphoproliferative Disorder of Donor Origin after Allogeneic Hematopoietic Stem Cell Transplantation.

ABSTRACT: Primary cutaneous posttransplant lymphoproliferative disorders (PTLDs) after allogeneic hematopoietic stem cell transplant (allo-HSCT) are exceedingly rare, with only 6 published cases, all of them consisting in T-cell neoplasms. In this report, we present for the first time a donor-derived B-cell PTLD consisting in a primary, cutaneous, B-cell, marginal zone, lymphoproliferative disorder (PCMZLPD). The patient, a 37-year-old woman with a history of Hodgkin lymphoma received an allo-HSCT from her healthy, matched, related father, achieving complete host chimerism in the bone marrow and peripheral blood. However, 8 years after the allo-HSCT, she presented asymptomatic skin lesions consisting in oval, well-defined, slightly raised erythematous plaques, located on the arms, trunk, and legs. Skin biopsies of 2 lesions demonstrated a class-switched IgG+, EBV-, PCMZLPD, showing kappa light chain restriction and monoclonal rearrangement of the IgH gene. Microsatellite genotyping and 2-color fluorescence in situ hybridization (X and Y chromosomes) confirmed that the origin of the neoplastic cells was the donor graft. The lesions showed an indolent behavior, good response to topical corticosteroids, and no need for systemic treatment. Our case broadens the spectrum of PTLD, a diverse group of lymphoid and/or plasmacytic proliferations with variable clinical presentations and histopathological features.

Analysis of Circulating Tumor DNA in Synchronous Metastatic Colorectal Cancer at Diagnosis Predicts Overall Patient Survival.

Sporadic colorectal cancer (sCRC) initially presents as metastatic tumors in 25-30% of patients. The 5-year overall survival (OS) in patients with metastatic sCRC is 50%, falling to 10% in patients presenting with synchronous metastatic disease (stage IV). In this study, we systematically analyzed the mutations of RAS, PIK3CA and BRAF genes in circulating tumor DNA (ctDNA) and tumoral tissue DNA (ttDNA) from 51 synchronous metastatic colorectal carcinoma (SMCC) patients by real-time PCR, and their relationship with the clinical, biological and histological features of disease at diagnosis. The highest frequency of mutations detected was in the KRAS gene, in tumor biopsies and plasma samples, followed by mutations of the PIK3CA, NRAS and BRAF genes. Overall, plasma systematically contained those genetic abnormalities observed in the tumor biopsy sample from the same subject, the largest discrepancies detected between the tumor biopsy and plasma from the same patient being for mutations in the KRAS and PIK3CA genes, with concordances of genotyping results between ttDNA and ctDNA at diagnosis of 75% and 84%, respectively. Of the 51 SMCC patients in the study, 25 (49%) showed mutations in at least 1 of the 4 genes analyzed in patient plasma. From the prognostic point of view, the presence and number of the most common mutations in the RAS, PIK3CA and BRAF genes in plasma from SMCC patients are independent prognostic factors for OS. Determination of the mutational status of ctDNA in SMCC could be a key tool for the clinical management of patients.

Genomic profiling of sporadic liver metastatic colorectal cancer.

Sporadic colorectal cancer (sCRC) is the third leading cause of cancer death in the Western world. Approximately, a quarter of sCRC patients present metastatic dissemination at the moment of diagnosis, the liver being the most frequently affected organ. Additionally, this group of CRC patients is characterized by a worse prognosis. In the last decades, significant technological developments for genome analysis have fostered the identification and characterization of genetic alterations involved in the pathogenesis of sCRC. However, genetic alterations involved in the metastatic process through which tumor cells are able to colonize other tissues with a different microenvironment, still remain to be fully identified. Here, we review current knowledge about the most relevant genomic alterations involved in the liver metastatic process of sCRC, including detailed information about the genetic profile of primary colorectal tumors vs. their paired liver metastases.

Contingent cfDNA Screening Implementation: Increasing Diagnostic Accuracy and Reducing Invasive Testing - 6 Years' Results in a Single Center.

OBJECTIVES: Universal screening for trisomy using cell-free DNA (cfDNA) has proven to be more effective than combined test, but it is not cost efficient currently. Contingent cfDNA screening on the results of the first-trimester combined test can improve the detection rate of the combined test and reduce the number of invasive tests at a lower cost than universal screening. In 2018, a contingent screening program was implemented in the community of Castilla y Leon (Spain). This study aims to compare the results achieved in Salamanca University Hospital during the first 3 years of contingent screening (2018-2020) with those of the previous 3 years (2015-2017) to assess the changes in the trisomy detection rate and the number of invasive tests. METHODS: A total of 9,903 singleton pregnancies without malformations nor nuchal translucency >p99 were included. 5,165 patients underwent combined screening and 4,738 had contingent screening based on the combined test risk. In the combined test group, women were offered an invasive test if the risk was ≥1:270, while risks under 1:270 were considered low risks, and no further testing was offered. In the contingent screening group, invasive testing was offered if the risk was ≥1:100 (≥1:50 from 2020 onwards), while cfDNA was offered if the combined test risk was between 1:100 and 1:1,000 (1:50-1:1,000 from 2020 onwards). When risk was <1:1,000, no further testing was offered. Aneuploidies detected by cfDNA were confirmed by invasive diagnostic testing. RESULTS: There were 33 cases of trisomy 21 (T21) throughout the 6 years of study. Four cases had low/intermediate risks and were spotted by cfDNA. Risk >1:1,000 threshold for contingent test detected 100% T21. There was a false-positive result for trisomy 13. There were no false-negative results. "No-call" cfDNA results were minimized by repeating blood collection 2 weeks later, as fetal fraction (FF) was doubled. Invasive testing had a drop rate of 84% after contingent screening implementation. DISCUSSION: The implementation of population-based contingent screening significantly reduces the number of invasive tests without lowering diagnostic accuracy. To achieve the maximum efficiency of the program, it is important to know the best cut-offs according to the population where the program is to be implemented. The number of uninformative results due to low FF can be reduced by repeating the test 2 weeks after the initial extraction: this increases the FF to twice the initial one, achieving informative results and avoiding unnecessary invasive tests.

Perineural Invasion as a Clue to Malignant Behavior in a Dermatofibroma.

Dermatofibroma (DF) represents one of the most common mesenchymal proliferations of the skin. Their recurrence rate, even when incompletely excised, is very low, whereas the atypical, aneurysmal, and cellular variants have recurrence rates of up to 20% each. Extraordinary rare malignant lesions with metastases to lymph nodes and/or lung have been described. We report a 64-year-old woman with a long history (years) of a skin lesion on her right arm that became painful during the last months. Histologically, it consisted of a conventional cellular DF in which perineural invasion was present. Subsequently, the lesion showed a clinically aggressive course with recurrences, sarcomatous transformation, and pulmonary metastases. Given that no predictive morphological features have been identified to separate classical benign DF from rare metastasizing forms, perineural invasion in an otherwise conventional DF could be a histopathologic clue for an adverse prognosis and should provoke a closer clinical follow-up.

Genetic alterations in colorectal cancer: implications for the prognosis and treatment of the disease.

In recent years, important advances have been achieved in the understanding of the genetic abnormalities present in colorectal tumors and their association with their ontogeny and progression. Accordingly, while the presence of mutations in the APC and/or KRAS genes is associated with neoplastic transformation, mutations in SMAD and DCC, different chromosomal abnormalities located at 7, 17p and 18q, and complex karyotypes are frequently linked with tumor progression. From a clinical point of view, the presence of microsatellite instability (MSI) is associated with lower relapse rates and a greater overall survival, as well as resistance to adjuvant treatment with fluoropyrimidines, whereas mutations in the BRAF gene have been associated with early relapse. At the molecular level, studies of intratumoral genetic heterogeneity associated with the metastatic process of colorectal cancer (CRC) have focused on analyzing mutations in the genes involved in the treatment of the disease. In fact, different mutational profiles have been observed among primary tumors, lymph node metastases and liver metastases in the same patient. In this sense, the genetic heterogeneity of CRC at the intratumor level may explain the high relapse rates reported and the refractory nature of tumors treated with monoclonal antibodies.

Ductus Venosus Agenesis as a Marker of Pallister-Killian Syndrome.

The ductus venosus (DV) is a shunt that allows the direct flow of well-oxygenated blood from the umbilical vein (UV) to the coronary and cerebral circulation through the foramen ovale. Its agenesis has been associated with chromosomal abnormalities and rare genetic syndromes, structural defects, intrauterine growth restriction (IUGR) and even antepartum fetal demise. Pallister-Killian Syndrome (PKS) is a rare sporadic disorder with specific tissue mosaic distribution of an extra 12p isochromosome (i(12p)). Its main clinical features are moderate to severe intellectual disability/neuromotor delay, skin pigmentation abnormalities, typical facial appearance, variable association with multiple congenital malformations and epilepsy. Though prenatal findings (including congenital diaphragmatic hernia, ventriculomegaly, congenital heart disease, polyhydramnios, and rhizomelic shortening) have been described in literature, prenatal diagnosis is difficult as there are no associated identification signs no distinctive or pathognomonic signs, and some of these malformations are hard to identify prenatally. The tissue mosaicism linked to this syndrome and the decrease of the abnormal clone carrier of the i(p12) after successive trypsinizations of cultured cells makes the diagnosis even more challenging. We present the case of a 27.5 weeks pregnant woman with a fetal ductus venosus agenesis (DVA) as the main guide marker. To our knowledge this is the first case published in literature reporting a DVA as a guide sign to diagnose a complex condition as Pallister-Killian syndrome. We also underscore the key role of new genetic techniques as microarrays to avoid misdiagnosis when only a subtle sonographic sign is present in complex conditions like this.

Detailed characterization of multiple myeloma circulating tumor cells shows unique phenotypic, cytogenetic, functional, and circadian distribution profile.

Circulating myeloma tumor cells (CTCs) as defined by the presence of peripheral blood (PB) clonal plasma cells (PCs) are a powerful prognostic marker in multiple myeloma (MM). However, the biological features of CTCs and their pathophysiological role in MM remains unexplored. Here, we investigate the phenotypic, cytogenetic, and functional characteristics as well as the circadian distribution of CTCs vs paired bone marrow (BM) clonal PCs from MM patients. Our results show that CTCs typically represent a unique subpopulation of all BM clonal PCs, characterized by downregulation (P < .05) of integrins (CD11a/CD11c/CD29/CD49d/CD49e), adhesion (CD33/CD56/CD117/CD138), and activation molecules (CD28/CD38/CD81). Fluorescence in situ hybridization analysis of fluorescence-activated cell sorter-sorted CTCs also unraveled different cytogenetic profiles vs paired BM clonal PCs. Moreover, CTCs were mostly quiescent and associated with higher clonogenic potential when cocultured with BM stromal cells. Most interestingly, CTCs showed a circadian distribution which fluctuates in a similar pattern to that of CD34(+) cells, and opposite to stromal cell-derived factor 1 plasma levels and corresponding surface expression of CXC chemokine receptor 4 on clonal PCs, suggesting that in MM, CTCs may egress to PB to colonize/metastasize other sites in the BM during the patients' resting period.

Involvement of primary mesenchymal precursors and hematopoietic bone marrow cells from chronic myeloid leukemia patients by BCR-ABL1 fusion gene.

For decades now, it is well established that chronic myeloid leukemia (CML) is a hematopoietic stem cell(HPC) disorder. However, it remains to be determined whether BCR-ABL1 gene rearrangement occurs in a HPC or at an earlier stem cell and whether the degree of involvement of hematopoiesis by the BCR-ABL1 fusion gene relates to the response to therapy. Here, we have investigated by interphase fluorescence in situ hybridization (iFISH) the distribution of BCR-ABL1 fusion gene in FACS-sorted bone marrow (BM) populations of mesenchymal precursor cells (MPC) and other hematopoietic cell populations from 18 newly diagnosed CML patients. Overall, our results showed systematic involvement at relatively high percentages of BM maturing neutrophils (97%615%), basophils (95%612%), eosinophils (90%68%), CD341 precursors cells (90%67%),monocytes (84%630%), nucleated red blood cells (87%624%), and mast cells (77%633%). By contrast, MPC(30%634%), B-cells (15%627%), T-lymphocytes (50%626%), and NK-cells (35%634%) were involved at lower percentages. In 8/18 CML patients, 2 tumor BCR-ABL11 subclones were detected by iFISH. Of note, all tumor cell subclones were systematically detected in CD341 cells, whereas MPC were only involved by the ancestral tumor cell subclone. In summary, here we confirm the presence at diagnosis of the BCR-ABL1 fusion gene inMPC, CD341 precursors, and other different BM hematopoietic myeloid cell lineages from CML patients,including also in a significant fraction of cases, a smaller percentage of T, B, and NK lymphocytes.Interestingly, involvement of MPC was restricted to the ancestral BCR-ABL11 subclone.

Improving the outcome of acute cholecystitis: the non-standardized treatment must no longer be employed.

PURPOSE: Therapeutic recommendations of acute cholecystitis are not consistently implemented, which generates greater patient morbidity and higher health care costs. The aim of this article is to evaluate the burden of acute cholecystitis, to detect potentially modifiable variables, and to propose a therapeutic strategy that will allow us to improve the quality of care. METHODS: We carried out a retrospective study of patients who were admitted to the hospital from January 2010 to December 2012 using a univariate analysis of parameters including the admitting department, age, treatment administered, and length of stay. RESULTS: A total of 967 patients were admitted to the hospital with a diagnosis of acute cholecystitis, 692 (72%) to the Surgery Department, 257 (26%) to Internal Medicine-Digestive, and 18 (2%) to other departments. Four hundred ninety-eight (51.5%) were operated on: 107 (21%) on an urgent basis, 111 (22%) at an early stage (<96 h at diagnosis), 152 (30%) at a late stage (>96 h at diagnosis), and 128 (26%) at a delayed date (other admission). Patients who were admitted into the surgery department were five times more likely to be operated on than patients admitted into other departments (p<0.01). Patients operated on at a late stage had a longer length of stay than early stage surgery patients (p<0.05) and than non-operated ones (p<0.05). Patients<74 years old were more frequently operated than older ones (p<0.05). CONCLUSIONS: The non-standardized treatment of acute cholecystitis causes high clinical and surgical variability, long average stay, more readmissions, and high hospital costs. Therefore, patients with a diagnosis of acute cholecystitis should be admitted to the Surgery Department, thereby increasing the probability of receiving definite treatment.

Intravascular schwannoma as an extremely unusual cause of vein obstruction: a case report.

The blood vessel lumen is an extremely rare location for a benign peripheral nerve sheath tumor like schwannoma. Less than 10 cases have been previously reported. In this report, we present a case of a 68-year-old woman who had a soft tissue nodule at the posterior calf of her left leg during a physical examination. Pathological examination was performed after complete surgical excision. The patient underwent follow-up for 12 months after surgery without evidence of recurrence or any other complication. This is the first case of intravascular schwannoma reported as a cause of vein obstruction. Microscopically, the tumor was composed of Schwann spindle cells that were immunoreactive for S100 protein and SOX10. This tumor was surrounded by a well-defined vascular smooth muscle wall. Prospective series are required to improve the knowledge on the underlying mechanisms of intravascular schwannoma development.

Cytogenetic profiles in multiple myeloma and monoclonal gammopathy of undetermined significance: a study in highly purified aberrant plasma cells.

Cytogenetic studies in clonal plasma cell disorders have mainly been done in whole bone marrow or CD138(+) microbead-enriched plasma cells and suggest that recurrent immunoglobulin heavy chain translocations - e.g. t(4;14) -are primary oncogenetic events. The aim of this study was to determine cytogenetic patterns of highly purified aberrant plasma cells (median purity ≥ 98%) in different clonal plasma cell disorders. We analyzed aberrant plasma cells from 208 patients with multiple myeloma (n=148) and monoclonal gammopathy of undetermined significance (n=60) for the presence of del(13q14), del(17p13) and t(14q32) using multicolor interphase fluorescence in situ hybridization. Additionally, immunoglobulin heavy chain gene arrangements were analyzed and complementarity determining region 3 was sequenced in a subset of patients and combined multicolor interphase fluorescence in situ hybridization/immunofluorescent protein staining analyses were performed in selected cases to confirm clonality and cytogenetic findings. At diagnosis, 96% of cases with multiple myeloma versus 77% of monoclonal gammopathy of undetermined significance cases showed at least one cytogenetic alteration and/or hyperdiploidy. The cytogenetic heterogeneity of individual cases reflected coexistence of cytogenetically-defined aberrant plasma cell clones, and led to the assumption that karyotypic alterations were acquired stepwise. Cases of multiple myeloma and monoclonal gammopathy of undetermined significance frequently showed different but related cytogenetic profiles when other cytogenetic alterations such as deletions/gains of the immunoglobulin heavy chain or the fibroblast growth factor receptor 3 were additionally considered. Interestingly, in 24% of multiple myeloma versus 62% of monoclonal gammopathy of undetermined significance patients with an immunoglobulin heavy chain translocation, aberrant plasma cells with and without t(14q32) coexisted in the same patient. Our data suggest that recurrent immunoglobulin heavy chain translocations might be absent in the primordial plasma cell clone in a significant proportion of patients with clonal plasma cell disorders carrying these cytogenetic alterations.

Transmembrane serine protease 2, a SARS-CoV-2 internalization protease, correlates with clinical outcome, molecular features, and immunotherapy response in colorectal cancer.

Background: Transmembrane serine protease 2 (TMPRSS2) mediates the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into host cells. The relevant research indicates the intestine to be a target of SARS-CoV-2 infection, and thus we aimed to investigate the correlation between TMPRSS2 expression and the prognosis, molecular features, and immunotherapy response in patients with colorectal cancer (CRC). Methods: The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases were used in this study and a total of 1,385 patients were identified. The CIBERSORT algorithms were used to evaluate the relative infiltration levels of immune cell types in the tumor microenvironment (TME). The correlation between TMPRSS2 expression and immunotherapy response rate was assessed in another 2 independent cohorts. Results: TMPRSS2 expression was significantly downregulated in cancer tissue compared to the adjacent normal tissue, and patients with CRC with lower TMPRSS2 expression showed notably poorer prognosis. Functional enrichment analysis found that low TMPRSS2 expression was significantly associated with cancer metastasis-related pathways. Further analysis based on the miRWalk tool and JASPAR database identified a list of microRNAs (miRNAs) and transcriptional factors targeting TMPRSS2. Distinct differences in immune cell infiltration and tumor purity reflected by estimate and mutant-allele tumor heterogeneity score were observed between patients with low and high TMPRSS2 expression levels. Interestingly, patients with a low TMPRSS2 expression level showed a higher response rate to immunotherapy. Conclusions: CRC cells may be more resistant to SARS-CoV-2 infection due to the decreased expression of TMPRSS2, which could be a newly identified biomarker for prognosis and immunotherapy response prediction in patients with CRC.

The Impact of Liquid Biopsies Positive for EGFR Mutations on Overall Survival in Non-Small Cell Lung Cancer Patients.

In recent years, non-small cell lung cancer treatment has been revolutionized. EGFR tyrosine kinase inhibitors and our improved understanding of its alterations have driven new diagnostic strategies. Liquid biopsies have emerged as a useful tool in these contexts, showing potential utility in early diagnosis combined with low-dose CT scans, as well as potential in monitoring treatment response and predicting the development of patients. We studied the circulating tumor DNA (ctDNA) of 38 EGFR-mutated non-small cell lung cancer patients at diagnosis in different moments of their disease by liquid biopsy techniques. Our results show that mean overall survival was significantly lower when a liquid biopsy was positive for the detection of EGFR mutations compared with wild-type patients in their liquid biopsy in both univariate (29 ± 4 vs. 104 ± 19 months; p = 0.004) and multivariate analysis (p = 0.008). Taking this into consideration, liquid biopsies could be key to improving the control of this disease.

Cerebellar Hemangioblastoma with Leptomeningeal Spread and a Fatal Outcome: A Rare Case Report with MDM2 and EGFR Alterations.

Hemangioblastoma (HB) is a Central Nervous System (CNS) tumor with a generally favorable behavior and prognosis, classified as WHO grade 1. Sporadic HB is not related to any inherited disease, and it usually appears in a single location. Sporadic or VHL-related HBs show variable patterns of growth velocity. Cases of growing HB can cause mild symptoms such as headache, but some cases develop serious complications such as accumulation of cerebrospinal fluid in the brain with secondary neurological damage sometimes being irreversible when early treatment is not started. Our case showed some clinical characteristics more frequently observed in VHL-related HB rather than sporadic HB, and the presence of alterations in MDM2 and EGFR that could be related to the oncogenesis of these tumors. Even when the treatment of choice for HB is surgery, the presence of these genetic alterations could open a new window for research aimed at assessing the possibility of new therapies with TKIs-EGFR and anti-MDM2 inhibitors in those HB cases with multifocal recurrences or cases with an adverse clinical behavior.

Prognostic Impact of EGFR Amplification and Visceral Pleural Invasion in Early Stage Pulmonary Squamous Cell Carcinomas Patients after Surgical Resection of Primary Tumor.

Over the last few decades, an increasing amount of information has been accumulated on biomarkers in non-small cell lung cancer (NSCLC). Despite these advances, most biomarkers have been identified in the adenocarcinoma histological subtype (AC). However, the application of molecular-targeted therapies in the prognosis and treatment of SCC in the clinical setting is very limited, becoming one of the main focus areas in research. Here, we prospectively analyzed the frequency of numerical/structural abnormalities of chromosomes 5, 7, 8, 9, 13 and 22 with FISH in 48 pulmonary SCC patients. From a total of 12 probes, only abnormalities of the 7p12 and 22q12 chromosomal regions were identified as unique genetic variables associated with the prognosis of the disease. The study for these two chromosomal regions was extended to 108 patients with SCC. Overall, chromosome losses were observed more frequently than chromosome gains, i.e., 61% versus 19% of all the chromosome abnormalities detected. The highest levels of genetic amplification were detected for the 5p15.2, 7p12, 8q24 and 22q11 chromosome bands, of which several genes are potentially involved in the pathogenesis of SCC, among others, include the EGFR gene at chromosome 7p12. Patients who displayed EGFR amplification (n = 13; 12%) were mostly older than 65 years (p = 0.07) and exclusively patients in early T-primary tumor stage (pT1−pT2; p = 0.03) with a significantly shortened overall survival (OS) (p ≤ 0.001). Regarding prognosis, the clinical, biological, and histopathologic characteristics of the disease that displayed a significant adverse influence on OS in the univariate analysis included patients older than 65 years (p = 0.02), the presence of lymph node involvement (p = 0.005), metastasis (p = 0.01) and, visceral pleural invasion (VPI) at diagnosis (p = 0.04). EGFR amplification also conferred an adverse impact on patient OS in the whole series (p = 0.02) and especially in patients in early stages (pT1−pT2; p = 0.01). A multivariate analysis of the prognostic factors for OS showed that the most informative combination of independent variables to predict an adverse outcome was the presence of VPI and/or EGFR amplification (p < 0.001). Based on these two variables, a scoring system was built to stratify patients into low- (no adverse features: score 0; n = 69), intermediate- (one adverse feature: score 1; n = 29) and high-risk (two adverse features: score 2; n = 5) groups, with significantly different (p = 0.001) OS rates at 50 months, which were as following: 32%, 28% and 0%, respectively. In the present study, we show that the presence of a high level of 7p12 (EGFR) amplification, exclusively detected in early stage SCC (pT1−pT2), is an independent adverse prognostic factor for OS. The identification of the EGFR gene copy number using FISH techniques may provide a more accurate diagnosis of high-risk populations after the complete resection of the primary tumor. When combined with VPI, three groups of pulmonary SCC were clearly identified that show the extent of the disease. This is of such importance that further prospective studies are necessary in larger series of SCC patients to be classified at the time of diagnosis. This could be achieved with the combined assessment of 7p12 amplification and VPI in primary tumor samples.

Dysregulated Expression of Three Genes in Colorectal Cancer Stratifies Patients into Three Risk Groups.

Despite advances in recent years in the study of the molecular profile of sporadic colorectal cancer (sCRC), the specific genetic events that lead to increased aggressiveness or the development of the metastatic process of tumours are not yet clear. In previous studies of the gene expression profile (GEP) using a high-density array (50,000 genes and 6000 miRNAs in a single assay) in sCRC tumours, we identified a 28-gene signature that was found to be associated with an adverse prognostic value for predicting patient survival. Here, we analyse the differential expression of these 28 genes for their possible association with tumour local aggressiveness and metastatic processes in 66 consecutive sCRC patients, followed for >5 years, using the NanoString nCounter platform. The global transcription profile (expression levels of the 28 genes studied simultaneously) allowed us to discriminate between sCRC tumours and nontumoral colonic tissues. Analysis of the biological and functional significance of the dysregulated GEPs observed in our sCRC tumours revealed 31 significantly altered canonical pathways. Among the most commonly altered pathways, we observed the increased expression of genes involved in signalling pathways and cellular processes, such as the PI3K-Akt pathway, the interaction with the extracellular matrix (ECM), and other functions related to cell signalling processes (SRPX2). From a prognostic viewpoint, the altered expression of BST2 and SRPX2 genes were the only independent variables predicting for disease-free survival (DFS). In addition to the pT stage at diagnosis, dysregulated transcripts of ADH1B, BST2, and FER1L4 genes showed a prognostic impact on OS in the multivariate analysis. Based on the altered expression of these three genes, a scoring system was built to stratify patients into low-, intermediate-, and high-risk groups with significantly different 5-year OS rates: 91%, 83%, and 52%, respectively. The prognostic impact was validated in two independent series of sCRC patients from the public GEO database (n = 562 patients). In summary, we show a strong association between the altered expression of three genes and the clinical outcome of sCRC patients, making them potential markers of suitability for adjuvant therapy after complete tumour resection. Additional prospective studies in larger series of patients are required to confirm the clinical utility of the newly identified biomarkers because the number of patients analysed remains small.

Flow cytometry immunophenotyping of fine-needle aspiration specimens: utility in the diagnosis and classification of non-Hodgkin lymphomas.

AIMS: To establish the utility of flow cytometry (FCM) for screening and diagnosis of B cell non-Hodgkin lymphoma (B-NHL) from lymphoid tissue samples obtained by fine-needle aspiration (FNA). METHODS AND RESULTS: We compared prospectively FCM versus cytology/histology analysis of FNA samples for the diagnostic screening and further World Health Organization (WHO) subclassification of B-NHL. FCM and cytology showed a high degree of agreement (93%); however, diagnosis of reactive processes (RP), B-NHL and T-NHL by FCM showed higher sensitivity than cytology (92-100% versus 64-94%, respectively), without false positive NHL cases. The antibody combination used did not allow a positive diagnosis of Hodgkin lymphoma as distinct from a RP. A high concordance rate was found between FCM and histopathology (74%) in subtyping B-NHL. In this regard, mantle-cell lymphoma and chronic lymphocytic leukaemia/small lymphocytic lymphoma showed the highest degree of agreement (100% concordant rates). In turn, FCM showed higher sensitivity/specificity in classifying follicular lymphoma (FL) and large B cell lymphomas, while the opposite occurred for marginal-zone and lymphoplasmacytic lymphomas. CONCLUSIONS: FCM enhances the diagnostic ability of FNA cytology, playing a crucial role in a rapid and accurate differential diagnosis between RP, B-NHL and T-NHL. In addition, immunophenotyping of FNA samples contributes to a more precise subclassification of B-NHL when combined with histopathology and genetic/molecular data.