Validation of a Novel Three-Dimensional (3D Fusion) Gross Sampling Protocol for Clear Cell Renal Cell Carcinoma to Overcome Intratumoral Heterogeneity: The Meet-Uro 18 Study

We aimed to overcome intratumoral heterogeneity in clear cell renal cell carcinoma (clearRCC). One hundred cases of clearRCC were sampled. First, usual standard sampling was applied (1 block/cm of tumor); second, the whole tumor was sampled, and 0.6 mm cores were taken from each block to construct a tissue microarray; third, the residual tissue, mapped by taking pieces 0.5 × 0.5 cm, reconstructed the entire tumor mass. Precisely, six randomly derived pieces of tissues were placed in each cassette, with the number of cassettes being based on the diameter of the tumor (called multisite 3D fusion). Angiogenic and immune markers were tested. Routine 5231 tissue blocks were obtained. Multisite 3D fusion sections showed pattern A, homogeneous high vascular density (10%), pattern B, homogeneous low vascular density (8%) and pattern C, heterogeneous angiogenic signatures (82%). PD-L1 expression was seen as diffuse (7%), low (33%) and absent (60%). Tumor-infiltrating CD8 scored high in 25% (pattern hot), low in 65% (pattern weak) and zero in 10% of cases (pattern desert). Grading was upgraded in 26% of cases (G3-G4), necrosis and sarcomatoid/rhabdoid characters were observed in, respectively, 11 and 7% of cases after 3D fusion (p = 0.03). CD8 and PD-L1 immune expressions were higher in the undifferentiated G4/rhabdoid/sarcomatoid clearRCC subtypes (p = 0.03). Again, 22% of cases were set to intermediate to high risk of clinical recurrence due to new morphological findings of all aggressive G4, sarcomatoid/rhabdoid features by using 3D fusion compared to standard methods (p = 0.04). In conclusion, we propose an easy-to-apply multisite 3D fusion sampling that negates bias due to tumor heterogeneity

MiT Family Translocation Renal Cell Carcinoma: from the Early Descriptions to the Current Knowledge

The new category of MiT family translocation renal cell carcinoma has been included into the World Health Organization (WHO) classification in 2016. The MiT family translocation renal cell carcinoma comprises Xp11 translocation renal cell carcinoma harboring TFE3 gene fusions and t(6;11) renal cell carcinoma harboring TFEB gene fusion. At the beginning, they were recognized in childhood; nevertheless, it has been demonstrated that these neoplasms can occur in adults as well. In the nineties, among Xp11 renal cell carcinoma, ASPL, PRCC, and SFPQ (PSF) were the first genes recognized as partners in TFE3 rearrangement. Recently, many other genes have been identified, and a wide spectrum of morphologies has been described. For this reason, the diagnosis may be challenging based on the histology, and the differential diagnosis includes the most common renal cell neoplasms and pure epithelioid PEComa/epithelioid angiomyolipoma of the kidney. During the last decades, many efforts have been made to identify immunohistochemical markers to reach the right diagnosis. To date, staining for PAX8, cathepsin K, and melanogenesis markers are the most useful identifiers. However, the diagnosis requires the demonstration of the chromosomal rearrangement, and fluorescent in situ hybridization (FISH) is considered the gold standard. The outcome of Xp11 translocation renal cell carcinoma is highly variable, with some patients surviving decades with indolent disease and others dying rapidly of progressive disease. Despite most instances of t(6;11) renal cell carcinoma having an indolent clinical course, a few published cases demonstrate aggressive behavior. Recently, renal cell carcinomas with TFEB amplification have been described in connection with t(6;11) renal cell carcinoma. Those tumors appear to be associated with a more aggressive clinical course. For the aggressive cases of MiT family translocation carcinoma, the optimal therapy remains to be determined; however, new target therapies seem to be promising, and the search for predictive markers is mandatory

Unbalanced IDO1/IDO2 endothelial expression and skewed keynurenine pathway in the pathogenesis of COVID-19 and post-COVID-19 pneumonia

Despite intense investigation, the pathogenesis of COVID-19 and the newly defined long COVID-19 syndrome are not fully understood. Increasing evidence has been provided of metabolic alterations characterizing this group of disorders, with particular relevance of an activated tryptophan/kynurenine pathway as described in this review. Recent histological studies have documented that, in COVID-19 patients, indoleamine 2,3-dioxygenase (IDO) enzymes are differentially expressed in the pulmonary blood vessels, i.e., IDO1 prevails in early/mild pneumonia and in lung tissues from patients suffering from long COVID-19, whereas IDO2 is predominant in severe/fatal cases. We hypothesize that IDO1 is necessary for a correct control of the vascular tone of pulmonary vessels, and its deficiency in COVID-19 might be related to the syndrome's evolution toward vascular dysfunction. The complexity of this scenario is discussed in light of possible therapeutic manipulations of the tryptophan/kynurenine pathway in COVID-19 and post-acute COVID-19 syndromes

Wide spetcrum mutational analysis of metastatic renal cell cancer : a retrospective next generation sequencing approach

Renal cell cancer (RCC) is characterized by histological and molecular heterogeneity that may account for variable response to targeted therapies. We evaluated retrospectively with a next generation sequencing (NGS) approach using a pre-designed cancer panel the mutation burden of 32 lesions from 22 metastatic RCC patients treated with at least one tyrosine kinase or mTOR inhibitor. We identified mutations in the VHL, PTEN, JAK3, MET, ERBB4, APC, CDKN2A, FGFR3, EGFR, RB1, TP53 genes. Somatic alterations were correlated with response to therapy. Most mutations hit VHL1 (31,8%) followed by PTEN (13,6%), JAK3, FGFR and TP53 (9% each). Eight (36%) patients were wild-type at least for the genes included in the panel. A genotype concordance between primary RCC and its secondary lesion was found in 3/6 cases. Patients were treated with Sorafenib, Sunitinib and Temsirolimus with partial responses in 4 (18,2%) and disease stabilization in 7 (31,8%). Among the 4 partial responders, 1 (25%) was wild-type and 3 (75%) harbored different VHL1 variants. Among the 7 patients with disease stabilization 2 (29%) were wild-type, 2 (29%) PTEN mutated, and single patients (14% each) displayed mutations in VHL1, JAK3 and APC/CDKN2A. Among the 11 non-responders 7 (64%) were wild-type, 2 (18%) were p53 mutated and 2 (18%) VHL1 mutated. No significant associations were found among RCC histotype, mutation variants and response to therapies. In the absence of predictive biomarkers for metastatic RCC treatment, a NGS approach may address single patients to basket clinical trials according to actionable molecular specific alterations.Peer reviewe

PD-L1 expression heterogeneity in non-small cell lung cancer: Evaluation of small biopsies reliability

Immunotherapy with checkpoint inhibitors, allowing recovery of effector cells function, has demonstrated to be highly effective in many tumor types and represents a true revolution in oncology. Recently, the anti-PD1 agent pembrolizumab was granted FDA approval for the first line treatment of patients with advanced non-small cell lung cancer (NSCLC) whose tumors show PD-L1 expression in \ue2\u89\ua5 50% of neoplastic cells and as a second line treatment for patients with NSCLC expressing PD-L1 in \ue2\u89\ua51% of neoplastic cells, evaluated with a validated assay. For the large majority of patients such evaluation is made on small biopsies. However, small tissue samples such as core biopsies might not be representative of tumors and may show divergent results given the possible heterogeneous immunoexpression of the biomarker. We therefore sought to evaluate PD-L1 expression concordance in a cohort of 239 patients using tissue microarrays (TMA) as surrogates of biopsies stained with a validated PD-L1 immunohistochemical assay (SP263) and report the degree of discordance among tissue cores in order to understand how such heterogeneity could affect decisions regarding therapy. We observed a discordance rate of 20% and 7.9% and a Cohen's \uce\uba value of 0.53 (moderate) and 0,48 (moderate) for \ue2\u89\ua5 1% and \ue2\u89\ua5 50% cutoffs, respectively. Our results suggest that caution must be taken when evaluating single biopsies from patients with advanced NSCLC eligible for immunotherapy; moreover, at least 4 biopsies are necessary in order to minimize the risk of tumor misclassification

Validation of 34betaE12 immunoexpression in clear cell papillary renal cell carcinoma as a sensitive biomarker

Clear cell papillary renal cell carcinoma (CCPRCC) is a recently recognised neoplasm with a broad spectrum of morphological characteristics, thus representing a challenging differential diagnosis, especially with the low malignant potential multicystic renal cell neoplasms and clear cell renal cell carcinoma. We selected 14 cases of CCPRCC with a wide spectrum of morphological features diagnosed on morphology and CK7 immunoreactivity and analysed them using a panel of immunohistochemical markers, focusing on 34 beta E12 and related CKs 1,5,10 and 14 and several molecular analyses such as fluorescence in situ hybridisation (FISH), array comparative genomic hybridisation (aCGH), VHL methylation, VHL and TCEB1 sequencing and multiplex ligation-dependent probe amplification (MLPA). Twelve of 13 (92%) CCPRCC tumours were positive for 34 beta E12. One tumour without 3p alteration by FISH revealed VHL mutation and 3p deletion at aCGH; thus, it was re-classified as clear cell RCC. We concluded that: (1) immunohistochemical expression of CK7 is necessary for diagnostic purposes, but may not be sufficient to identify CCPRCC, while 34 beta E12, in part due to the presence of CK14 antigen expression, can be extremely useful for the recognition of this tumour; and (2) further molecular analysis of chromosome 3p should be considered to support of CCPRCC diagnosis, when FISH analysis does not evidence the common loss of chromosome 3p.Peer reviewe

Racial differences in the outcome of patients with urothelial carcinoma of the upper urinary tract: an international study

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/86897/1/j.1464-410X.2011.10188.x.pd

The Local Complement Activation on Vascular Bed of Patients with Systemic Sclerosis : A Hypothesis-Generating Study

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LC3B and ph-S6K are both expressed in epithelioid and classic renal angiomyolipoma: a rationale tissue-based evidence for combining use of autophagic and mTOR targeted drugs

Background: Targeted drugs to the autophagy processes are emerging in clinical trials. The aim of this work is to assess the magnitude of autophagic expression in renal angiomyolipoma. Methods: Fourteen cases of renal angiomyolipoma were recruited. Anti-LC3B-II and anti-phospho-S6K were detected by Western blot analysis. For immunohistochemical staining, sections were stained with the antibodies LC3B-II and cathepsin-K. LC3B-II was also analyzed by immunofluorescence. We have also carried out electron microscopy analysis on tumor cells. Results: 13 classic and 1 epithelioid renal angiomyolipoma were recruited. The Western-blot LC3B-II analysis shows increasing in protein expression in all cases, however quantitative protein expression ranged from 1 to 15 (mean 5). The autophagosome protein LC3B-I also significantly increased in all tumor extraction. The expression of LC3B-II protein was confirmed in tumoral samples by immunofluorescence. The lysosomal marker cathepsin-K was observed by immunohistochemistry on all tumours. The Western-blot ph-S6K analysis showed significant protein overexpression along all cases after evaluation of the quantitative S6K/Ponceaus ratio. In 6/14 (52%) the expression was high, with a quantitative increase of 653 fold induction in 4 angiomyolipoma compared to normal tissue. At electron microscopy, cancer cells evidenced round or oval electron-dense granules associated with membranes and granules with double membrane. Conclusion: Both autophagic LC3B-II and ph-S6K molecules are over-represented in both epithelioid and classic renal angiomyolipoma and a combined use of inhibitors to the autophagic and mTOR processes may be designed in clinical trials, when enrolling patients affected by tumours in tuberous sclerosis or angiomyolipoma at risk of bledding

True 3q Chromosomal Amplification in Squamous Cell Lung Carcinoma by FISH and aCGH Molecular Analysis: Impact on Targeted Drugs

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