Comparison of matched formalin-fixed paraffin embedded and fresh frozen meningioma tissue reveals bias in proteomic profiles.

Tissue biopsies are most commonly archived in a paraffin block following tissue fixation with formaldehyde (FFPE) or as fresh frozen tissue (FFT). While both methods preserve biological samples, little is known about how they affect the quantifiable proteome. We performed a 'bottom-up' proteomic analysis (N = 20) of short and long-term archived FFPE surgical samples of human meningiomas and compared them to matched FFT specimens. FFT facilitated a similar number of proteins assigned by MetaMorpheus compared with matched FFPE specimens (5378 vs. 5338 proteins, respectively (p = 0.053), regardless of archival time. However, marked differences in the proteome composition were apparent between FFPE and FFT specimens. Twenty-three percent of FFPE-derived peptides and 8% of FFT-derived peptides contained at least one chemical modification. Methylation and formylation were most prominent in FFPE-derived peptides (36% and 17% of modified FFPE peptides, respectively) while, most of phosphorylation and iron modifications appeared in FFT-derived peptides (p < 0.001). A mean 14% (± 2.9) of peptides identified in FFPE contained at least one modified Lysine residue. Importantly, larger proteins were significantly overrepresented in FFT specimens, while FFPE specimens were enriched with smaller proteins.

Divergence of mutational signatures in association with breast cancer subtype.

Abnormal molecular processes occurring throughout the genome leave distinct somatic mutational patterns termed mutational signatures. Exploring the associations between mutational signatures and clinicopathological features can unravel potential mechanisms driving tumorigenic processes. We analyzed whole genome sequencing (WGS) data of tumor and peripheral blood samples from 37 primary breast cancer (BC) patients receiving neoadjuvant chemotherapy. Comprehensive clinico-pathologic features were correlated with genomic profiles and mutational signatures. Somatic mutational landscapes were highly concordant with known BC data sets. Remarkably, we observed a divergence of dominant mutational signatures in association with BC subtype. Signature 5 was overrepresented in hormone receptor positive (HR+) patients, whereas triple-negative tumors mostly lacked Signature 5, but expectedly overrepresented Signature 3. We validated these findings in a large WGS data set of BC, demonstrating dominance of Signature 5 in HR+ patients, mostly in luminal A subtype. We further investigated the association between Signature 5 and gene expression signatures, and identified potential networks, likely related to estrogen regulation. Our results suggest that the yet elusive Signature 5 represents an alternative mechanism for mutation accumulation in HR+ BC, independent of the homologous recombination repair machinery related to Signature 3. This study provides theoretical basis for further elucidating the processes promoting hormonal breast carcinogenesis.

Immunotherapy response modeling by ex-vivo organ culture for lung cancer.

One of the major hurdles for the advancement of cancer immunotherapy is lack of robust, accessible experimental models. We aimed to produce an ex-vivo organ culture (EVOC) model of immunotherapy for non-small cell lung cancer (NSCLC). Freshly resected early stage tumors were collected from the operating room, fragmented to clusters < 450 µm and cultured with fetal calf serum and human autologous serum. The resulting EVOC includes cancer epithelial cells within tumor tissue clusters and immune cells. Original tissue features are reflected in the EVOCs. The response to immune checkpoint inhibitors (ICI) was assessed by IFNγ gene induction. Interestingly, IFNγ EVOC induction was numerically higher when anti-CTLA4 was added to anti-PD-L1 treatment, supporting the notion that anti-CTLA4 impacts cancer partly through tumor-resident immune cells. In parallel, immunohistochemistry (IHC) for key immune-related proteins was performed on the formalin-fixed paraffin embedded (FFPE) corresponding tumors. EVOC IFNγ induction by ICI correlated with basal non-induced IFNγ, CD8, CD4 and FOXP3 mRNA levels within EVOCs and with tumor-FFPE-IHC for CD8 and granzyme B. A weaker correlation was seen with tumor-FFPE-IHC for CD3, CD4, CD68, FOXP3 and tumor-PD-L1. Tertiary lymphoid structure density was also correlated with the ICI response. Our study provides novel data about biomarkers that correlate with ICI-induced response of early stage NSCLC. Retention of the microenvironment and minimal addition of exogenous factors suggest this model to reliably represent the original tumor. The cluster-based EVOC model we describe can provide a valuable, yet simple and widely applicable tool for the study of immunotherapy in NSCLC.

Congenital Sucrase-isomaltase Deficiency: A Novel Compound Heterozygous Mutation Causing Aberrant Protein Localization.

OBJECTIVES: Congenital diarrheal disorders is a group of inherited enteropathies presenting in early life and requiring parenteral nutrition. In most cases, genetics may be the key for precise diagnosis. We present an infant girl with chronic congenital diarrhea that resolved after introduction of fructose-based formula but had no identified mutation in the SLC5A1 gene. Using whole exome sequencing (WES) we identified other mutations that better dictated dietary adjustments. METHODS: WES of the patient and her parents was performed. The analysis focused on recessive model including compound heterozygous mutations. Sanger sequencing was used to validate identified mutations and to screen the patient's newborn sister and grandparents. Expression and localization analysis were performed in the patient's duodenal biopsies using immunohistochemistry. RESULTS: Using WES we identified a new compound heterozygote mutation in sucrase-isomaltase (SI) gene; a maternal inherited known V577G mutation, and a novel paternal inherited C1531W mutation. Importantly, the newborn offspring carried similar compound heterozygous mutations. Computational predictions suggest that both mutations highly destabilize the protein. SI expression and localization studies determined that the mutated SI protein was not expressed on the brush border membrane in the patient's duodenal biopsies, verifying the diagnosis of congenital sucrase-isomaltase deficiency (CSID). CONCLUSIONS: The novel compound heterozygote V577G/C1531W SI mutations lead to lack of SI expression in the duodenal brush border, confirming the diagnosis of CSID. These cases of CSID extend the molecular spectrum of this condition, further directing a more adequate dietary intervention for the patient and newborn sibling.

Congenital protein losing enteropathy: an inborn error of lipid metabolism due to DGAT1 mutations.

Protein-losing enteropathy (PLE) is a clinical disorder of protein loss from the gastrointestinal system that results in hypoproteinemia and malnutrition. This condition is associated with a wide range of gastrointestinal disorders. Recently, a unique syndrome of congenital PLE associated with biallelic mutations in the DGAT1 gene has been reported in a single family. We hypothesize that mutations in this gene are responsible for undiagnosed cases of PLE in infancy. Here we investigated three children in two families presenting with severe diarrhea, hypoalbuminemia and PLE, using clinical studies, homozygosity mapping, and exome sequencing. In one family, homozygosity mapping using SNP arrays revealed the DGAT1 gene as the best candidate gene for the proband. Sequencing of all the exons including flanking regions and promoter regions of the gene identified a novel homozygous missense variant, p.(Leu295Pro), in the highly conserved membrane-bound O-acyl transferase (MBOAT) domain of the DGAT1 protein. Expression studies verified reduced amounts of DGAT1 in patient fibroblasts. In a second family, exome sequencing identified a previously reported splice site mutation in intron 8. These cases of DGAT1 deficiency extend the molecular and phenotypic spectrum of PLE, suggesting a re-evaluation of the use of DGAT1 inhibitors for metabolic disorders including obesity and diabetes.

Genetic mutation screen in early non--small-cell lung cancer (NSCLC) specimens.

BACKGROUND: Testing for genetic abnormalities in epithelial growth factor receptor (EGFR), anaplastic lymphoma receptor tyrosine kinase (ALK), and potentially additional genes is a critical tool in the care of advanced NSCLC. There is conflicting evidence for the role of such tests in early NSCLC. We report a single-institute Sequenom testing for a wide range of mutations and their clinical correlations in early-resected NSCLC specimens. MATERIALS AND METHODS: Early NSCLC paraffin-embedded, formalin-fixed (FFPE) specimens were collected, DNA extracted, and using Sequenom-based matrix-assisted laser desorption/ionization-time of flight analysis, mutations in 22 oncogenes and tumor suppressor genes were evaluated. Clinical data was collected retrospectively. RESULTS: The technique was found to be feasible. Thirty-six of 96 patients (37.5%) had any genetic abnormality identified, and 8 (8.3%) had 2 or more mutations. Kirsten rat sarcoma viral oncogene homolog (KRAS) and EGFR were the most common genes to appear mutated (15.6%); phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) was the gene to be found most commonly in tumors with co-mutations. Transversions were found mostly in KRAS gene mutations and to be nonprognostic. No difference in the spectrum of mutations was found between squamous-cell and non-squamous-cell lung cancers. Ever-smokers showed a trend for worse prognosis, with a similar spectrum of mutations. CONCLUSION: Sequenom-based mutation screen is feasible using FFPE samples. More than a third of the patients were found to harbor some genetic abnormality, and 8% were found to have more than a single mutated gene. Wide-range gene screens using large sample depositories are required for further insight into the important genes at play in early NSCLC.

Structural basis for hyperpermeability of tumor vessels in advanced lung adenocarcinoma complicated by pleural effusion.

BACKGROUND: Malignant pleural effusion (MPE) has a profound impact on quality of life and survival in patients with lung cancer. Identification of the factors within the tumor and its environment that mediate MPE is still lacking. PATIENTS AND METHODS: Intratumoral microvessel density (MVD), endothelial cell and pericyte (PC) capillary coverage, endothelial cell (EC)-PC relationship, lymphatic endothelium integrity, and the expression of receptor tyrosine kinases were all assessed immunohistochemically in pleural tumor biopsy specimens from 24 patients with lung adenocarcinoma (ADC) with and without pleural disease, with the aim to evaluate the involvement with MPE. RESULTS: In the effusion-positive⁺ specimens, MVD values were found to be significantly higher, and a number of vessels were noted to lack immunoreactivity for ECs (CD31). Likewise, PC α-smooth muscle actin (αSMA) expression was also less extensive in the MPE⁺ cases. The observation of only sporadic staining of PCs can also explain the findings regarding platelet-derived growth factor receptors (PDGFRs), the expression of which, although more prominent in MPE⁺ samples, were almost exclusively detected on tumor stromal cells and not on vascular PCs. Conversely, vascular endothelial growth factor receptors (VEGFRs) appeared on both kinds of cells. With respect to lymphatic vessels, lymphatic intraluminal tumor cells were occasionally found in MPE⁺ specimens. CONCLUSION: Our study suggests that disturbed vessel wall integrity, as well as abnormalities of fluid clearance by the lymphatic system, together with overexpression of growth factors, may take part in the pleural fluid accumulation in lung ADCs. Results of the decreased PC capillary coverage and PDGFR expression in MPE are discussed.