Numerous software tools exist for data-independent acquisition (DIA) analysis of clinical samples, necessitating their comprehensive benchmarking. We present a benchmark dataset comprising real-world ...inter-patient heterogeneity, which we use for in-depth benchmarking of DIA data analysis workflows for clinical settings. Combining spectral libraries, DIA software, sparsity reduction, normalization, and statistical tests results in 1428 distinct data analysis workflows, which we evaluate based on their ability to correctly identify differentially abundant proteins. From our dataset, we derive bootstrap datasets of varying sample sizes and use the whole range of bootstrap datasets to robustly evaluate each workflow. We find that all DIA software suites benefit from using a gas-phase fractionated spectral library, irrespective of the library refinement used. Gas-phase fractionation-based libraries perform best against two out of three reference protein lists. Among all investigated statistical tests non-parametric permutation-based statistical tests consistently perform best.
Metastasis is the major cause of cancer-associated death. Partial activation of the epithelial-to-mesenchymal transition program (partial EMT) was considered a major driver of tumour progression from ...initiation to metastasis. However, the role of EMT in promoting metastasis has recently been challenged, in particular concerning effects of the Snail and Twist EMT transcription factors (EMT-TFs) in pancreatic cancer. In contrast, we show here that in the same pancreatic cancer model, driven by Pdx1-cre-mediated activation of mutant Kras and p53 (KPC model), the EMT-TF Zeb1 is a key factor for the formation of precursor lesions, invasion and notably metastasis. Depletion of Zeb1 suppresses stemness, colonization capacity and in particular phenotypic/metabolic plasticity of tumour cells, probably causing the observed in vivo effects. Accordingly, we conclude that different EMT-TFs have complementary subfunctions in driving pancreatic tumour metastasis. Therapeutic strategies should consider these potential specificities of EMT-TFs to target these factors simultaneously.
Intratumoral heterogeneity is a critical frontier in understanding how the tumor microenvironment (TME) propels malignant progression. Here, we deconvolute the human pancreatic TME through ...large-scale integration of histology-guided regional multiOMICs with clinical data and patient-derived preclinical models. We discover “subTMEs,” histologically definable tissue states anchored in fibroblast plasticity, with regional relationships to tumor immunity, subtypes, differentiation, and treatment response. “Reactive” subTMEs rich in complex but functionally coordinated fibroblast communities were immune hot and inhabited by aggressive tumor cell phenotypes. The matrix-rich “deserted” subTMEs harbored fewer activated fibroblasts and tumor-suppressive features yet were markedly chemoprotective and enriched upon chemotherapy. SubTMEs originated in fibroblast differentiation trajectories, and transitory states were notable both in single-cell transcriptomics and in situ. The intratumoral co-occurrence of subTMEs produced patient-specific phenotypic and computationally predictable heterogeneity tightly linked to malignant biology. Therefore, heterogeneity within the plentiful, notorious pancreatic TME is not random but marks fundamental tissue organizational units.
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•PDAC regional heterogeneity stems from sub-tumor microenvironments (subTMEs)•SubTMEs exhibit distinct immune phenotypes and CAF differentiation states•SubTMEs execute distinct tumor-promoting and chemoprotective functions•Intratumoral subTME co-occurrence links stromal heterogeneity to patient outcome
Intratumoral heterogeneity in the human pancreatic tumor microenvironment is not random but originates in well-definable regional tissue states. The underlying sub-tumor microenvironments shape regional epithelial and immune phenotypes and influence key clinical metrics of disease progression.
The developmental process of local and distant metastases represents the major and defining trait of malignant tumors, whereby tumor cells sustain the capability to migrate from the initial tumor ...site, seed, and grow at a location other than that of the initial tumor ...
In this retrospective study, we analyzed the association between tumor budding and perineural invasion as well as their prognostic role in pancreatic ductal adenocarcinoma. A total of
N
= 119 ...patients resected for pancreatic ductal carcinoma from 1996 to 2015 were included. Clinical and standard histopathological parameters were retrieved from the patient’s records. One representative hematoxylin and eosin section from the tumor region was examined for perineural invasion and tumor budding using light microscopy. Tumor budding was assessed independently using two different methods: in the first approach, the number of buds was counted over three fields of 0.237 mm
2
at 40-fold magnification; in the second approach, tumor budding was quantified according to the recommendation of the International Tumor Budding Consensus Conference (ITBCC) over a field of 0.785 mm
2
at 20-fold magnification. Linear and logistic regression was applied to delineate association between perineural invasion, tumor budding, and other parameters; Kaplan-Meier and Cox regression were used in the survival analysis. Regardless of the quantification approach, high tumor budding was a significant negative prognostic factor in the univariable Cox regression (> 5 buds/0.237 mm
2
, hazard ratio (HR) 1.66, 95% confidence interval (CI) 1.06–2.61,
p
= 0.027; ≥ 10 buds/0.785 mm
2
, HR 1.68, 95% CI 1.07–2.64,
p
= 0.024). In the multivariable model adjusting for stage and standard histopathological parameters, lymph vessel invasion (HR = 2.43, 95% CI 1.47–4.03,
p
= 0.001) and tumor budding > 5 buds/0.237 mm
2
(HR = 1.70, 95% CI 1.07–2.7,
p
= 0.026) were independent negative prognostic factors, while adjuvant therapy was a positive prognostic factor (HR = 0.54, 95% CI 0.33–0.86,
p
= 0.009). No significant prognostic value could be delineated for perineural invasion. In conclusion, tumor budding is an independent negative prognostic factor in pancreatic ductal adenocarcinoma associated with lymph node metastasis. The prognostic role of perineural invasion remains uncertain.
Colorectal adenocarcinomas (CRC) are one of the most commonly diagnosed tumors worldwide. Colorectal adenocarcinomas primarily metastasize into the liver and (less often) into the peritoneum. ...Patients suffering from CRC-liver metastasis (CRC-LM) typically present with a dismal overall survival compared to non-metastasized CRC patients. The metastasis process and metastasis-promoting factors in patients with CRC are under intensive debate. However, CRC studies investigating the proteome biology are lacking. Formalin-fixed paraffin-embedded (FFPE) tissue specimens provide a valuable resource for comprehensive proteomic studies of a broad variety of clinical malignancies. The presented pilot study compares the proteome of primary CRC and patient-matched CRC-LM. The applied protocol allows a reproducible and straightforward identification and quantification of over 2,600 proteins within the dissected tumorous tissue. Subsequent unsupervised clustering reveals distinct proteome biologies of the primary CRC and the corresponding CRC-LM. Statistical analysis yields multiple differentially abundant proteins in either primary CRC or their corresponding liver metastases. A more detailed analysis of dysregulated biological processes suggests an active immune response in the liver metastases, including several proteins of the complement system. Proteins with structural roles, e.g. cytoskeleton organization or cell junction assembly appear to be less prominent in liver metastases as compared to primary CRC. Immunohistochemistry corroborates proteomic high expression levels of metabolic proteins in CRC-LM. We further assessed how the in vitro inhibition of two in CRC-LM enriched metabolic proteins affected cell proliferation and chemosensitivity. The presented proteomic investigation in a small clinical cohort promotes a more comprehensive understanding of the distinct proteome biology of primary CRC and their corresponding liver metastases.
Raman spectroscopy offers label-free assessment of bladder tissue for in vivo and ex vivo intraoperative applications. In a retrospective study, control and cancer specimens were prepared from ten ...human bladder resectates. Raman microspectroscopic images were collected from whole tissue samples in a closed chamber at 785 nm laser excitation using a 20× objective lens and 250 µm step size. Without further preprocessing, Raman images were decomposed by the hyperspectral unmixing algorithm vertex component analysis into endmember spectra and their abundancies. Hierarchical cluster analysis distinguished endmember Raman spectra that were assigned to normal bladder, bladder cancer, necrosis, epithelium and lipid inclusions. Interestingly, Raman spectra of microplastic particles, pigments or carotenoids were detected in 13 out of 20 specimens inside tissue and near tissue margins and their identity was confirmed by spectral library surveys. Hypotheses about the origin of these foreign materials are discussed. In conclusion, our Raman workflow and data processing protocol with minimal user interference offers advantages for future clinical translation such as intraoperative tumor detection and label-free material identification in complex matrices.
Background Pancreatic ductal adenocarcinoma (PDAC) is characterized by an aggressive biology and poor prognosis. Experimental evidence has suggested a role for the transcriptional repressor Zinc ...finger E-box binding homeobox 1 (ZEB1) in epithelial-mesenchymal transition, invasion, and metastasis in PDAC. ZEB1 expression has been observed in cancer cells as well as stromal fibroblasts. Our study aimed to evaluate the prognostic value of ZEB1 expression in PDAC tissue. Methods Patient baseline and follow-up data were extracted from a prospectively maintained database. After clinicopathologic re-review, serial sliced tissue slides were immunostained for ZEB1, E-cadherin, vimentin, and pan-cytokeratin. ZEB1 expression in cancer cells and adjacent stromal fibroblasts was graded separately and correlated to routine histopathologic parameters and survival after resection. Results A total of 117 cases of PDAC were included in the study. High ZEB1 expression in cancer cells and in stromal cancer-associated fibroblasts was associated with poor prognosis. There was also a trend for poor prognosis with a lymph node ratio of greater than 0.10. In line with its role as an inducer of epithelial-mesenchymal transition, ZEB1 expression in cancer cells was positively correlated with Vimentin expression and negatively with E-Cadherin expression. In multivariate analysis, stromal ZEB1 expression grade was the only independent factor of survival after resection. Conclusion Our data suggest that ZEB1 expression in cancer cells as well as in stromal fibroblasts are strong prognostic factors in PDAC. Stromal ZEB1 expression is identified for the first time as an independent predictor of survival after resection of PDAC. This observation suggests that therapies targeting ZEB1 and its downstream pathways could hit both cancer cells and supporting cancer-associated fibroblasts.
Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease. Circulating tumor cells (CTC) in the blood are hypothesized as the means of systemic tumor spread. Blood obtained from healthy donors ...and patients with PDAC was therefore subject to size-based CTC-isolation (ISET). We additionally compared Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations in pancreatic CTC and corresponding tumors, and evaluated their significance as prognostic markers. Samples from 68 individuals (58 PDAC patients, 10 healthy donors) were analyzed; CTCs were present in patients with UICC stage IA-IV tumors and none of the controls (p < 0.001). Patients with >3 CTC/ml had a trend for worse median overall survival (OS) than patients with 0.3-3 CTC/ml (P = 0.12). Surprisingly, CTCs harbored various KRAS mutations in codon 12 and 13. Patients with a KRAS
mutation in their CTC (n = 14) had a trend to better median OS (24.5 months) compared to patients with other (10 months), or no detectable KRAS mutations (8 months; P = 0.04). KRAS mutations in CTC and corresponding tumor were discordant in 11 of 26 "tumor-CTC-pairs" (42%), while 15 (58%) had a matching mutation; survival was similar in both groups (P = 0.36). Genetic characterization, including mutations such as KRAS, may prove useful for prognosis and understanding of tumor biology.
Accurate determination of resection margins in breast specimens is important as complete removal of malignancy is a prerequisite for patients' outcome. Mammography (DM) as 2D-technique provides only ...limited value in margin assessment. Therefore, we investigated whether cone-beam computed tomography (CBCT) or digital breast tomosynthesis (DBT) has incremental value in assessing margins to microcalcifications. Three independent readers investigated breast specimens for presence of microcalcifications and the smallest distance to margins. Histopathology served as gold standard. Microcalcifications were detected in 15 out of 21 included specimens (71%). Pooled sensitivity for DM, DBT and CBCT for microcalcifications compared to preoperative DM was 0.98 (CI 0.94-0.99), 0.83 (CI 0.73-0.94) and 0.94 (CI 0.87-0.99), pooled specificity was 0.99 (CI 0.99-0.99), 0.73 (CI 0.51-0.96) and 0.60 (CI 0.35-0.85). Mean measurement error for margin determination for DM, DBT and CBCT was 10 mm, 14 mm and 6 mm (p = 0.002) with significant difference between CBCT and the other devices (p < 0.03). Mean reading time required by the readers to analyze DM, DBT and CBCT, was 36, 43 and 54 s (p < 0.001). Although DM allows reliable detection of microcalcifications, measurement of resection margin was significantly more accurate with CBCT. Thus, a combination of methods or improved CBCT might provide a more accurate determination of disease-free margins in breast specimens.
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