Combining proteogenomics with laser capture microdissection (LCM) in cancer research offers a targeted way to explore the intricate interactions between tumor cells and the different microenvironment components. This is especially important for immuno-oncology (IO) research where improvements in the predictability of IO-based drugs are sorely needed, and depends on a better understanding of the spatial relationships involving the tumor, blood supply, and immune cell interactions, in the context of their associated microenvironments. LCM is used to isolate and obtain distinct histological cell types, which may be routinely performed on complex and heterogeneous solid tumor specimens. Once cells have been captured, nucleic acids and proteins may be extracted for in-depth multimodality molecular profiling assays. Optimizing the minute tissue quantities from LCM captured cells is challenging. Following the isolation of nucleic acids, RNA-seq may be performed for gene expression and DNA sequencing performed for the discovery and analysis of actionable mutations, copy number variation, methylation profiles, etc. However, there remains a need for highly sensitive proteomic methods targeting small-sized samples. A significant part of this protocol is an enhanced liquid chromatography mass spectrometry (LC-MS) analysis of micro-scale and/or nano-scale tissue sections. This is achieved with a silver-stained one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) approach developed for LC-MS analysis of fresh-frozen tissue specimens obtained via LCM. Included is a detailed in-gel digestion method adjusted and specifically designed to maximize the proteome coverage from amount-limited LCM samples to better facilitate in-depth molecular profiling. Described is a proteogenomic approach leveraged from microdissected fresh frozen tissue. The protocols may also be applicable to other types of specimens having limited nucleic acids, protein quantity, and/or sample volume.

The combination of single-cell RNA sequencing and microdissection techniques that preserves positional information has become a major tool for spatial transcriptome analyses. However, high costs and time requirements, especially for experiments at the single cell scale, make it challenging for this approach to meet the demand for increased throughput. Therefore, we proposed combinational DNA barcode (CDB)-seq as a medium-throughput, multiplexed approach combining Smart-3SEQ and CDB magnetic microbeads for transcriptome analyses of microdissected tissue samples. We conducted a comprehensive comparison of conditions for CDB microbead preparation and related factors and then applied CDB-seq to RNA extracts, fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) mouse brain tissue samples. CDB-seq transcriptomic profiles of tens of microdissected samples could be obtained in a simple, cost-effective way, providing a promising method for future spatial transcriptomics.

Metabolites reflect the biological state of cells and tissue, and metabolomics is therefore a field of high interest both to understand normal physiological functions and disease development. When studying heterogeneous tissue samples, mass spectrometry imaging (MSI) is a valuable tool as it conserves the spatial distribution of analytes on tissue sections. A large proportion of metabolites are, however, small and polar, making them vulnerable to delocalizing through diffusion during sample preparation. Here we present a sample preparation method optimized to limit diffusion and delocalization of small polar metabolites in fresh frozen tissue sections. This sample preparation protocol includes cryosectioning, vacuum frozen storage, and matrix application. The methods described were primely developed for matrix-assisted laser desorption/ionization (MALDI) MSI, but the protocol describing cryosectioning and vacuum freezing storage can also be applied before desorption electrospray ionization (DESI) MSI. Our vacuum drying and vacuum packing approach offers a particular advantage to limit delocalization and safe storage.

Breast cancer (BC) tissues have been proved to harbor microorganisms, which could potentially contribute to oncogenesis. Formalin-fixed paraffin-embedded (FFPE) tissues are the most widespread clinical samples in BC research. To verify the potential of FFPE tissues in microbiological analysis, we analyzed the microbial communities of FFPE and fresh frozen (FF) tumor samples from 30 participants diagnosed with BC deploying 16S rRNA sequencing. The operational taxonomic units (OTUs) analysis showed that 78.55% of OTUs in FFPE samples were consistent with FF samples. The composition of core bacteria did not change much, and there is also no difference in alpha diversity between FFPE and FF (without unclassified bacteria). Taxonomic variation results show that Firmicutes and Bacteroidota phyla, and their major classes, maintained the same proportion under two preservation methods. In addition, the major class Gammaproteobacteria, as well as its dominant orders Burkholderiales and Pseudomonadales all showed no significant difference in paired analysis. Moreover, the Proteobacteria and Actinobacteriota phyla showed no significant difference between FFPE and FF samples after subtracting unclassified bacteria. Therefore, premised with the intrinsic tumor heterogeneity and unclassified bacteria, there are potential values of FFPE tissues for intratumoral microbiome analysis in breast cancer.

Formalin-fixed paraffin-embedded (FFPE) tissue is the most common source of archived material for genomic medicine. However, FFPE tissue is suboptimal for high-throughput analyses, such as RNA sequencing, because the quality of nucleic acids in FFPE tissues is low. We compared RNA-seq with the nCounter system to evaluate use of FFPE tissue for genomic medicine. Twelve fresh frozen bladder cancer samples were analyzed by both RNA sequencing and nCounter, and matched FFPE samples, by nCounter. Gene-expression values obtained by these two platforms were compared by calculating Pearson correlation coefficients for each sample (across the set of matched genes) and for each matched gene (across the set of samples). For each sample, gene-expression levels measured by RNA sequencing highly correlated with those measured by nCounter (all Pearson\'s R > 0.8, P < 0.0001), as seen by hierarchical clustering. RNA sequencing results for fresh frozen tissues positively correlated with nCounter results for FFPE tissues (R ranged from 0.675 to 0.873, all P < 0.0001). Correlation and hierarchical-clustering analyses of nCounter data from the two specimens demonstrated a strong positive correlation between each group (R ranged from 0.779 to 0.977, all P < 0.0001). Our findings suggest that the nCounter system is useful for assaying archived-FFPE samples and that the gene-expression signatures obtained from FFPE samples represent those from fresh frozen tissues.

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.

Biobanking tissue of high quality and fidelity is imperative for cancer genomics research. Since it is a challenging process, we sought to develop a protocol that improves the fidelity and quantity of biobanked primary prostate cancer (CaP) tissue.
We conducted a pilot study evaluating pathologic concordance of biobanked tissue and the radical prostatectomy specimen using either standard protocol (SP) vs. next-generation protocol (NGP).
There were no significant differences in clinical and pathologic characteristics (age, BMI, preoperative PSA, prostate weight, race, final prostatectomy Gleason score, or pathologic tumor and nodal stages) between the two protocol arms. Utilization of the NGP compared to the standard protocol resulted in a significantly higher rate of pathologic concordance between the biobanked and RP specimens (61.8% vs. 37.9%, P = 0.0231) as well as a nearly two-fold increase in the amount of biobanked tumor tissue (330 mm3 vs. 174 mm3, P < 0.001). When looking at relevant clinical and pathologic characteristics, NGP was associated with pathologic concordance on both univariate [OR 2.65 (95% CI 1.13-6.21), P = 0.025] and multivariate analysis [OR 3.11 (95% CI 1.09-8.88), P = 0.034].
Our study validates the NGP as a multidisciplinary approach for improving the fidelity and amount of biobanked primary CaP tissue for future studies. Given the challenges to banking tissue from primary CaP as tumors are often difficult to visualize grossly and are frequently multifocal, optimizing the fidelity and volume of biobanked tissue is an important step forward to improve the generalizability of genomic data as we move towards precision medicine.

UNASSIGNED: To evaluate the potential effect of embedding with carboxylmethyl cellulose (CMC) and embedding with optimal cutting temperature (OCT) compound followed by washing with PBS (OCT-W) on the analysis of breast cancer tissue samples with desorption electrospray ionization mass spectrometry imaging (DESI-MSI).
UNASSIGNED: DESI-MSI of fresh frozen (FF) tissue samples, OCT-embedded samples, CMC-embedded samples, and OCT-W samples from the same breast cancer tumor tissue were performed. The ratio of maximum abundance ion was used to assess the reproducibility of DESI-MSI analysis. In addition, the effects of the treatment of each group were examined by comparing the characteristic ion species and the ion signal intensity detected by DESI-MSI.
UNASSIGNED: DESI-MSI of continuous sections of FF samples showed that the coefficient of variation (CV) of the pair-to-pair ratios of m/ z 281.25, m/ z 309.28 and m/ z 279.23 ions, the three ions with the highest intensity in the tumor region, were 19.61%, 20.74% and 10.18%, respectively. The characteristic ion species detected by DESI-MSI of CMC embedded tissue and the OCT-W tissue were almost the same, compared with those of the FF tumor tissue. However, ion species detected in OCT embedded samples were less than 50% of the FF samples. In terms of ion signal intensity, the CMC embedded tissue was not affected overall, while the signal of most of the characteristic ions of the OCT-W group showed decreased intensity (P<0.05).
UNASSIGNED: FF tissue sections and CMC-embedded samples can be used for DESI-MSI routine analysis. OCT embedding affects the feasibility of sample analysis whether or not the sample undergoes washing with PBS. CMC embedding agent is recommended if the tissue sections need to be fixated and supported due to small sample size, fragility, or other problems.

Purpose: Currently, formalin-fixed paraffin-embedded (FFPE) tissue specimens are the conventional material for gene testing for non-small cell lung cancer (NSCLC) patients. In our study, we aimed to develop a quick gene testing procedure using fresh core needle biopsy samples from NSCLC patients. Methods: In total, 77 fresh NSCLC samples obtained from core needle biopsy were evaluated by frozen section examination. If the NSCLC diagnosis and adequate tumor cell counts were confirmed by histopathology, the fresh tissues were used to extract DNA and subsequent gene testing by ARMS-PCR. Meanwhile, the paired FFPE core needle biopsy samples from 30 NSCLC patients also underwent gene testing. Results: In total, 77 fresh samples showed an EGFR mutation rate of 61.0%, higher than the levels in the Asian. Following a comparison of gene testing results with fresh tissues and paired FFPE tissues from the 30 patients, no significant difference in the DNA concentration extracted from fresh tissues and FFPE tissues was found. However, DNA purity was significantly higher in fresh tissues than that in FFPE tissues. Gene testing detected the same gene mutations in 93.3% of cases in fresh tissues and paired FFPE tissues. The gene testing procedure using fresh biopsy samples greatly shortens the waiting time of patients. Conclusion: The multi-gene mutation testing using fresh core needle biopsy samples from NSCLC patients is a reasonable, achievable, and quick approach. Fresh tissues may serve as a potential alternative to FFPE tissues for gene testing in NSCLC patients.

UNASSIGNED: Patients with locally advanced or metastatic pancreatic ductal adenocarcinoma (A-PDAC) are not candidates for surgical resection and are often offered palliative chemotherapy. The ready availability of a safe and effective tumor sampling technique to provide material for both diagnosis and comprehensive genetic profiling is critical for informing precision medicine in A-PDAC, thus potentially increasing survival. The aim of this study is to examine the feasibility and benefits of routine comprehensive genomic profiling (CGP) of A-PDAC using EUS-FNA material.
UNASSIGNED: This is a prospective cohort study to test the clinical utility of fresh frozen or archival EUS-FNA samples in providing genetic material for CGP. The results of the CGP will be reviewed at a molecular tumor board. The proportion of participants that have a change in their treatment recommendations based on their individual genomic profiling will be assessed. Correlations between CGP and stage, prognosis, response to treatment and overall survival will also be investigated. This study will open to recruitment in 2020, with a target accrual of 150 A-PDAC patients within 36 months, with a 2-year follow-up. It is expected that the majority of participants will be those who have already consented for their tissue to be biobanked in the Victorian Pancreatic Cancer Biobank at the time of diagnostic EUS-FNA. Patients without archival or biobanked material that is suitable for CGP may be offered a EUS-FNA procedure for the purposes of obtaining fresh frozen material.
UNASSIGNED: This trial is expected to provide crucial data regarding the feasibility of routine CGP of A-PDAC using EUS-FNA material. It will also provide important information about the impact of this methodology on patients\' survival.