Identification of proteomic biomarkers predicting prostate cancer aggressiveness and lethality despite biopsy-sampling error.

BACKGROUND: Key challenges of biopsy-based determination of prostate cancer aggressiveness include tumour heterogeneity, biopsy-sampling error, and variations in biopsy interpretation. The resulting uncertainty in risk assessment leads to significant overtreatment, with associated costs and morbidity. We developed a performance-based strategy to identify protein biomarkers predictive of prostate cancer aggressiveness and lethality regardless of biopsy-sampling variation. METHODS: Prostatectomy samples from a large patient cohort with long follow-up were blindly assessed by expert pathologists who identified the tissue regions with the highest and lowest Gleason grade from each patient. To simulate biopsy-sampling error, a core from a high- and a low-Gleason area from each patient sample was used to generate a 'high' and a 'low' tumour microarray, respectively. RESULTS: Using a quantitative proteomics approach, we identified from 160 candidates 12 biomarkers that predicted prostate cancer aggressiveness (surgical Gleason and TNM stage) and lethal outcome robustly in both high- and low-Gleason areas. Conversely, a previously reported lethal outcome-predictive marker signature for prostatectomy tissue was unable to perform under circumstances of maximal sampling error. CONCLUSIONS: Our results have important implications for cancer biomarker discovery in general and development of a sampling error-resistant clinical biopsy test for prediction of prostate cancer aggressiveness.

Thymosin beta 15: a novel regulator of tumor cell motility upregulated in metastatic prostate cancer.

The Dunning rat prostatic carcinoma is a model system where cell motility closely correlates with the metastatic phenotype. We have identified a novel gene, upregulated in the highly motile and metastatic Dunning cancer cell lines, that represents a new member of the thymosin-beta family, thymosin beta 15. Transfection of antisense thymosin beta 15 constructs into rat prostatic carcinoma cells demonstrates that this molecule positively regulates cell motility, a critical component of the metastatic pathway. Thymosin beta 15 levels are elevated in human prostate cancer and correlate positively with the Gleason tumor grade. Thymosin beta 15 may represent a potential new biochemical marker for human prostate cancer progression.

Increased proteasome-dependent degradation of the cyclin-dependent kinase inhibitor p27 in aggressive colorectal carcinomas.

The cell-cycle inhibitor p27 is a potential tumor suppressor, but its gene has never been found inactivated in human tumors. Because cell-cycle regulation of p27 cellular abundance occurs at the post-transcriptional level, we analyzed p27 protein expression and degradation in human colorectal carcinomas. Proteasome-mediated degradation activity of p27 was compared with its protein levels in a subset of tumor samples. We found that carcinomas with low or absent p27 protein displayed enhanced proteolytic activity specific for p27, suggesting that low p27 expression can result from increased proteasome-mediated degradation rather than altered gene expression. Patients whose tumors expressed p27 had a median survival of 151 months, whereas patients who lacked p27 (10%) had a median survival of 69 months. By multivariate analysis, p27 was found to be an independent prognostic marker. Lack of p27 was associated with poor prognosis (2.9 risk ratio for death; P = 0.003). The absence of p27 protein expression is thus a powerful negative prognostic marker in colorectal carcinomas, particularly in stage II tumors, and thereby may help in the selection of patients who will benefit from adjuvant therapy. These data suggest that aggressive tumors may result from the selection of a clone or clones that lack p27 due to increased proteasome-mediated degradation.

Prognostic significance of AMP-activated protein kinase expression and modifying effect of MAPK3/1 in colorectal cancer.

BACKGROUND: AMP-activated protein kinase (AMPK, PRKA) has central roles in cellular metabolic sensing and energy balance homeostasis, and interacts with various pathways (e.g., TP53 (p53), FASN, MTOR and MAPK3/1 (ERK)). AMP-activated protein kinase activation is cytotoxic to cancer cells, supporting AMPK as a tumour suppressor and a potential therapeutic target. However, no study has examined its prognostic role in colorectal cancers. METHODS: Among 718 colon and rectal cancers, phosphorylated AMPK (p-AMPK) and p-MAPK3/1 expression was detected in 409 and 202 tumours, respectively, by immunohistochemistry. Cox proportional hazards model was used to compute mortality hazard ratio (HR), adjusting for clinical and tumoral features, including microsatellite instability, CpG island methylator phenotype, LINE-1 methylation, and KRAS, BRAF and PIK3CA mutations. RESULTS: Phosphorylated AMPK expression was not associated with survival among all patients. Notably, prognostic effect of p-AMPK significantly differed by p-MAPK3/1 status (P(interaction)=0.0017). Phosphorylated AMPK expression was associated with superior colorectal cancer-specific survival (adjusted HR 0.42; 95% confidence interval (CI), 0.24-0.74) among p-MAPK3/1-positive cases, but not among p-MAPK3/1-negative cases (adjusted HR 1.22; 95% CI: 0.85-1.75). CONCLUSION: Phosphorylated AMPK expression in colorectal cancer is associated with superior prognosis among p-MAPK3/1-positive cases, but not among p-MAPK3/1-negative cases, suggesting a possible interaction between the AMPK and MAPK pathways influencing tumour behaviour.

Improving the yield of circulating tumour cells facilitates molecular characterisation and recognition of discordant HER2 amplification in breast cancer.

BACKGROUND: Circulating tumour cells (CTCs) offer a non-invasive approach to obtain and characterise metastatic tumour cells, but their usefulness has been limited by low CTC yields from conventional isolation methods. METHODS: To improve CTC yields and facilitate their molecular characterisation we compared the Food and Drug Administration-approved CellSearch Epithelial Kit (CEK) to a simplified CTC capture method, CellSearch Profile Kit (CPK), on paired blood samples from patients with metastatic breast (n=75) and lung (n=71) cancer. Molecular markers including Human Epidermal growth factor Receptor 2 (HER2) were evaluated on CTCs by fluorescence in situ hybridisation (FISH) and compared to patients' primary and metastatic cancer. RESULTS: The median cell count from patients with breast cancer using the CPK was 117 vs 4 for CEK (P<0.0001). Lung cancer samples were similar; CPK: 145 cells vs CEK:4 cells (P<0.0001). Recovered CTCs were relatively pure (60-70%) and were evaluable by FISH and immunofluorescence. A total of 10 of 30 (33%) breast cancer patients with HER2-negative primary and metastatic tissue had HER2-amplified CTCs. CONCLUSION: The CPK method provides a high yield of relatively pure CTCs, facilitating their molecular characterisation. Circulating tumour cells obtained using CPK technology demonstrate that significant discordance exists between HER2 amplification of a patient's CTCs and that of the primary and metastatic tumour.

CDC25 phosphatases as potential human oncogenes.

Cyclin-dependent kinases (CDKs) are activated by CDC25 phosphatases, which remove inhibitory phosphate from tyrosine and threonine residues. In human cells, CDC25 proteins are encoded by a multigene family, consisting of CDC25A, CDC25B, and CDC25C. In rodent cells, human CDC25A or CDC25B but not CDC25C phosphatases cooperate with either Ha-RASG12V or loss of RB1 in oncogenic focus formation. Such transformants were highly aneuploid, grew in soft agar, and formed high-grade tumors in nude mice. Overexpression of CDC25B was detected in 32 percent of human primary breast cancers tested. The CDC25 phosphatases may contribute to the development of human cancer.

miR-29b expression is associated with disease-free survival in patients with ovarian serous carcinoma.

Micro-RNAs are a group of small noncoding RNAs approximately 22 nucleotides in length. Recent work has shown differential expression of mature micro-RNAs in human cancers. We characterized the alteration in expression of miR-29b in ovarian serous carcinoma. miR-29b expression was analyzed using quantitative stem-loop reverse transcriptase polymerase chain reaction on a set of 50 formalin-fixed, paraffin-embedded ovarian serous carcinoma samples. Protein expression of p53, estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2, Ki-67, and insulinlike growth factor 1 was quantified in the corresponding tissue microarray. The expression profile of miR-29b was correlated with clinicopathological and patient survival data. We provide definitive evidence that miR-29b is down-regulated in a significant proportion of ovarian serous carcinomas and is associated with specific clinicopathological features, most notably high miR-29b expression being associated with reduced disease-free survival.

BRAF(V600E) mutation and the biology of papillary thyroid cancer.

BRAF((V600E)) mutation is the most frequent genetic alteration in papillary thyroid carcinomas (PTCs) that are 80-90% of all thyroid cancers. We evaluated the relationship between BRAF((V600E)) and tumor, host, and environmental factors in PTCs from all geographical areas of Sicily. By PCR, BRAF((V600E)) was investigated in a series of 323 PTCs diagnosed in 2002-2005. The correlation between clinicopathological tumor, host, and environmental characteristics and the presence of BRAF((V600E)) were evaluated by both univariate and multivariate analyses. BRAF((V600E)) was found in 38.6% PTCs, with a 52% frequency in the classical PTCs and 26.4% in the tall cell variant. Univariate analysis indicated that BRAF((V600E)) was associated with greater tumor size (P=0.0048), extra-thyroid invasion (P<0.0001), and cervical lymph nodal metastases (P=0.0001). Multivariate logistic regression analysis confirmed that BRAF((V600E)) was an independent predictor of extra-thyroid invasion (P=0.0001) and cervical lymph nodal metastasis (P=0.0005). The association between BRAF((V600E)) and extra-thyroid invasion was also found in micro-PTCs (P=0.006). In 60 classical PTCs, BRAF((V600E)) was positively correlated with matrix metalloproteinase-9 expression (P=0.0047), suggesting a possible mechanism for BRAF((V600E)) effect on PTC invasiveness. No association was found between BRAF((V600E)) and patient age, gender, or iodine intake. In contrast, a strong association was found with residency in Eastern Sicily (P<0.0001 compared with Western Sicily). These results indicate that BRAF((V600E)) mutation is a marker of aggressive disease in both micro- and macro-PTCs. Moreover, for the first time, a possible link between BRAF((V600E)) mutation and environmental carcinogens is suggested.

Role of the Cdc25A phosphatase in human breast cancer.

The phosphatase Cdc25A plays an important role in cell cycle regulation by removing inhibitory phosphates from tyrosine and threonine residues of cyclin-dependent kinases, and it has been shown to transform diploid murine fibroblasts in cooperation with activated Ras. Here we show that Cdc25A is overexpressed in primary breast tumors and that such overexpression is correlated with higher levels of cyclin-dependent kinase 2 (Cdk2) enzymatic activity in vivo. Furthermore, in the breast cancer cell line MCF-7, Cdc25A activity is necessary for both the activation of Cdk2 and the subsequent induction of S-phase entry. Finally, in a series of small (< 1 cm) breast carcinomas, overexpression of Cdc25A was found in 47% of patients and was associated with poor survival. These data suggest that overexpression of Cdc25A contributes to the biological behavior of primary breast tumors and that both Cdc25A and Cdk2 are suitable therapeutic targets in early-stage breast cancer.

Forkhead transcription factors are critical effectors of cell death and cell cycle arrest downstream of PTEN.

PTEN acts as a tumor suppressor, at least in part, by antagonizing phosphoinositide 3-kinase (PI3K)/Akt signaling. Here we show that Forkhead transcription factors FKHRL1 and FKHR, substrates of the Akt kinase, are aberrantly localized to the cytoplasm and cannot activate transcription in PTEN-deficient cells. Restoration of PTEN function restores FKHR to the nucleus and restores transcriptional activation. Expression of a constitutively active form of FKHR that cannot be phosphorylated by Akt produces the same effect as reconstitution of PTEN on PTEN-deficient tumor cells. Specifically, activated FKHR induces apoptosis in cells that undergo PTEN-mediated cell death and induces G(1) arrest in cells that undergo PTEN-mediated cell cycle arrest. Furthermore, both PTEN and constitutively active FKHR induce p27(KIP1) protein but not p21. These data suggest that Forkhead transcription factors are critical effectors of PTEN-mediated tumor suppression.

Her-2-neu expression and progression toward androgen independence in human prostate cancer.

BACKGROUND: Human prostate cancers are initially androgen dependent but ultimately become androgen independent. Overexpression of the Her-2-neu receptor tyrosine kinase has been associated with the progression to androgen independence in prostate cancer cells. We examined the expression of Her-2-neu in normal and cancerous prostate tissues to assess its role in the progression to androgen independence. METHODS: Prostate cancer tissue sections were obtained from 67 patients treated by surgery alone (UNT tumors), 34 patients treated with total androgen ablation therapy before surgery (TAA tumors), and 18 patients in whom total androgen ablation therapy failed and who developed bone metastases (androgen-independent [AI] disease). The sections were immunostained for Her-2-neu, androgen receptor (AR), prostate-specific antigen (PSA), and Ki-67 (a marker of cell proliferation) protein expression. Messenger RNA (mRNA) levels and gene amplification of Her-2-neu were examined by RNA in situ hybridization and fluorescent in situ hybridization(FISH), respectively, in a subset of 27 tumors (nine UNT, 11 TAA, and seven AI). All statistical tests were two-sided. RESULTS: Her-2-neu protein expression was statistically significantly higher in TAA tumors than in UNT tumors with the use of two different scoring methods (P =.008 and P =.002). The proportion of Her-2-neu-positive tumors increased from the UNT group (17 of 67) to the TAA group (20 of 34) to the AI group (14 of 18) (P<.001). When compared with UNT tumors, tumor cell proliferation was higher in AI tumors (P =.014) and lower in TAA tumors (P<.001). All tumors expressed AR and PSA proteins. Although Her-2-neu mRNA expression was high in TAA and AI tumors, no Her-2-neu gene amplification was detected by FISH in any of the tumor types. CONCLUSIONS: Her-2-neu expression appears to increase with progression to androgen independence. Thus, therapeutic targeting of this tyrosine kinase in prostate cancer may be warranted.

Thrombopoietin receptor expression in human cancer cell lines and primary tissues.

c-mpl is the receptor for the recently identified megakaryocyte growth and differentiation factor thrombopoietin. Thrombopoietin has been shown to be capable of raising platelet counts in animals and is about to enter clinical trials in humans. In anticipation of its likely use in the care of patients receiving cancer chemotherapy, we evaluated the expression of human c-mpl by reverse transcription PCR on 39 human cell lines and 20 primary human tissue samples derived from both normal and malignant sources. c-mpl transcripts were found in all megakaryocytic cell lines tested (CMK, CMK-2B, CMK-2D, SO, and DAMI), the CD34+ leukemia cell line KMT-2, and a hepatocellular carcinoma cell line (Hep3B). Among primary tissues, fetal liver cells and brain had detectable levels of c-mpl message, and among primary tumors, none were found to express c-mpl. These data support the conclusion that c-mpl has restricted expression that is primarily, but not exclusively, related to megakaryocytopoiesis. These observations suggest that thrombopoietin is unlikely to have direct effects on other malignant or normal tissue should it have a clinical role in the treatment of chemotherapy-induced thrombocytopenia.

Identification of genes expressed differentially by LNCaP or PC-3 prostate cancer cell lines.

The transition from androgen-dependent to aggressive androgen-independent growth is an important development in prostate cancer, and the identification of genes expressed differentially between these two phenotypes could provide new therapeutic targets as well as prognostic markers. Using a subtractive approach designated linker capture subtraction, we have now isolated several genes expressed differentially between the human prostate carcinoma cell lines LNCaP and PC-3, which are androgen-dependent and androgen-independent, respectively. In addition to 10 known genes that may be involved in signal transduction, tumor growth, tumor invasion, and metastasis, 3 novel genes were isolated. One of these, designated NPG-1, was expressed by the majority of primary tumors examined. Moreover, the degree of NPG-1 expressed correlated with an aggressive histopathology.

Loss of PTEN expression in paraffin-embedded primary prostate cancer correlates with high Gleason score and advanced stage.

The tumor suppressor gene PTEN/MMAC-1/TEP-1 (referred to hereafter as PTEN) maps to chromosome 10q23 and encodes a dual specificity phosphatase. The PTEN protein negatively regulates cell migration and cell survival and induces a G1 cell cycle block via negative regulation of the phosphatidylinositol 3'-kinase/protein kinase B/Akt signaling pathway. PTEN is frequently mutated or deleted in both prostate cancer cell lines and primary prostate cancers. A murine polyclonal antiserum was raised against a glutathione S-transferase fusion polypeptide of the COOH terninus of PTEN. Archival paraffin tissue sections from 109 cases of resected prostate cancer were immunostained with the antiserum, using DU145 and PC-3 cells as positive and negative controls, respectively. PTEN expression was seen in the secretory cells. Cases were considered positive when granular cytoplasmic staining was seen in all tumor cells, mixed when areas of both positive and negative tumor cell clones were seen, and negative when adjacent benign prostate tissue but not tumor tissue showed positive staining. Seventeen cases (15.6%) of prostate cancer were positive, 70 cases (64.2%) were mixed, and 22 cases (20.2%) were negative. Total absence of PTEN expression correlated with the Gleason score (P = 0.0081) and correlated more significantly with a Gleason score of 7 or higher (P = 0.0004) and with advanced pathological stage (American Joint Committee on Cancer stages T3b and T4; P = 0.0078). Thus, loss of PTEN protein is correlated with pathological markers of poor prognosis in prostate cancer.

Prognostic role of the cyclin-dependent kinase inhibitor p27 in non-small cell lung cancer.

Despite its potential role as a tumor suppressor, p27 gene, a member of the Cip/Kip family of cyclin-dependent kinase inhibitor genes, has never been found mutated in human tumors. We investigated p27 protein expression in a series of 108 non-small cell lung cancers (57.4% stage 1, 16.7% stage 2, and 25.9% stage 3) to determine whether the lack or altered expression of this protein correlates with neoplastic transformation and/or progression. We performed immunohistochemistry and Western blot analysis of each specimen. We found that tumors expressing low to undetectable levels of p27 contained high p27 degradation activity. When we evaluated the outcome of the patients in relationship to p27 expression, we found p27 to be a prognostic factor correlating with the overall survival times (P = 0.0012). The possibility of a simple assay, such as the immunohistochemical analysis of p27 expression on routinely formalin-fixed, paraffin-embedded specimens, has considerable value for the prognosis of patients who undergo surgical resection. In addition, confirmation of the involvement of the proteasome-mediated proteolysis in p27 degradation should stimulate new strategies of nonsurgical treatments of non-small cell lung cancer.

Loss or altered subcellular localization of p27 in Barrett's associated adenocarcinoma.

The cyclin-dependent kinase inhibitor p27 is a negative regulator of the cell division cycle. It is expressed at the highest levels during the quiescent (G0) and prereplicative (G1) phases, and its degradation is required for entry into the S phase. Because lack of p27 is associated with aggressive behavior in a variety of tumors of epithelial and lymphoid origin, we used immunohistochemistry and in situ hybridization to evaluate the expression of p27 in metaplastic and dysplastic Barrett's epithelium and to assess its prognostic significance in Barrett's associated adenocarcinoma (BAA) of the esophagus. In metaplastic Barrett's epithelium, p27 protein and mRNA were restricted to the superficial third of glands in all cases and extended to the lower third in 4 cases. In contrast, expression of p27 message and protein was both increased and full-thickness, in the 23 cases with high-grade dysplasia adjacent to BAA and in carcinoma in situ. Although all invasive carcinomas had elevated levels of p27 mRNA, 45 (83%) of 54 invasive carcinomas had low p27 protein levels (<50% positive tumor cells). Low p27 protein correlated with higher histological grade (P < 0.0001), depth of invasion (P = 0.0120), presence of lymph node metastasis (P = 0.05), and survival (P = 0.0197). In addition to the nuclear staining, cytoplasmic staining of p27 was noted in 11 of 23 (48%) of cases of dysplasia and in 14 of 54 (26%) adenocarcinomas and confirmed, in a subset of cases, by subcellular fractionation of protein lysates obtained from fresh tumor tissues. Cytoplasmic localization of p27 was also associated with decreased survival (P = 0.0239). Loss of p27 conferred poor prognosis independently of proliferative index, as assessed by Ki-67 (MIB-1) immunostaining, which was not significantly different in survivors versus nonsurvivors. These results show that: (a) distribution of p27 message and protein parallel one another in metaplastic and dysplastic Barrett's epithelium, suggesting transcriptional regulation of the gene in the nonneoplastic setting; (b) p27 is inactivated in the majority of BAA as a result of either post-transcriptional modification or altered subcellular localization; and (c) loss of the cell cycle inhibitor p27 is associated with parameters of aggressive behavior and unfavorable outcome in BAA.

Methylation of the hMLH1 promoter correlates with lack of expression of hMLH1 in sporadic colon tumors and mismatch repair-defective human tumor cell lines.

Somatic mutations in DNA mismatch repair genes have been observed in sporadic tumors as well as cell lines and xenografts derived from such tumors implicating genetic defects of mismatch repair genes in the development of such tumors. However, the proportion of sporadic tumors in which mismatch repair genes have been inactivated has not been determined accurately. We have analyzed 66 sporadic colorectal tumors for the expression of hMLH1 by immunohistochemistry and identified 4 tumors that do not express hMLH1. These four colorectal tumors, a colon tumor cell line (SW48) and an endometrial tumor cell line (AN3CA), did not express hMLH1, despite the absence of mutations in its coding sequence. Cytosine methylation of the hMLH1 promoter region was found in these four colorectal tumors, whereas cytosine methylation of the hMLH1 promoter region was absent in adjacent normal tissue or in nine tumors that expressed hMLH1. In addition, cytosine methylation of the hMLH1 promoter region was observed in the SW48 and AN3CA cell lines that do not express hMLH1 but not in four tumor cell lines known to express hMLH1 mRNA. Our data indicate that DNA methylation is likely to be a common mode of mismatch repair gene inactivation in sporadic tumors.

The cell cycle inhibitor p27 is an independent prognostic marker in small (T1a,b) invasive breast carcinomas.

Breast carcinomas < or = 1 cm in size (T1a,b) are being detected more frequently as a result of screening. Because traditional prognostic parameters are either lacking (tumor size) or rare (nodal metastases), a marker(s) is needed to identify the subset of patients who could benefit from adjuvant therapy. A retrospective series of 202 patients with stage T1a,b invasive breast carcinomas was evaluated. The clinicopathological features (age, histological grade, extensive in situ carcinoma, hormone receptor status, and nodal metastasis) as well as microvessel density and the expression of c-erb-B2, p53, MIB-1/Ki-67, and cdc25B were assessed. In addition, expression of the cell cycle inhibitor p27 was evaluated. Nineteen patients (18% of patients who had axillary dissection) had locoregional lymph node metastases. Forty-two % of them died of disease (median survival, 112 months), whereas mortality was 11% in node-negative patients (median survival, 168 months; P = 0.0055). Patients with low p27 expression had a median survival of 139 months (17% mortality) versus 174 months (9% mortality) in the group with high p27 expression (P = 0.0233). Lack of p27 was associated with poor prognosis when node-positive patients were excluded (P = 0.0252). Nodal status and low p27 were found to be the only independent prognostic parameters by both univariate and multivariate analysis, with relative risks of dying of disease of 4.9 (P = 0.001) and 3.4 (P = 0.0306), respectively. Assessment of p27, which yields prognostic information in node-negative patients, could be useful to identify patients with small, invasive breast carcinomas who might benefit from adjuvant therapy.

HRad17, a human homologue of the Schizosaccharomyces pombe checkpoint gene rad17, is overexpressed in colon carcinoma.

Using the palindromic PCR-cDNA display method, we have cloned a novel gene overexpressed by human colon carcinoma relative to normal colon. Among normal tissues examined, only testis expresses it at a high level. Sequence analysis revealed its extensive homology with checkpoint genes rad17 of Schizosaccharomyces pombe and RAD24 of Saccharomyces cerevisiae. This novel gene designated as hRad17 is localized to chromosome 5q12,13.1, a region known to be deleted in a variety of human cancers. Promoter region and one pseudogene of hRad17 have been identified. Whereas the increased expression of hRad17 by human colon carcinomas may be related to the known resistance of these cells to DNA-damaging agents during therapy, the deletion of hRad17 in a variety of cancers may predispose them to increased rate of mutation and heightened sensitivity to DNA-damaging agents, including radiation and anticancer drugs.

Profiling the tumor microenvironment proteome in prostate cancer using laser capture microdissection coupled to LCâ¿¿MSâ¿¿A technical report.

Laser capture microdissection (LCM) allows microscopic procurement of specific cell types from tissue sections. Here, we present an optimized workflow for coupling LCM to LCâ¿¿MS/MS including: sectioning of tissue, a standard LCM workflow, protein digestion and advanced LCâ¿¿MS/MS. Soluble proteins extracted from benign epithelial cells, their associated stroma, tumor epithelial cells and their associated stromal cells from a single patient tissue sample were digested and profiled using advanced LCâ¿¿MS/MS. The correlation between technical replicates was R2 = 0.99 with a mean % CV of 9.55% ± 8.73. The correlation between sample replicates was R2 = 0.97 with a mean % CV of 13.83% ± 10.17. This represents a robust, systematic approach for profiling of the tumor microenvironment using LCM coupled to label-free LCâ¿¿MS/MS.