Reverse transcription-quantitative polymerase chain reaction: description of a RIN-based algorithm for accurate data normalization

<p>Abstract</p> <p>Background</p> <p>Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the gold standard technique for mRNA quantification, but appropriate normalization is required to obtain reliable data. Normalization to accurately quantitated RNA has been proposed as the most reliable method for in vivo biopsies. However, this approach does not correct differences in RNA integrity.</p> <p>Results</p> <p>In this study, we evaluated the effect of RNA degradation on the quantification of the relative expression of nine genes (<it>18S</it>, <it>ACTB</it>, <it>ATUB</it>, <it>B2M</it>, <it>GAPDH</it>, <it>HPRT</it>, <it>POLR2L</it>, <it>PSMB6</it> and <it>RPLP0</it>) that cover a wide expression spectrum. Our results show that RNA degradation could introduce up to 100% error in gene expression measurements when RT-qPCR data were normalized to total RNA. To achieve greater resolution of small differences in transcript levels in degraded samples, we improved this normalization method by developing a corrective algorithm that compensates for the loss of RNA integrity. This approach allowed us to achieve higher accuracy, since the average error for quantitative measurements was reduced to 8%. Finally, we applied our normalization strategy to the quantification of <it>EGFR</it>, <it>HER2 </it>and <it>HER3 </it>in 104 rectal cancer biopsies. Taken together, our data show that normalization of gene expression measurements by taking into account also RNA degradation allows much more reliable sample comparison.</p> <p>Conclusion</p> <p>We developed a new normalization method of RT-qPCR data that compensates for loss of RNA integrity and therefore allows accurate gene expression quantification in human biopsies.</p

Prognostic relevance of nuclear receptors in relation to peritumoral inflammation and tumor infiltration by lymphocytes in breast cancer

The prognostic impact of tumor-infiltrating lymphocytes (TILs) is intensively investigated in breast cancer (BC). It is already known that triple-negative breast cancer (TNBC), the most aggressive type of BC, has the highest percentage of TILs. In addition, there is an influence of steroid hormone receptor expression (type I nuclear receptors) on TIL subpopulations in breast cancer tissue. The link between type II nuclear receptors and the level of TILs is unclear. Therefore, the aim of this study was to quantify TILs in a panel of 264 sporadic breast cancers and investigate the correlation of TIL levels with type I and II nuclear receptors expression. TIL levels were significantly increased in the subgroup of TNBC. By contrast, they decreased in estrogen (ER)- or progesterone receptor (PR)-positive cases. Moreover, TIL levels were correlated with type II nuclear receptors, including PPARγ, with a significant inverse correlation of the nuclear form (r = −0.727, p 15% showed a significantly decreased overall survival. In addition, peritumoral inflammation was also quantified in BC tissue samples. In our cohort, although the level of peritumoral inflammation was not correlated with OS, it determined the prognostic value of ER, PR, and PPARγ in BC. Altogether, the present study provides a differentiated overview of the relations between nuclear receptor expression, TIL levels, peritumoral inflammation, and prognosis in BC

Correlation of the TIGIT-PVR immune checkpoint axis with clinicopathological features in triple-negative breast cancer

BackgroundT cell immunoreceptor with Ig and ITIM domains (TIGIT) interacts with poliovirus receptor (PVR) to contribute to cancer immune escape. Recently, TIGIT and PVR have been identified as promising immunotherapy targets. Their gene expression is upregulated in many solid tumors, but their protein expression level is not well documented, particularly in triple negative breast cancer (TNBC), the breast cancer subtype that most benefit from immunotherapy.MethodsTIGIT and PVR expression levels were assessed by immunohistochemistry in 243 surgically resected localized TNBC and then their relationship with clinical-pathological features and clinical outcome was analyzed.ResultsTIGIT expression was observed in immune cells from the tumor microenvironment, whereas PVR was mainly expressed by tumor cells. High TIGIT expression was significantly associated with age (p=0.010), histological grade (p=0.014), non-lobular histology (p=0.024), adjuvant chemotherapy (p=0.006), and various immune cell populations (tumor infiltrating lymphocytes (TILs), CD3+, CD8+, PD-1+ cells; all p&lt;0.0001), PD-L1+ tumor cells (p&lt;0.0001), and PD-L1+ stromal cells (p=0.003). Infiltration by TIGIT+ cells tended to be higher in non-molecular apocrine tumors (p=0.088). PVR was significantly associated with histological grade (p&lt;0.0001), the basal-like (p=0.003) and non-molecular apocrine phenotypes (p=0.039), high TILs infiltration (p=0.011), CD3+ (p=0.002), CD8+ (p=0.024) T cells, and PD-L1 expression in tumor (p=0.003) and stromal cells (p=0.001). In univariate analysis, only known prognostic factors (age, tumor size, lymph node status, adjuvant chemotherapy, TILs and CD3+ T-cell infiltrate) were significantly associated with relapse-free survival (RFS) and overall survival. High TIGIT and PVR expression levels tended to be associated with longer RFS (p=0.079 and 0.045, respectively). The analysis that included only non-molecular apocrine TNBC revealed longer RFS for tumors that strongly expressed TIGIT or PVR (p=0.025 for TIGIT and 0.032 for PVR).ConclusionsThese results indicated that in TNBC, TIGIT+ cells can easily interact with PVR to exert their inhibitory effects. Their wide expression in TNBC and their association with other immune checkpoint components suggest the therapeutic interest of the TIGIT-PVR axis

Assessment of epidermal growth factor receptor (EGFR) expression in primary colorectal carcinomas and their related metastases on tissue sections and tissue microarray

Metastatic colorectal carcinomas (CRC) resistant to chemotherapy may benefit from targeting monoclonal therapy cetuximab when they express the epidermal growth factor receptor (EGFR). Because of its clinical implications, we studied EGFR expression by immunohistochemistry on tissue sections of primary CRC (n=32) and their related metastases (n=53). A tissue microarray (TMA) was generated from the same paraffin blocks to determine whether this technique could be used for EGFR screening in CRC. On tissue sections, 84% of the primary CRC and 94% of the metastases were EGFR-positive. When matched, they showed a concordant EGFR-positive status in 78% of the cases. Moreover, staining intensity and extent of EGFR-positive cells in the primary CRC correlated with those observed in the synchronous metastases. On TMA, 65% of the primary CRC, 66% of the metastases, and 43% of the matched primary CRC metastases were EGFR-positive. There was no concordant EGFR status between the primary and the metastatic sites. A strong discrepancy of EGFR status was noted between TMA and tissue sections. In conclusion, EGFR expression measured in tissue sections from primary CRC and their related metastases was found to be similar and frequent, but it was significantly underestimated by the TMA technique

CXCR2 Levels Correlate with Immune Infiltration and a Better Prognosis of Triple-Negative Breast Cancers

International audienceChemokines and their receptors are key players in breast cancer progression and outcome. Previous studies have shown that the chemokine receptor CXCR2 was expressed at higher levels by cells of the tumor microenvironment in triple-negative breast cancers (TNBCs). The aim of this study was to focus our attention on a retrospective cohort of 290 TNBC cases and analyze the involvement of CXCR2, CD11b (a marker of granulocytes) and CD66b (a marker of neutrophils) and their link with immune infiltration and immune checkpoint markers. We report that high densities of CXCR2-, CD11b- and CD66b-positive cells were associated with high-grade tumors. Moreover, molecular apocrine TNBCs, defined here as tumors that express both AR and FOXA1 biomarkers, exhibited low levels of CXCR2 and CD11b. High CXCR2 and CD11b levels were correlated with elevated density of tumor-infiltrating lymphocytes (TILs), CD8+ cytotoxic lymphocytes, expression of PD-L1 by tumor and stromal cells and of PD-1 by stromal cells. On the other hand, CD66b levels were associated only with CD8+, stromal PD-L1 and PD-1 expression. In univariate analysis, low levels of CXCR2 were correlated with poor OS and RFS. In multivariate analysis, low levels of CXCR2 were associated with poor OS. Finally, in TNBC treated with adjuvant chemotherapy, CXCR2 density was associated with longer RFS. Overall, our data highlight the potential beneficial association of high levels of CXCR2 with a subgroup of TNBC patients characterized by a better prognosis

In situ hybridization for the assessment of urokinase plasminogen activator and plasminogen activator inhibitor type‑1 in formalin‑fixed paraffin‑embedded breast cancer specimens

International audienceUrokinase plasminogen activator (uPA) and its inhibitor, plasminogen activator inhibitor type 1 (PAI‑1), have been reported as prognostic and predictive biomarkers in breast cancer, particularly in patients with node‑negative tumors. uPA and PAI‑1 expression levels classify patients into a poor‑prognosis subgroup, requiring adjuvant chemotherapy and a favorable‑prognosis subgroup, which can be considered for de‑escalation. However, the clinical use of these two biomarkers remains limited, since fresh‑frozen/fresh tumor samples are currently required for their quantification. The aim of the present study was to compare PLAU and SERPINE1 mRNA expression levels (corresponding to uPA and PAI‑1 proteins, respectively), assessed using in situ hybridization in 83 formalin‑fixed paraffin‑embedded (FFPE) breast tumor samples, with uPA and PAI‑1 protein expression assessed using immunometric assay with paired fresh‑frozen breast cancer samples. The results from the two methods significantly correlated as regards uPA quantification; however, >30% of the samples were discordant, according to the clinically validated threshold. Concordance between the two analytical methods was less prominent for PAI‑1 protein and SERPINE1 mRNA. Taken together, the results of the present study indicate that although PLAU and SERPINE1 mRNA may be reliably detected in FFPE samples using in situ hybridization, this technology cannot be used as a substitute for the replacement of the immunometric assay‑derived quantification on fresh‑frozen samples

Prognostic Value of CXCR2 in Breast Cancer

International audienceThe tumor microenvironment appears essential in cancer progression and chemokines are mediators of the communication between cancer cells and stromal cells. We have previously shown that the ligands of the chemokine receptor CXCR2 were expressed at higher levels in triple-negative breast cancers (TNBC). Our hypothesis was that CXCR2 expression could also be altered in breast cancer. Here, we have analyzed the potential role of CXCR2 in breast cancer in a retrospective cohort of 105 breast cancer patients. Expression of CXCR2, CD11b (a marker of granulocytes) and CD66b (a marker of neutrophils) was analyzed by immunohistochemistry on tumor samples. We demonstrated that CXCR2 stained mainly stromal cells and in particular neutrophils. CXCR2, CD11b and CD66b expression were correlated with high grade breast cancers. Moreover, TNBC displayed a higher expression of CXCR2, CD11b and CD66b than hormone receptor positive or Her2 positive tumors. High levels of CXCR2 and CD11b, but not CD66b, were associated with a higher infiltration of T lymphocytes and B lymphocytes. We also observed a correlation between CXCR2 and AP-1 activity. In univariate analyses, CXCR2, but not CD11b or CD66b, was associated with a lower risk of relapse; CXCR2 remained significant in multivariate analysis. Our data suggest that CXCR2 is a stromal marker of TNBC. However, higher levels of CXCR2 predicted a lower risk of relapse

Immunohistochemical staining for p16 and BRAFV600E is useful to distinguish between sporadic and hereditary (Lynch syndrome-related) microsatellite instable colorectal carcinomas

International audienceDNA mismatch repair (MMR) protein analysis by immunohistochemistry (IHC) can identify colorectal cancer (CRC) with microsatellite instability (MSI). As MLH1-deficient CRC can be hereditary or sporadic, markers to distinguish between them are needed. MLH1 promoter methylation assay is the reference method; however, sometimes, it is challenging on formalin-fixed paraffin-embedded tissue samples. We assessed by IHC the expression of BRAFV600E, p16, MGMT, and CDX2 in 55 MLH1-deficient MSI CRC samples (of which 8 had a germline MLH1 mutation) to determine whether this panel differentiates between sporadic and hereditary CRCs. We also analyzed MLH1 promoter methylation by methylation-specific PCR and pyrosequencing and BRAF status by genotyping. None of the hereditary CRCs showed MLH1 methylation, BRAF mutation, BRAFV600E-positive immunostaining, or loss of p16 expression. We detected MLH1 promoter methylation in 67 % and a BRAF mutation in 42 % of CRC, all showing MLH1 promoter methylation. BRAFV600E IHC and BRAF genotyping gave concordant results in all but two samples. Loss of expression of p16 was found in 30 % of CRC with methylation of the MLH1 promoter, but its expression was retained in all non-methylated and part of MLH1-methylated tumors (100 % specificity, 30 % sensitivity). CDX2 and MGMT expression was not associated with MLH1 status. Thus, BRAFV600E and p16 IHC may help in differentiating sporadic from hereditary MLH1-deficient CRC with MSI. Specifically, p16 IHC might be used as a surrogate marker for MLH1 promoter methylation, because all p16-negative CRCs displayed MLH1 methylation, whereas hereditary CRCs were all p16-positive

Prognostic Value of HER2-Low Expression in Non-Metastatic Triple-Negative Breast Cancer and Correlation with Other Biomarkers

International audienceHER2-low breast cancer (i.e., HER 1+ or 2+, without gene amplification) is an emerging subtype for which very few data are available, especially within the triple-negative breast cancer (TNBC) group. Our aim was to evaluate HER2 expression and its prognostic value in a large retrospective series of patients with non-metastatic TNBC (median age: 57.7 years; range: 28.5–98.6). Among the 296 TNBC samples, 83.8% were HER2 0, 13.5% were HER2 1+, and 2.7% were HER2 2+ (HercepTestTM and 2018 ASCO/CAP guidelines for HER2 scoring). CK5/6 and/or EGFR-expressing androgen receptors and FOXA1-expressing tumors were classified as basal-like (63.8%) and molecular apocrine-like (MA, 40.2%), respectively. Compared with HER2 0 tumors, HER2 1+/2+ tumors exhibited a lower histological grade (1/2) (35.4% vs. 18.2%, p = 0.007) and MA profile (57.5% vs. 36.7%, p = 0.008). Moreover, patients with HER2 1+/2+ tumors were older (p = 0.047). After a median follow-up of 9.7 years, HER2 2+ tumors (compared with HER2 0/1+ tumors) were associated with worse relapse-free survival (RFS) (HR = 3.16, 95% CI [1.27; 7.85], p = 0.034) in a univariate analysis. Overall survival (OS) and RFS were not different in the HER2 0 and 1+/2+ groups. HER2 levels were not significantly associated with OS or RFS in a multivariate analysis

High PTPN13 expression in high grade serous ovarian carcinoma is associated with a better patient outcome

International audienceBackground:Chromosome 4q loss of heterozygosity (LOH) is frequently observed in high-grade serous ovarian carcinoma (HGSOC). However, this LOH has not been clearly associated with the inactivation of any tumor suppressor gene(s). As the tumor suppressor gene PTPN13 is located on chromosome 4q21, we investigated its expression in HGSOC.Methods:PTPN13 protein expression was investigated by immunohistochemistry (IHC) in normal ovary epithelium and in 30 HGSOC samples, whereas PTPN13 mRNA expression was quantified by RT-PCR in another independent cohort of 28 HGSOC samples. Patients in both cohorts were followed for more than 8.5 years.Results:PTPN13 protein expression was lower in one third of HGSOC samples compared with normal ovary epithelium. In both cohorts, high PTPN13 expression level (mRNA or protein) in the tumor was associated with favorable outcome and significantly longer survival (HR=0.27; p=0.0087 and HR=0.42; p=0.03, respectively).Conclusion:This study demonstrates, for the first time, that high PTPN13 expression level is a prognostic indicator of favorable outcome in patients with HGSOC. This finding, in conjunction with our previous mechanistic studies, suggests that PTPN13 loss, possibly by 4q LOH, enhances HGSOC aggressiveness and highlight the interest of studying PTPN13 signaling in HGSOC to identify new potential therapeutic targets