Maud Milder

Human MAIT cells are xenobiotic-resistant, tissue-targeted, CD161hi IL-17-secreting T cells.

Mucosal-associated invariant T (MAIT) cells are very abundant in humans and have antimicrobial specificity, but their functions remain unclear. MAIT cells are CD161(hi)IL-18Rα(+) and either CD4(-)CD8(-) (DN) or CD8αβ(int) T cells. We now show that they display an effector-memory phenotype (CD45RA(-)CD45RO(+)CD95(hi)CD62L(lo)), and their chemokine receptor expression pattern (CCR9(int)CCR7(-)CCR5(hi)CXCR6(hi)CCR6(hi)) indicates preferential homing to tissues and particularly the intestine and the liver. MAIT cells can represent up to 45% of the liver lymphocytes. They produce interferon-γ and Granzyme-B as well as high levels of interleukin-17 after phorbol myristate acetate + ionomycin stimulation. Most MAIT cells are noncycling cells (< 1% are Ki-67(+)) and express the multidrug resistance transporter (ABCB1). As expected from this phenotype, MAIT cells are more resistant to chemotherapy than other T-cell populations. These features might also allow MAIT cells to resist the xenobiotics potentially secreted by the gut bacteria. We also show that this population does not appear to have antiviral specificity and that CD8 MAIT cells include almost all the ABCB1(+)CD161(hi) CD8 T cells. Together with their already known abundance and antimicrobial specificity, the gut-liver homing characteristics, high expression of ABCB1, and ability to secrete interleukin-17 probably participate in the antibacterial properties of MAIT cells.

Circulating tumor DNA and circulating tumor cells in metastatic triple negative breast cancer patients.

Circulating tumor DNA (ctDNA) is a new circulating tumor biomarker which might be used as a prognostic biomarker in a way similar to circulating tumor cells (CTCs). Here, we used the high prevalence of TP53 mutations in triple negative breast cancer (TNBC) to compare ctDNA and CTC detection rates and prognostic value in metastatic TNBC patients. Forty patients were enrolled before starting a new line of treatment. TP53 mutations were characterized in archived tumor tissues and in plasma DNA using two next generation sequencing (NGS) platforms in parallel. Archived tumor tissue was sequenced successfully for 31/40 patients. TP53 mutations were found in 26/31 (84%) of tumor samples. The same mutation was detected in the matched plasma of 21/26 (81%) patients with an additional mutation found only in the plasma for one patient. Mutated allele fractions ranged from 2 to 70% (median 5%). The observed correlation between the two NGS approaches (R2 = 0.903) suggested that ctDNA levels data were quantitative. Among the 27 patients with TP53 mutations, CTC count was ≥1 in 19 patients (70%) and ≥5 in 14 patients (52%). ctDNA levels had no prognostic impact on time to progression (TTP) or overall survival (OS), whereas CTC numbers were correlated with OS (p = 0.04) and marginally with TTP (p = 0.06). Performance status and elevated LDH also had significant prognostic impact. Here, absence of prognostic impact of baseline ctDNA level suggests that mechanisms of ctDNA release in metastatic TNBC may involve, beyond tumor burden, biological features that do not dramatically affect patient outcome.

Detection rate and prognostic value of circulating tumor cells and circulating tumor DNA in metastatic uveal melanoma

Circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) have been recently investigated in several cancer types, but their respective clinical significance remains to be determined. In our prospective study, we compared the detection rate and the prognostic value of these two circulating biomarkers in patients with metastatic uveal melanoma. GNAQ/GNA11 mutations were characterized in archived tumor tissue. Using a highly sensitive and mutation‐specific bidirectional pyrophosphorolysis‐activated polymerization (bi‐PAP) technique, GNAQ c.626A>T, GNAQ c.626A>C and GNA11 c.626A>T copy numbers were quantified in plasma from 12 mL of blood. CTCs were detected at the same time in 7.5 mL of blood by the CellSearch® technique. Patient characteristics and outcome were prospectively collected. CTCs (≥1) were detected in 12 of the 40 included patients (30%, range 1–20). Among the 26 patients with known detectable mutations, ctDNA was detected and quantified in 22 (84%, range 4–11,421 copies/mL). CTC count and ctDNA levels were associated with the presence of miliary hepatic metastasis (p = 0.004 and 0.03, respectively), with metastasis volume (p = 0.005 and 0.004) and with each other (p < 0.0001). CTC count and ctDNA levels were both strongly associated with progression‐free survival (p = 0.003 and 0.001) and overall survival (p = 0.0009 and <0.0001). In multivariate analyses, ctDNA appeared to be a better prognostic marker than CTC. In conclusion, ctDNA and CTC are correlated and both have poor prognostic significance. CTC detection can be performed in every patient but, in patients with detectable mutations, ctDNA was more frequently detected than CTC and has possibly more prognostic value.

Pyrophosphorolysis-activated polymerization detects circulating tumor DNA in metastatic uveal melanoma.

Purpose: To develop a molecular tool to detect circulating tumor–derived DNA (ctDNA) in the plasma from patients with uveal melanoma as a marker of tumor burden and monitor treatment efficacy. Experimental Design: A real-time PCR was developed on the basis of bidirectional pyrophosphorolysis-activated polymerization (bi-PAP) for the quantification of ctDNA using 3′blocked primer pairs specific for the 3 recurrent mutually exclusive mutations of Gα subunits GNAQ and GNA11. Results: Sensitivity and specificity of bi-PAP were assessed on serial dilutions of tumor DNA in normal DNA for the 3 recurrent mutations. Each assay could detect a single mutated molecule per reaction, whereas 104 copies of normal DNA were not detected. The ctDNA was readily detected in plasma of mice bearing uveal melanoma xenografts in amounts proportional to circulating human DNA. Finally, plasma was almost always found positive (20 of 21 tested patients) in a prospective analysis of patients with metastatic uveal melanoma. Conclusions: Bi-PAP assays detect and quantify ctDNA in patients with metastatic uveal melanoma. A prospective study is ongoing to assess the clinical usefulness of ctDNA level in uveal melanoma. Clin Cancer Res; 18(14); 3934–41. ©2012 AACR.

Patient-Specific Circulating Tumor DNA Detection during Neoadjuvant Chemotherapy in Triple-Negative Breast Cancer.

BACKGROUND In nonmetastatic triple-negative breast cancer (TNBC) patients, we investigated whether circulating tumor DNA (ctDNA) detection can reflect the tumor response to neoadjuvant chemotherapy (NCT) and detect minimal residual disease after surgery. METHODS Ten milliliters of plasma were collected at 4 time points: before NCT; after 1 cycle; before surgery; after surgery. Customized droplet digital PCR (ddPCR) assays were used to track tumor protein p53 (TP53) mutations previously characterized in tumor tissue by massively parallel sequencing (MPS). RESULTS Forty-six patients with nonmetastatic TNBC were enrolled. TP53 mutations were identified in 40 of them. Customized ddPCR probes were validated for 38 patients, with excellent correlation with MPS (r = 0.99), specificity (≥2 droplets/assay), and sensitivity (at least 0.1%). At baseline, ctDNA was detected in 27/36 patients (75%). Its detection was associated with mitotic index (P = 0.003), tumor grade (P = 0.003), and stage (P = 0.03). During treatment, we observed a drop of ctDNA levels in all patients but 1. No patient had detectable ctDNA after surgery. The patient with rising ctDNA levels experienced tumor progression during NCT. Pathological complete response (16/38 patients) was not correlated with ctDNA detection at any time point. ctDNA positivity after 1 cycle of NCT was correlated with shorter disease-free (P < 0.001) and overall (P = 0.006) survival. CONCLUSIONS Customized ctDNA detection by ddPCR achieved a 75% detection rate at baseline. During NCT, ctDNA levels decreased quickly and minimal residual disease was not detected after surgery. However, a slow decrease of ctDNA level during NCT was strongly associated with shorter survival.

High Numbers of Differentiated Effector CD4 T Cells Are Found in Patients with Cancer and Correlate with Clinical Response after Neoadjuvant Therapy of Breast Cancer

CD4(+) T cells influence tumor immunity in complex ways that are not fully understood. In this study, we characterized a population of human differentiated effector CD4(+) T cells that is defined by low levels of the interleukin (IL)-2 and IL-7 receptors (CD25(-)CD127(-)). We found that this cell population expands in patients with various types of cancer, including breast cancer, to represent 2% to 20% of total CD4(+) blood T lymphocytes as compared with only 0.2% to 2% in healthy individuals. Notably, these CD25(-)CD127(-)CD4 T cells expressed effector markers such as CD244 and CD11b with low levels of CD27, contrasting with the memory phenotype dominating this population in healthy individuals. These cells did not cycle in patients, nor did they secrete IL-10 or IL-17, but instead displayed cytotoxic features. Moreover, they encompassed oligoclonal expansions paralleling an expansion of effector CD8(+) T cells that included tumor antigen-specific T cells. During neoadjuvant chemotherapy in patients with breast cancer, we found that the increase in CD25(-)CD127(-) CD4(+) T cells correlated with tumor regression. This observation suggested that CD4(+) T cells included tumor antigen-specific cells, which may be generated by or participate in tumor regressions during chemotherapy. In summary, our results lend support to the hypothesis that CD4(+) T cells are involved in human antitumor responses.

A GWAS in uveal melanoma identifies risk polymorphisms in the CLPTM1L locus

Uveal melanoma, a rare malignant tumor of the eye, is predominantly observed in populations of European ancestry. A genome-wide association study of 259 uveal melanoma patients compared to 401 controls all of European ancestry revealed a candidate locus at chromosome 5p15.33 (region rs421284: OR = 1.7, CI 1.43–2.05). This locus was replicated in an independent set of 276 cases and 184 controls. In addition, risk variants from this region were positively associated with higher expression of CLPTM1L. In conclusion, the CLPTM1L region contains risk alleles for uveal melanoma susceptibility, suggesting that CLPTM1L could play a role in uveal melanoma oncogenesis.Cancer: Risk allele identified for melanoma of the eyeResearchers have discovered an important genetic risk variant linked to uveal melanoma, a rare malignant tumor of the eye. Marc-Henri Stern from Institut Curie in Paris, France, and colleagues compared more than 860,000 single DNA variants covering the entire genome, from the genomes of 259 people with uveal melanoma and 401 healthy controls, all of whom were of European ancestry. The researchers found that a series of closely linked gene variants on the short arm of chromosome 5 were significantly more common in the melanoma patients. They confirmed the association between this genomic region and disease risk in an independent cohort of 276 cancer cases and 184 controls. Expression analyses showed that the CLPTM1L gene contained in this region was more expressed in people with the risk variants, pointing to CLPTM1L playing a role in tumor development.

Multiple Hotspot Mutations Scanning by Single Droplet Digital PCR

BACKGROUND Progress in the liquid biopsy field, combined with the development of droplet digital PCR (ddPCR), has enabled noninvasive monitoring of mutations with high detection accuracy. However, current assays detect a restricted number of mutations per reaction. ddPCR is a recognized method for detecting alterations previously characterized in tumor tissues, but its use as a discovery tool when the mutation is unknown a priori remains limited. METHODS We established 2 ddPCR assays detecting all genomic alterations within KRAS exon 2 and EGFR exon 19 mutation hotspots, which are of clinical importance in colorectal and lung cancer, with use of a unique pair of TaqMan® oligoprobes. The KRAS assay scanned for the 7 most common mutations in codons 12/13 but also all other mutations found in that region. The EGFR assay screened for all in-frame deletions of exon 19, which are frequent EGFR-activating events. RESULTS The KRAS and EGFR assays were highly specific and both reached a limit of detection of <0.1% in mutant allele frequency. We further validated their performance on multiple plasma and formalin-fixed and paraffin-embedded tumor samples harboring a panel of different KRAS or EGFR mutations. CONCLUSIONS This method presents the advantage of detecting a higher number of mutations with single-reaction ddPCRs while consuming a minimum of patient sample. This is particularly useful in the context of liquid biopsy because the amount of circulating tumor DNA is often low. This method should be useful as a discovery tool when the tumor tissue is unavailable or to monitor disease during therapy.

Lymph node invasion by tumor cells modifies the distribution of dendritic cell subsets and memory T cell profiles in human cancer patients

In human breast cancer, the invasion of tumor-draining lymph nodes (TDLNs) is an important step in disease progression and has predictive value [1]. TDLN dendritic cells (DCs), which are comprised of lymphoid-organ-resident and skin-derived migratory DCs, present tumor antigens to the naive T cells and induce their activation and polarization into different functional subsets (Th1, Th2, Th17, Th22, Tfh, regulatory T cells) that will lead to antitumor T cell responses or to tolerance [2].

Circulating tumor DNA (ctDNA) in metastatic uveal melanoma (MUM): Correlation with outcome in 87 patients (pts) from Institut Curie

Purpose CtDNA can be detected in the plasma of MUM pts using a real-time PCR based on the pyrophosphorolysis-activated polymerization (bi-PAP) targeting the most frequent GNAQ and GNA11 mutations present in 85% of UM (Madic et al, 2012). Methods May 2011-March 2013:87 MUM pts were included in 3 prospective studies to assess the bi-PAP assay and analyse clinical and outcome correlations. Results Median (med) age 57, primary tumor med diameter 15 mm; enucleation 32, proton beam 52, iodine disk 3. Enucleated eyes showed mostly mixed histotype and genomic high risk by array-CGH: 8q gain and/or 3p loss. With a med disease free interval of 39 months (mo), 83 pts developed liver metastases first, with radiological miliary disease in 55; 4 had extra hepatic lesions without liver involvement. 35 pts were enrolled at the time of metastasis diagnostic. With a med follow-up of 8 mo, 28 patients had disease progression and 34 died of metastasis. The med survival was 13 mo. Tumor samples were available in 82 pts, genotyping is ongoing for 8. GNAQ 626A>T or A>C, GNA11 626A>T and rare mutations were found in 9, 19, 26 is and 6 cases respectively; 14 were wild-type tumors. CtDNA was detected in 51/54 samples (med 30 copies/ml, range 1-11421) and correlated with the metastatic tumor burden as assessed by liver MRI (med 64 cm3, range 0.2-7384). Correlation with progression-free and overall survival will be presented. Conclusion ctDNA is a promising tool to assess the tumor burden and the micrometastatic dissemination of uveal melanoma, and a potential biomarker to evaluate the efficacy of targeted therapies in pts carrying GNA Q/11 mutated tumors.