Pilot Feasibility Study: 18 F-DPA-714 PET/CT Macrophage Imaging in Triple-Negative Breast Cancers (EITHICS).

ABSTRACT: Tumor-associated macrophages are targets of interest in triple-negative breast cancer (TNBC). The translocator protein 18 kDa (TSPO) is a sensitive marker for macrophages and holds potential relevance in TNBC stratification. This pilot prospective study (EITHICS, NCT04320030) aimed to assess the potential of TSPO PET/CT imaging using 18 F-DPA-714 in primary TNBC, compared with immunohistochemistry, autoradiography, and TSPO polymorphism. PATIENTS AND METHODS: Thirteen TNBC patients were included. They underwent TSPO genotyping (HAB, MAB, LAB), 18 F-FDG PET/CT, and breast MRI. Semiquantitative PET parameters were computed. VOIs were defined on the tumor lesion, healthy breast tissue, and pectoral muscle to obtain SUV, tumor-to-background ratio (TBR), and time-activity curves (TACs). Additionally, immunohistochemistry, 3 H-DPA-714, and 3 H-PK-11195 autoradiography were conducted. RESULTS: The majority of TNBC tumors (11/13, 84%) had a preponderance of M2-polarized macrophages with a median proportion of 82% (range, 44%-94%). 18 F-DPA-714 PET/CT clearly identified TNBC tumors with an excellent TBR. Three distinct patterns of 18 F-DPA-714 TACs were identified, categorized as "above muscular," "equal to muscular," and "below muscular" with reference to the muscular background. For the "above muscular" group (2 HAB and 2 MAB), "equal muscular" group (3 HAB, 3 MAB, and 1 LAB), and "below muscular" group (1 LAB and 1 MAB), tumor TACs showed a 18 F-DPA-714 accumulation slope of 1.35, 0.62, and 0.22, respectively, and a median SUV mean of 4.02 (2.09-5.31), 1.66 (0.93-3.07), and 0.61 (0.43-1.02). CONCLUSIONS: This study successfully demonstrated TNBC tumor targeting by 18 F-DPA-714 with an excellent TBR, allowing to stratify 3 patterns of uptake potentially influenced by the TSPO polymorphism status. Further studies in larger populations should be performed to evaluate the prognostic value of this new biomarker.

A de novo pathogenic variant in the MSH6 gene in a 52 years-old woman.

Lynch syndrome (LS) is a condition which predisposes individuals primarily to early-onset colorectal and endometrial cancer. LS is characterized by a germline pathogenic variant in one of the MMR (MisMatch Repair) gene, inducing a phenotype of microsatellite instability in the tumor, which may be associated with a loss of expression of MMR proteins detected by standard immunohistochemistry on tumor tissue. Most of the time, LS is inherited from a parent in whom the condition may not be known due to incomplete penetrance, but de novo pathogenic variant is a rare occurrence. Here, we describe the case of a 52-year-old woman with no family history of LS, referred to the genetics department for colorectal cancer at the age of 50. Genetic analysis revealed a de novo germline pathogenic variant in the MSH6 gene. To date, this case is only the second report of a de novo pathogenic variant in the MSH6 gene in Lynch syndrome. De novo mutations have been extensively studied over the past years, but little is known about their origin and mechanism of occurrence in MMR genes. However, knowledge of mutation status allows better cancer risk management for the patient and an appropriate genetic testing and counseling for her family.

Retrorectal Mucinous Adenocarcinoma Arising from a Tailgut Cyst: A Case Report.

We report the case of a 57-year-old woman who presented with local invasion of the anal canal by mucinous adenocarcinoma, the malignant transformation of a long-term preexisting retrorectal tailgut cyst. This progression is infrequent and justifies preemptive surgical treatment of retrorectal cysts.

Development of a Prognostic Tool to Guide the Decision to Extend Adjuvant Aromatase Inhibitors for up to Ten Years in Postmenopausal Early Breast Cancer Patients.

Background: The selection of women with hormone receptor-positive (HR+) early breast cancer (EBC) at high risk of relapse after five years (yrs.) of adjuvant aromatase inhibitors (AIs) is crucial, as the benefit of extending AIs is counterbalanced by toxicity. We developed a clinicopathological tool to estimate the residual risk of relapse after five years of adjuvant AIs. Methods: The Institut de Cancérologie de l'Ouest (ICO) database was used to determine a prognostic score of post-five-year AI relapse. Cox regression models estimated our score's prognostic performance. Results: In total, 1105 women were included. Median follow-up was 44 months (IQR = 21-70) post-AI treatment. From the Cox models, we designed a dichotomous prognostic score including the number of macrometastases, age (>70 yrs. vs. ≤70 yrs.), tumor size (≥T2 vs. not), and mitotic activity (≥2 vs. not). Overall, 77.5% of patients were classified as being at low risk and 22.5% at high risk of late recurrence. Low-risk patients had a five- to ten-year local or distant recurrence risk of 7.6% (95% CI, 5.4% to 10.6%) as compared with 26.9% (95% CI, 19.9% to 35.7%) for the high-risk roup. Conclusion: In this study, we developed a simple tool to identify women at high risk of relapse despite completing five years of AIs.

STING-dependent paracriny shapes apoptotic priming of breast tumors in response to anti-mitotic treatment.

A fascinating but uncharacterized action of antimitotic chemotherapy is to collectively prime cancer cells to apoptotic mitochondrial outer membrane permeabilization (MOMP), while impacting only on cycling cell subsets. Here, we show that a proapoptotic secretory phenotype is induced by activation of cGAS/STING in cancer cells that are hit by antimitotic treatment, accumulate micronuclei and maintain mitochondrial integrity despite intrinsic apoptotic pressure. Organotypic cultures of primary human breast tumors and patient-derived xenografts sensitive to paclitaxel exhibit gene expression signatures typical of type I IFN and TNFα exposure. These cytokines induced by cGAS/STING activation trigger NOXA expression in neighboring cells and render them acutely sensitive to BCL-xL inhibition. cGAS/STING-dependent apoptotic effects are required for paclitaxel response in vivo, and they are amplified by sequential, but not synchronous, administration of BH3 mimetics. Thus anti-mitotic agents propagate apoptotic priming across heterogeneously sensitive cancer cells through cytosolic DNA sensing pathway-dependent extracellular signals, exploitable by delayed MOMP targeting.

The inhibitory receptor CD94/NKG2A on CD8+ tumor-infiltrating lymphocytes in colorectal cancer: a promising new druggable immune checkpoint in the context of HLAE/ß2m overexpression.

We previously demonstrated that HLA-E/ß2m overexpression by tumor cells in colorectal cancers is associated with an unfavorable prognosis. However, the expression of its specific receptor CD94/NKG2 by intraepithelial tumor-infiltrating lymphocytes, their exact phenotype and function, as well as the relation with the molecular status of colorectal cancer and prognosis remain unknown. Based on a retrospective cohort of 234 colorectal cancer patients, we assessed the expression of HLA-E, ß2m, CD94, CD8, and NKp46 by immunohistochemistry on tissue microarray. The expression profile of HLA-E/ß2m on tumor cells and the density of tumor-infiltrating lymphocytes were correlated to the clinicopathological and molecular features (Microsatellite status, BRAF and RAS mutations). Then, from the primary tumors of 27 prospective colorectal cancers, we characterized by multiparameter flow cytometry the nature (T and/or NK cells) and the co-expression of the inhibitory NKG2A or activating NKG2C chain of ex vivo isolated CD94+ tumor-infiltrating lymphocytes. Their biological function was determined using an in vitro redirected cytolytic activity assay. Our results showed that HLA-E/ß2m was preferentially overexpressed in microsatellite instable tumors compared with microsatellite stable ones (45% vs. 19%, respectively, p = 0.0001), irrespective of the RAS or BRAF mutational status. However, HLA-E/ß2m+ colorectal cancers were significantly enriched in CD94+ intraepithelial tumor-infiltrating lymphocytes in microsatellite instable as well as in microsatellite stable tumors. Those CD94+ tumor-infiltrating lymphocytes mostly corresponded to CD8+ αß T cells, and  to a lesser extent to NK cells, and mainly co-expressed a functional inhibitory NKG2A chain. Finally, a high number of CD94+ intraepithelial tumor-infiltrating lymphocytes in close contact with tumor cells was independently associated with a worse overall survival. In conclusion, these findings strongly suggest that HLA-E/ß2m-CD94/NKG2A represents a new druggable inhibitory immune checkpoint, preferentially expressed in microsatellite instable tumors, but also in a subgroup of microsatellite stable tumors, leading to a new opportunity in colorectal cancer immunotherapies.

Intra-abdominal recurrence from colorectal carcinoma: Differences and similarities between local and peritoneal recurrence.

BACKGROUND: Peritoneal recurrences from colo-rectal cancer can be isolated (PR) or associated with local recurrences (LR). The purpose of this study was to analyze patterns and outcomes of LR and PR. METHODS: Analyze from a prospective database of 108 patients treated with CCS plus HIPEC at two cancer centers between 2008 and 2015. RESULTS: The population was divided into an LPR group (presence of LR with or without PR, n = 56) and a PR group (isolated PR, n = 52). The patients characteristics (age, sex, Charlson score, PCI) or perioperative treatments were comparable between the groups. The median number of resected organs for tumor involvement (respectively, 2 vs 1; p < 0.001), the percentage of patients with metastatic lymph nodes (LN+) from the resected specimen (respectively, 25% vs 7%; p = 0.016) and the mortality rate (respectively, 9% vs 0%; p = 0.023) were significantly higher in the LPR group. After a median follow-up of 32 (1-108) months, median overall survival was comparable between the two groups (respectively, 46 vs 42 months; p = 0.262). CONCLUSIONS: LR is associated with a higher incidence of organ invasion, LN involvement (25%) and postoperative mortality. Optimal surgical resection of LR with systematic lymphadenectomy of invaded organs seems mandatory.

Significance of lymph node involvement in local recurrence of colorectal cancer.

BACKGROUND: There are few data on lymphatic spread concomitant to local recurrence (LR) of colorectal cancer (CRC). The objectives of this study were to determine variables associated with lymphatic spread, to analyze the distribution of LN+, and understand the underlying mechanisms. METHODS: A total of 76 patients underwent resection of LR of CRC between January 2007 and December 2018 at Institut cancérologique de l'Ouest and were retrospectively reviewed. RESULTS: Twenty-five (32.9%) patients had lymph node (LN) involvement with LR. Lymphatics from the mesocolon-rectum and aorto-iliac compartments were involved in 21%, 20.3% and 18.1%, 20.3% for pelvic and retroperitoneal LRs, respectively. In multivariate analysis, the only predictive factor for LN invasion (LN+) was a primary positive LN status (odds ratio, 5.3; P = .007). Despite a trend toward a worse median overall survival in the LN+ group, the difference was not significant in comparison with the LN- group (46 vs. 57 months; P = 0.31) or with the LN- plus LN not assessed groups (46 months vs not reached; P = .07). CONCLUSIONS: LN invasion with LR from CRC is a frequent occurrence without significant impact on survival. The only predictive factor is a primary positive nodal status.

Identification of three subtypes of triple-negative breast cancer with potential therapeutic implications.

BACKGROUND: Heterogeneity and lack of targeted therapies represent the two main impediments to precision treatment of triple-negative breast cancer (TNBC), and therefore, molecular subtyping and identification of therapeutic pathways are required to optimize medical care. The aim of the present study was to define robust TNBC subtypes with clinical relevance. METHODS: Gene expression profiling by means of DNA chips was conducted in an internal TNBC cohort composed of 238 patients. In addition, external data (n = 257), obtained by using the same DNA chip, were used for validation. Fuzzy clustering was followed by functional annotation of the clusters. Immunohistochemistry was used to confirm transcriptomics results: CD138 and CD20 were used to test for plasma cell and B lymphocyte infiltrations, respectively; MECA79 and CD31 for tertiary lymphoid structures; and UCHL1/PGP9.5 and S100 for neurogenesis. RESULTS: We identified three molecular clusters within TNBC: one molecular apocrine (C1) and two basal-like-enriched (C2 and C3). C2 presented pro-tumorigenic immune response (immune suppressive), high neurogenesis (nerve infiltration), and high biological aggressiveness. In contrast, C3 exhibited adaptive immune response associated with complete B cell differentiation that occurs in tertiary lymphoid structures, and immune checkpoint upregulation. External cohort subtyping by means of the same approach proved the robustness of these results. Furthermore, plasma cell and B lymphocyte infiltrates, tertiary lymphoid structures, and neurogenesis were validated at the protein levels by means of histological evaluation and immunohistochemistry. CONCLUSION: Our work showed that TNBC can be subcategorized in three different subtypes characterized by marked biological features, some of which could be targeted by specific therapies.

Molecular apocrine tumours in EORTC 10994/BIG 1-00 phase III study: pathological response after neoadjuvant chemotherapy and clinical outcomes.

BACKGROUND: We explored, within the EORTC10994 study, the outcomes for patients with molecular apocrine (MA) breast cancer, and defined immunohistochemistry (IHC) as androgen-receptor (AR) positive, oestrogen (ER) and progesterone (PR) negative. We also assessed the concordance between IHC and gene expression arrays (GEA) in the identification of MA cancers. METHODS: Centrally assessed biopsies for AR, ER, PR, HER2 and Ki67 by IHC were classified into six subtypes: MA, triple-negative (TN) basal-like, luminal A, luminal B HER2 negative, luminal B HER2 positive and "other". The two main objectives were the pCR rates and survival outcomes in the overall MA subtype (and further divided by HER2 status) and the remaining five subtypes. RESULTS: IHC subtyping was obtained in 846 eligible patients. Ninety-three (11%) tumours were classified as the MA subtype. Both IHC and GEA data were available for 64 patients. In this subset, IHC concordance was 88.3% in identifying MA tumours compared with GEA. Within the MA subtype, pCR was observed in 33.3% of the patients (95% CI: 29.4-43.9) and the 5-year recurrence-free interval was 59.2% (95% CI: 48.2-68.6). Patients with MA and TN basal-like tumours have lower survival outcomes. CONCLUSIONS: Irrespective of their HER2 status, the prognosis for MA tumours remains poor and adjuvant trials evaluating anti-androgens should be considered.

SALL4 expression in gestational trophoblastic tumors: a useful tool to distinguish choriocarcinoma from placental site trophoblastic tumor and epithelioid trophoblastic tumor.

SALL4 has important functions in embryonic stem cells. The aim of this study was to investigate SALL4 expression in gestational trophoblastic neoplasia. We hypothesized that it could help to distinguish choriocarcinoma, the presumed most primitive form of gestational trophoblastic neoplasia, from placental site trophoblastic tumor and epithelioid trophoblastic tumor, which would be more differentiated variants. This study included 31 gestational trophoblastic neoplasias: 19 choriocarcinomas, 9 placental site trophoblastic tumors, 1 epithelioid trophoblastic tumor, and 2 mixed tumors comprising a placental site trophoblastic tumor and an epithelioid trophoblastic tumor. Unlike usual markers of gestational trophoblastic neoplasia (p63, human chorionic gonadotrophin and human placental lactogen), SALL4 was expressed in 100% of choriocarcinomas and it was not detected in any placental site trophoblastic tumor and epithelioid trophoblastic tumor. However, the proportion of positive cells varied in a wide range, from 10% to 70%, reflecting the fact that SALL4 was specifically present in mononuclear cells consistent with neoplastic cytotrophoblast. So, SALL4 may be helpful in the differential diagnosis of gestational trophoblastic neoplasias.

Clinical practice guidelines from the French College of Gynecologists and Obstetricians (CNGOF): benign breast tumors - short text.

Screening with breast ultrasound in combination with mammography is needed to investigate a clinical breast mass (Grade B), colored single-pore breast nipple discharge (Grade C), or mastitis (Grade C). The BI-RADS system is recommended for describing and classifying abnormal breast imaging findings. For a breast abscess, a percutaneous biopsy is recommended in the case of a mass or persistent symptoms (Grade C). For mastalgia, when breast imaging is normal, no MRI or breast biopsy is recommended (Grade C). Percutaneous biopsy is recommended for a BI-RADS category 4-5 mass (Grade B). For persistent erythematous nipple or atypical eczema lesions, a nipple biopsy is recommended (Grade C). For distortion and asymmetry, a vacuum core-needle biopsy is recommended due to the risk of underestimation by simple core-needle biopsy (Grade C). For BI-RADS category 4-5 microcalcifications without any ultrasound signal, a minimum 11-G vacuum core-needle biopsy is recommended (Grade B). In the absence of microcalcifications on radiography cores additional samples are recommended (Grade B). For atypical ductal hyperplasia, atypical lobular hyperplasia, lobular carcinoma in situ, flat epithelial atypia, radial scar and mucocele with atypia, surgical excision is commonly recommended (Grade C). Expectant management is feasible after multidisciplinary consensus. For these lesions, when excision margins are not clear, no new excision is recommended except for LCIS characterized as pleomorphic or with necrosis (Grade C). For grade 1 phyllodes tumor, surgical resection with clear margins is recommended. For grade 2 phyllodes tumor, 10mm margins are recommended (Grade C). For papillary breast lesions without atypia, complete disappearance of the radiological signal is recommended (Grade C). For papillary breast lesions with atypia, complete surgical excision is recommended (Grade C).

A Sex Cord-stromal Tumor, Specifically a Fibroma, Arising From the Uterine Corpus: A Case Report.

A 12-yr-old girl presented with lordosis and an intraperitoneal mass that revealed a tumor attached to the uterine fundus. The fallopian tubes and ovaries were spared. The mass was completely excised, and a patch of the uterine fundus and the proximal one third of the fallopian tubes were resected. The lesion was composed of bland spindle cells that were positive for sex cord-stromal markers, with particularly strong staining for inhibin and CD56, as well as patchy staining for calretinin, WT1, and steroidogenic factor 1. Thus, the patient was diagnosed with a sex cord-stromal tumor, specifically a fibroma, arising from the uterine corpus. The pathogenesis of this tumor is unclear. An ovarian origin in the context of adherence or a tumor arising from sex cord-stromal ectopic tissues cannot be excluded, but seem unlikely. The tumor might appear as a particular form of uterine tumor resembling an ovarian sex cord tumor. However, this tumor would differ from the presently described classical form of uterine tumor resembling an ovarian sex cord tumor owing to a pure stromal differentiation instead of a pure sex cord differentiation. Finally, because of the low risk for recurrence, long-term follow-up was prescribed for the patient.

Assessment of the specificity of a new folate-targeted photosensitizer for peritoneal metastasis of epithelial ovarian cancer to enable intraperitoneal photodynamic therapy. A preclinical study.

BACKGROUND: Ovarian cancer's prognosis remains dire after primary therapy. Recurrence rate is disappointingly high as 60% of women with epithelial ovarian cancer considered in remission will develop recurrent disease within 5 years. Special attention to undetected peritoneal metastasis during surgery is necessary as they are the main predictive factors of recurrences. Folate Receptor α (FRα) shows promising prospects in targeting ovarian cancerous cells and intraperitoneal photodynamic therapy (PDT) could be a solution in addition to macroscopic cytoreductive surgery to treat peritoneal micrometastasis. The aim of this preclinical study is to assess the specificity of a folate-targeted photosensitizer for ovarian peritoneal micrometastasis. METHODS: We used the NuTu-19 epithelial ovarian cancer cell line to induce peritoneal carcinomatosis in female Fischer 344 rats. Three groups of 6 rats were studied (Control (no photosensitizer)/Non-conjugated photosensitizer (Porph)/Folate-conjugated photosensitizer (Porph-s-FA)). Four hours after the administration of the photosensitizer, animals were sacrificed and intraperitoneal organs tissues were sampled. FRα tissue expression was evaluated by immunohistochemistry. Tissue incorporation of photosensitizers was assessed by confocal microscopy and tissue quantification. RESULTS: FRα is overexpressed in tumor, ovary, and liver whereas, peritoneum, colon, small intestine, and kidney do not express it. Cytoplasmic red endocytosis vesicles observed by confocal microscopy are well correlated to FRα tissue expression. Photosensitizer tissue quantification shows a mean tumor-to-normal tissue ratio of 9.6. CONCLUSION: We demonstrated that this new generation folate-targeted photosensitizer is specific of epithelial ovarian peritoneal metastasis and may allow the development of efficient and safe intraperitoneal PDT procedure.

Fischer 344 Rat: A Preclinical Model for Epithelial Ovarian Cancer Folate-Targeted Therapy.

OBJECTIVE: Ovarian cancer prognosis remains dire after primary therapy. Recurrence rates are disappointingly high as 60% of women with advanced epithelial ovarian cancer considered in remission will develop recurrent disease within 5 years. Special attention to undetected peritoneal metastasis and residual tumorous cells during surgery is necessary as they are the main predictive factors of recurrences. Folate receptor α (FRα) shows promising prospects in targeting ovarian cancerous cells. Our aim was to determine if the Fischer model described by Rose et al could be used to evaluate folate-targeted therapies in preclinical studies. METHODS: NuTu-19 epithelial ovarian cancer cell line was used to induce peritoneal carcinomatosis in female Fischer 344 rats. FRα expression by NuTu-19 cells was assessed in vitro by immunofluorescence using "Cytospin®" protocol. In vitro folate-targeted compound uptake by NuTu-19 cells was evaluated by incubation of FRα-positive ovarian cancer cell lines (NuTu-19/SKOV-3/OVCAR-3/IGROV-1) with or without (control) a folate-targeted photosensitizer. Intracellular incorporation was assessed by confocal microscopy. Determination of in vivo FRα tissue expression by several organs of the peritoneal cavity was studied by immunohistochemistry. RESULTS: NuTu-19 cells express FRα which allows intracellular incorporation of folate-targeted compound by endocytosis. FRα is expressed in tumor tissue, ovary, and liver. Peritoneum, colon, small intestine, and kidney do not express the receptor. CONCLUSIONS: Female Fischer 344 rat is an inexpensive reproducible and efficient preclinical model to study ovarian peritoneal carcinomatosis folate-targeted therapies.

Shear Wave Elastography (SWE): An Analysis of Breast Lesion Characterization in 83 Breast Lesions.

Qualitative and quantitative shear wave elastography (SWE) criteria were assessed to differentiate between malignant and benign breast lesions. This prospective study included 83 lesions. SWE features measured included maximal stiffness values inside the lesion (E(lesion)) and in the peri-lesion area (E(perilesion)) and ratio values (R(lesion) and R(perilesion)) according to the formula E(lesion) or E(perilesion)/E(fat), with E(fat) corresponding to normal fatty tissue. We compared ultrasonography (B-mode), SWE and histologic sizes. With qualitative and quantitative SWE analysis, sensitivity was 94% and specificity 73%. Malignant lesions appeared more heterogeneous, with higher stiffness and ratio values than benign lesions (p < 0.001). For malignant lesions, SWE size was better correlated to histologic size than B-mode size. Using benign SWE signs to selectively downgrade category 4a and 4b lesions, the specificity improved from 13% to 51% without loss in sensitivity (100%) compared to ultrasound.

Implications of a two-step procedure in surgical management of patients with early-stage endometrioid endometrial cancer.

OBJECTIVE: Since European Society for Medical Oncology (ESMO) recommendations and French guidelines, pelvic lymphadenectomy should not be systematically performed for women with early-stage endometrioid endometrial cancer (EEC) preoperatively assessed at presumed low- or intermediate-risk. The aim of our study was to evaluate the change of our surgical practices after ESMO recommendations, and to evaluate the rate and morbidity of second surgical procedure in case of understaging after the first surgery. METHODS: This retrospective single-center study included women with EEC preoperatively assessed at presumed low- or intermediate-risk who had surgery between 2006 and 2013. Two periods were defined the times before and after ESMO recommendations. Demographics characteristics, surgical management, operative morbidity, and rate of understaging were compared. The rate of second surgical procedure required for lymph node resection during the second period and its morbidity were also studied. RESULTS: Sixty-one and sixty-two patients were operated for EEC preoperatively assessed at presumed low-or intermediate-risk before and after ESMO recommendations, respectively. Although immediate pelvic lymphadenectomy was performed more frequently during the first period than the second period (88.5% vs. 19.4%; p<0.001), the rate of postoperative risk-elevating or upstaging were comparable between the two periods (31.1% vs. 27.4%; p=0.71). Among the patients requiring second surgical procedure during the second period (21.0%), 30.8% did not undergo the second surgery due to their comorbidity or old age. For the patients who underwent second surgical procedure, mean operative time of the second procedure was 246.1±117.8 minutes. Third operation was required in 33.3% of them because of postoperative complications. CONCLUSION: Since ESMO recommendations, second surgical procedure for lymph node resection is often required for women with EEC presumed at low- or intermediate-risk. This reoperation is not always performed due to age/comorbidity of the patients, and presents a significant morbidity.

Lipidomics for clinical diagnosis: Dye-Assisted Laser Desorption/Ionization (DALDI) method for lipids detection in MALDI mass spectrometry imaging.

Lipid-based biomarkers for research and diagnosis are rapidly emerging to unpack the basis of person-to-person and population variations in disease susceptibility, drug and nutritional responses, to name but a few. Hence, with the advent of MALDI Mass Spectrometry Imaging, lipids have begun to be investigated intensively. However, lipids are highly mobile during tissue preparation, and are soluble in the solvent used for matrix preparation or in the fixing fluid such as formalin, resulting in substantial delocalization. In the present article, we investigated as another alternative, the possibility of using specific dyes that can absorb UV wavelengths, in order to desorb the lipids specifically from tissue sections, and are known to immobilize them in tissues. Indeed, after lipid insolubilization with chromate solution or chemical fixation with osmium tetroxide, heterocyclic-based dyes can be directly used without matrix. Taking into account the fact that some dyes have this matrix-free capability, we identified particular dyes dedicated to histological staining of lipids that could be used with MALDI mass spectrometry imaging. We stained tissue sections with either Sudan Black B, Nile Blue A, or Oil Red O. An important advantage of this assay relies on its compatibility with usual practices of histopathological investigation of lipids. As a new method, DALDI stands for Dye-Assisted Laser Desorption Ionization and allows for future clinical and histopathological applications using routine histological protocols. Additionally, this novel methodology was validated in human ovarian cancer biopsies to demonstrate its use as a suitable procedure, for histological diagnosis in lipidomics field.

HFIP extraction followed by 2D CTAB/SDS-PAGE separation: a new methodology for protein identification from tissue sections after MALDI mass spectrometry profiling for personalized medicine research.

Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) and profiling technology have become the easiest methods for quickly accessing the protein composition of a tissue area. Unfortunately, the demand for the identification of these proteins remains unmet. To overcome this bottleneck, we combined several strategies to identify the proteins detected via MALDI profiling including on-tissue protein extraction using hexafluoroIsopropanol (1,1,1,3,3,3-hexafluoro-2-propanol, HFIP) coupled with two-dimensional cetyl trimethylammonium bromide/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D CTAB/SDS-PAGE) for separation followed by trypsin digestion and MALDI-MS analyses for identification. This strategy was compared with an on-tissue bottom-up strategy that we previously developed. The data reflect the complementarity of the approaches. An increase in the number of specific proteins identified has been established. This approach demonstrates the potential of adapted extraction procedures and the combination of parallel identification approaches for personalized medicine applications. The anatomical context provides important insight into identifying biomarkers and may be considered a first step for tissue-based biomarker research, as well as the extemporaneous examination of biopsies during surgery.