Histopathological growth patterns of liver metastasis: updated consensus guidelines for pattern scoring, perspectives and recent mechanistic insights.

The first consensus guidelines for scoring the histopathological growth patterns (HGPs) of liver metastases were established in 2017. Since then, numerous studies have applied these guidelines, have further substantiated the potential clinical value of the HGPs in patients with liver metastases from various tumour types and are starting to shed light on the biology of the distinct HGPs. In the present guidelines, we give an overview of these studies, discuss novel strategies for predicting the HGPs of liver metastases, such as deep-learning algorithms for whole-slide histopathology images and medical imaging, and highlight liver metastasis animal models that exhibit features of the different HGPs. Based on a pooled analysis of large cohorts of patients with liver-metastatic colorectal cancer, we propose a new cut-off to categorise patients according to the HGPs. An up-to-date standard method for HGP assessment within liver metastases is also presented with the aim of incorporating HGPs into the decision-making processes surrounding the treatment of patients with liver-metastatic cancer. Finally, we propose hypotheses on the cellular and molecular mechanisms that drive the biology of the different HGPs, opening some exciting preclinical and clinical research perspectives.

Clinico-metabolic characterization improves the prognostic value of histological growth patterns in patients undergoing surgery for colorectal liver metastases.

BACKGROUND AND OBJECTIVES: The histological growth pattern (HGP) represents a strong prognostic factor in patients undergoing surgery for colorectal liver metastases (CRLM). We evaluated whether the combination of HGP with clinico-metabolic parameters could improve its prognostic value. METHODS: In a series of 108 patients undergoing resection of CRLM, the HGP of CRLM was scored according to international guidelines. Baseline clinico-metabolic clinical status was evaluated using a metabolic-Clinical Risk Score (mCRS), combining traditional Memorial Sloan Kettering-CRS parameters with the tumor-to-liver glucose uptake ratio as measured with 18 Fluorodeoxyglucose/positron emission tomography. RESULTS: In patients with desmoplastic HGP (DHGP) CRLM (20% of all patients), 5- and 10-years overall survival (OS) and disease free survival (DFS) were 66% and 43% and 37% and 24.5%, as compared with 35% and 21% and 11% and 11% in the non-DHGP group (p = 0.07 and 0.054). Among DHGP patients, those with a low-risk mCRS had improved postoperative outcomes, 5- and 10-years OS and DFS reaching 83.3% and 62.5% and 50% and 33%, as compared with 18% and 0% and 0% and 0% in high-risk mCRS patients (p = 0.007 and 0.003). In contrast, mCRS did not influence postoperative survivals in non-DHGP patients. CONCLUSIONS: Combining the clinico-metabolic characteristics with the HGP may improve prognostication in patients undergoing surgery for CRLM.

ICG-fluorescence imaging for detection of peritoneal metastases and residual tumoral scars in locally advanced ovarian cancer: A pilot study.

BACKGROUND AND OBJECTIVES: No intraoperative imaging techniques exist for detecting tumor nodules or tumor scar tissues in patients treated with upfront or interval cytoreductive surgery (CS) after neoadjuvant chemotherapy (NAC). The aims of this study were to evaluate the role of indocyanine green (ICG) fluorescence imaging (FI) for the detection of peritoneal metastases (PM) and evaluate whether it can be used to detect remnant tumor cells in scar tissue. METHODS: Patients with PM from ovarian cancer admitted for CS were included. ICG, at 0.25 mg per kg of patient weight, was injected intraoperatively after explorative laparotomy before CS. RESULTS: A total of 108 peritoneal lesions, including 25 scars, were imaged in 20 patients. Seventy-three were malignant (67.6%) and 35 benign (32.4%). The mean Tumor to Background Ratio (ex vivo) was 1.8 (SD 1.3) in malignant and 1.0 (SD 0.79) in benign nodules (P = 0.007). Of 25 post-NAC scars, the mean Tumor to Background Ratio (TBR) (in vivo) was 2.06 (SD 1.15) in malignant and 1.21 (SD 0.50) in benign nodules (P = 0.26). The positive predictive value of ICG-FI to detect tumor cells in scars was 57.1%. CONCLUSIONS: ICG-FI is accurate to demonstrate PM in ovarian cancer but unable to discriminate between benign and malignant post-NAC.

Fluorescence Imaging After Indocyanine Green Injection for Detection of Peritoneal Metastases in Patients Undergoing Cytoreductive Surgery for Peritoneal Carcinomatosis From Colorectal Cancer: A Pilot Study.

OBJECTIVE: The aim of this study was to evaluate the role of fluorescence imaging (FI) using an intraoperative injection of free indocyanine green (ICG) in the detection of peritoneal metastases (PM) due to colorectal cancer (CRC). BACKGROUND: A large proportion of patients undergoing cytoreductive surgery and hyperthermic intraperitoneal chemotherapy will have local recurrence. This is, in part, related to the presence of small undetected nodules in the peritoneal cavity. Near-infrared FI-guided surgery has provided new opportunities for detection of nonvisible lesions during cancer surgery. METHODS: Patients with PM from CRC admitted for cytoreductive surgery and hyperthermic intraperitoneal chemotherapy were selected for participation in this study (NCT02032485). Free ICG, at 0.25 mg/kg of patient weight, was intravenous (IV)-injected intraoperatively. Tumor-to-background ratio was calculated for all suspect resected PM. RESULTS: Sixty-three of 78 peritoneal resected nodules in 14 patients were evaluated for fluorescence, among them, 53 were malignant (84%) and 10 benign (16%). Twenty-six were hypofluorescent, 16 moderately hyperfluorescent, and 21 hyperfluorescent. Amongst the 42 nodules of the 9 patients with nonmucinous adenocarcinoma, the mean tumor-to-background ratio was 1.92 (SD 0.67) in malignant and 1.02 (SD 0.06) in benign nodules (P = 0.0099). In 4 of 14 patients (29%), the surgery was modified by intraoperative ICG-FI, which detected additional PM not found using visualization and palpation. CONCLUSIONS: This pilot study demonstrates that non-mucinous PM of CRC can be visualized intraoperatively using ICG-FI. Furthermore, ICG-FI findings resulted in modification of the planned surgery in 29% of patients.

Ex vivo detection of tumoral lymph nodes of colorectal origin with fluorescence imaging after intraoperative intravenous injection of indocyanine green.

BACKGROUND AND OBJECTIVES: The aim of this study was to investigate the potential role of indocyanine green (ICG) fluorescence imaging after intraoperative intravenous (IV) injection for the "ex vivo" detection of metastatic lymph nodes (mLNs) of colorectal cancer origin. METHODS: Fresh-fixed LNs in cassettes and/or paraffin-embedded LNs of patients included in a study that evaluated the role of ICG in the detection of peritoneal metastases of colorectal origin (Protocol NCT-01995591) were further explored with a dedicated near-infrared camera system for their fluorescence. An IV injection of ICG was delivered intraoperatively at 0.25 mg/kg. Signal to background ratios (SBRs) were calculated. RESULTS: LNs on operative specimens were evaluated for 12 patients (5 males, 7 females). A total of 182 LNs were analyzed. The mean LN number per patient was 15.2 (median: 15.5; range 3-22). SBRs of mLNs were significantly more fluorescent than benign LNs, 1.41 versus 1.04 arbitrary units (P < 0.0002). On univariate analysis, fluorescence was statistically correlated with LN surface area (>20 mm(2) ) (P < 0.0004). CONCLUSION: Ex vivo ICG fluorescence imaging after intraoperative IV injection represents a potential method for detecting invaded LN's of colorectal cancer origin on operative specimens. Further clinical studies are needed to better define optimal techniques. J. Surg. Oncol. 2016;114:348-353. © 2016 Wiley Periodicals, Inc.

Transcriptomic characterization of the histopathological growth patterns in breast cancer liver metastases.

Metastatic breast cancer (mBC) remains incurable and liver metastases (LM) are observed in approximately 50% of all patients with mBC. In some cases, surgical resection of breast cancer liver metastases (BCLM) is associated with prolonged survival. However, there are currently no validated marker to identify these patients. The interactions between the metastatic cancer cells and the liver microenvironment result in two main histopathological growth patterns (HGP): replacement (r-HGP), characterized by a direct contact between the cancer cells and the hepatocytes, and desmoplastic (d-HGP), in which a fibrous rim surrounds the tumor cells. In patients who underwent resection of BCLM, the r-HGP is associated with a worse postoperative prognosis than the d-HGP. Here, we aim at unraveling the biological differences between these HGP within ten patients presenting both HGP within the same metastasis. The transcriptomic analyses reveal overexpression of genes involved in cell cycle, DNA repair, vessel co-option and cell motility in r-HGP while angiogenesis, wound healing, and several immune processes were found overexpressed in d-HGP LM. Understanding the biology of the LM could open avenues to refine treatment of BC patients with LM.

Association between primary tumor characteristics and histopathological growth pattern of liver metastases in colorectal cancer.

INTRODUCTION: The microarchitecture of liver metastases (LMs), or histopathological growth pattern (HGP), has been demonstrated to be a significant prognostic factor in patients undergoing resection of colorectal liver metastases (CRLMs). Currently, however, HGP can be only determined on the operative specimen. Therefore, the development of new tools to predict the HGP of CRLMs before surgery and to understand the mechanisms that drive these patterns is important for improving individualization of therapeutic management. In this study, we analyzed data from a retrospective series of patients who underwent surgery for CRLMs to compare primary tumor characteristics, including markers of local aggressiveness and migratory capacity, and HGP of liver metastases. METHODS: Data from a retrospective series of 167 patients who underwent curative-intent resection of CRLMs and in whom pathological samples from both primary tumor and liver metastases were available were reviewed. At the primary tumor level, KRAS mutational status, grade of differentiation, and tumor budding were assessed. HGP was scored in each resected CRLM, according to consensus guidelines, and classified as desmoplastic (dHGP) or non-desmoplastic (non-dHGP). Associations between primary tumor characteristics and HGP of CRLMs were evaluated using a binary logistic regression model. Overall survival and disease-free survival were evaluated using Kaplan-Meier and multivariable Cox regression analyses. RESULTS: CRLMs were classified as dHGP in 36% of the patients and as non-dHGP in 64%. Higher rates of moderately or poorly differentiated primary tumors were observed in the non-dHGP CRLM group (80%), as compared with the dHGP group (60%) (OR = 3.6; 95%CI: 1.6-7.05; p = 0.001). Higher rates of tumor budding were observed in the non-dHGP CRLM group, with a median tumor budding value of 4 as compared with 2.5 in the dHGP group (p = 0.042). In the entire series, 5-year overall and disease-free survival were 43% and 32.5%, respectively. The non-dHGP CRLM group had worse post-hepatectomy survival, with 5-year overall and disease-free survival of 32.2% and 24.6%, respectively, as compared with 60.8% and 45.9%, respectively, for the dHGP group (p = 0.02). CONCLUSION: Colorectal tumors with moderate or poor differentiation and those with high tumor budding are more frequently associated with CRLMs with a non-dHGP. This suggests that primary tumor characteristics of local aggressiveness and migratory capacity could preferentially promote the development of CRLMs with an infiltrating pattern and that these parameters should be considered as part of new scores for predicting HGP before surgery. This finding may stimulate new lines of research for more individualized therapeutic decision in patients with CRLM candidate to surgery.

Tumor biology reflected by histological growth pattern is more important than surgical margin for the prognosis of patients undergoing resection of colorectal liver metastases.

INTRODUCTION: The histological growth pattern (HGP) of colorectal liver metastases (CRLMs) reflects tumor biology and local infiltrating behavior. In patients undergoing surgery for CRLMs, we investigated whether HGP and surgical margin status interact when influencing prognosis. METHODS: Clinicopathological data, margin status, and HGP were reviewed in patients who underwent resection of CRLMs. R1 margin was defined when cancer cells were present at any point along the margin. HGPs were scored according to international guidelines, identifying patients with desmoplastic (DHGP) or non-desmoplastic (non-DHGP) CRLMs. RESULTS: Among 299 patients, 16% had R1 resection and 81% had non-DHGP CRLMs. Non-DHGP was the only predictive factor for R1 resection (18.7% versus 7.4% in DHGP, p = 0.04). Poorer 5-year overall survival was observed in both R1 and non-DHGP groups in univariate analysis (27.6% in R1 versus 45.6% in R0, p = 0.026, and 37.2% in non-DHGP versus 59.2% in DHGP, p = 0.013), whereas non-DHGP but not R1 remained associated with worse prognosis in multivariate analysis. In patients with non-DHGP, R1 margin has no prognostic impact. CONCLUSIONS: In patients undergoing resection of CRLMs, the prognostic value of poor tumor biology, such as in patients with non-DHGP, exceeds that of surgical radicality.

Histopathological growth patterns and tumor-infiltrating lymphocytes in breast cancer liver metastases.

Liver is the third most common organ for breast cancer (BC) metastasis. Two main histopathological growth patterns (HGP) exist in liver metastases (LM): desmoplastic and replacement. Although a reduced immunotherapy efficacy is reported in patients with LM, tumor-infiltrating lymphocytes (TIL) have not yet been investigated in BCLM. Here, we evaluate the distribution of the HGP and TIL in BCLM, and their association with clinicopathological variables and survival. We collect samples from surgically resected BCLM (n = 133 patients, 568 H&E sections) and post-mortem derived BCLM (n = 23 patients, 97 H&E sections). HGP is assessed as the proportion of tumor liver interface and categorized as pure-replacement ('pure r-HGP') or any-desmoplastic ('any d-HGP'). We score the TIL according to LM-specific guidelines. Associations with progression-free (PFS) and overall survival (OS) are assessed using Cox regressions. We observe a higher prevalence of 'any d-HGP' (56%) in the surgical samples and a higher prevalence of 'pure r-HGP' (83%) in the post-mortem samples. In the surgical cohort, no evidence of the association between HGP and clinicopathological characteristics is observed except with the laterality of the primary tumor (p value = 0.049) and the systemic preoperative treatment before liver surgery (p value = .039). TIL is less prevalent in 'pure r-HGP' as compared to 'any d-HGP' (p value = 0.001). 'Pure r-HGP' predicts worse PFS (HR: 2.65; CI: (1.45-4.82); p value = 0.001) and OS (HR: 3.10; CI: (1.29-7.46); p value = 0.011) in the multivariable analyses. To conclude, we demonstrate that BCLM with a 'pure r-HGP' is associated with less TIL and with the worse outcome when compared with BCLM with 'any d-HGP'. These findings suggest that HGP could be considered to refine treatment approaches.

Use of fluorescence imaging during lymphatic surgery: A Delphi survey of experts worldwide.

BACKGROUND: Fluorescence imaging with indocyanine green is increasingly used during lymphedema patient management. However, to date, no guidelines exist on when it should and should not be used or how it should be performed. Our objective was to have an international panel of experts identify areas of consensus and nonconsensus in current attitudes and practices in fluorescence imaging with indocyanine green use during lymphedema surgery patient management. METHODS: A 2-round Delphi study was conducted involving 18 experts in the use of fluorescence imaging during lymphatic surgery, all asked to vote on 49 statements on patient preparation and contraindications (n = 7 statements), indocyanine green dosing and administration (n = 10), fluorescence imaging uses and potential advantages (n = 16), and potential disadvantages and training needs (n = 16). RESULTS: Consensus ultimately was reached on 40/49 statements, including consistent consensus regarding the value of fluorescence imaging with indocyanine green in almost all facets of lymphedema patient management, including early detection, assessing disease extent, preoperative work-up, surgical planning, intraoperative guidance, monitoring short- and longer-term outcomes, quality control, and resident training. All experts felt it was very safe, while 94% felt it should be part of routine care and that indocyanine green was superior to colored dyes and ultrasound. Nonetheless, there also was consensus that limited high-quality evidence remains a barrier to its widespread use and that patients should still be provided with specific information and asked to sign specific consent for both fluorescence imaging and indocyanine green. CONCLUSION: Fluorescence imaging with or without indocyanine green appears to have several roles in lymphedema prevention, diagnosis, assessment, and treatment.

Near-Infrared Fluorescence Imaging of Breast Cancer and Axillary Lymph Nodes After Intravenous Injection of Free Indocyanine Green.

BACKGROUND: Near-infrared fluorescence imaging (NIRFI) of breast cancer (BC) after the intravenous (IV) injection of free indocyanine green (fICG) has been reported to be feasible. However, some questions remained unclarified. OBJECTIVE: To evaluate the distribution of fICG in BC and the axillary lymph nodes (LNs) of women undergoing surgery with complete axillary LN dissection (CALND) and/or selective lymphadenectomy (SLN) of sentinel LNs (NCT no. 01993576 and NCT no. 02027818). METHODS: An intravenous injection of fICG (0.25 mg/kg) was administered to one series of 20 women undergoing treatment with mastectomy, the day before surgery in 5 (group 1) and immediately before surgery in 15 (group 2: tumor localization, 25; and pN+ CALND, 4) as well as to another series of 20 women undergoing treatment with tumorectomy (group 3). A dedicated NIR camera was used for ex vivo fluorescence imaging of the 45 BC lesions and the LNs. RESULTS: In group 1, two of the four BC lesions and one large pN+ LN exhibited fluorescence. In contrast, 24 of the 25 tumors in group 2 and all of the tumors in group 3 were fluorescent. The sentinel LNs were all fluorescent, as well as some of the LNs in all CALND specimens. Metastatic cells were found in the fluorescent LNs of the pN+ cases. Fluorescent BC lesions could be identified ex vivo on the surface of the lumpectomy specimen in 14 of 19 cases. CONCLUSIONS: When fICG is injected intravenously just before surgery, BC can be detected using NIRFI with high sensitivity, with metastatic axillary LNs also showing fluorescence. Such a technical approach seems promising in the management of BC and merits further investigation.

Absence of residual fluorescence in the surgical bed at near-infrared fluorescence imaging predicts negative margins at final pathology in patients treated with breast-conserving surgery for breast cancer.

INTRODUCTION: Positive margins after breast-conserving surgery (BCS) for breast cancer (BC) remain a major concern. In this study we investigate the feasibility and accuracy of indocyanine green (ICG) fluorescence imaging (FI) for the in vivo assessment of surgical margins during BCS. MATERIALS AND METHODS: Patients with BC admitted for BCS from October 2015 to April 2016 were proposed to be included in the present study (NCT02027818). ICG (0.25 mg/kg) was intravenously injected at induction anesthesia and ICG-FI of the surgical beds was correlated with final pathology results. RESULTS: Fifty patients consented to participate and thirty-five patients were retained for final analysis, 15 patients having been excluded for, respectively, incomplete video records data for signal to background ratio (SBR) calculation (11) and in situ tumors (4). The final pathological assessment of 35 breast specimens identified 5 (14.7%) positive margins. Intraoperative ICG-FI revealed hyperfluorescent signals in 15 (42.9%) patients and an absence of fluorescent signals in 20 (57.1%). Median SBR in patients with involved margins was 1.8 (SD 0.7) and was 1.25 (SD 0.6) in patients with clear margins (p = 0.05). The accuracy, specificity, positive and negative predictive value of ICG-FI for breast surgical margin assessment were 71%, 60%, 29% and 100%, respectively. CONCLUSION: ICG-FI of BC surgical beds has a high negative predictive value for surgical margin assessment during BCS. The absence of residual fluorescence in the surgical bed of patients with fluorescent tumors predicts negative margins at final pathology and allows the surgeon to avoid further intraoperative analysis.

Systemic Sentinel Lymph Node Detection Using Fluorescence Imaging After Indocyanine Green Intravenous Injection in Colorectal Cancer: Protocol for a Feasibility Study.

BACKGROUND: Nodal staging is a major concern in colorectal cancer as it is an important prognostic factor. Several techniques that could potentially improve patient treatment and prognosis have been developed to increase the accuracy of nodal staging. Sentinel lymph node detection has been shown to accurately reflect nodal status in various tumors and has become the standard procedure in nodal staging of breast cancer and melanoma. However, in colorectal cancer, sentinel lymph node detection techniques are still controversial as the sensitivity reported in the literature varies from one study to another. Recently, indocyanine green fluorescence-guided surgery has been reported to be a useful technique for detection of macroscopic and microscopic metastatic deposits in lymph nodes after intravenous administration of indocyanine green dye. However, no studies have focused on the potential role of sentinel lymph node detection after systemic administration of indocyanine green dye, so-called systemic sentinel lymph nodes, or on the correspondence between the identification of the sentinel lymph node by standard local injection techniques and the detection of fluorescent lymph nodes with this new approach. OBJECTIVE: The aim of this protocol is to validate the concept of sentinel lymph nodes identified by fluorescence imaging after intravenous injection of indocyanine green dye and to compare the sentinel lymph nodes identified by fluorescence imaging with sentinel lymph nodes detected by the standard blue dye technique. METHODS: This study (SeLyNoFI; Sentinel Lymph Nodes Fluorescence Imaging) is a diagnostic, single-arm, open-label feasibility study, including patients with colorectal adenocarcinoma with or without metastatic disease who are admitted for elective colorectal resection of the primary tumor. This study evaluates the feasibility of a new approach for improving the accuracy of nodal staging using fluorescence imaging after intravenous administration of indocyanine green dye. Sensitivity, positive predictive value, and accuracy of the classical blue dye technique and of the investigatory fluorescence imaging technique will be calculated. Translational research will be proposed, if applicable. RESULTS: As of June 2020, this study has been registered. Submission for ethical review is planned for September 2020. CONCLUSIONS: The potential correlation between the two different approaches to detect sentinel lymph nodes offers new strategies for improving the accuracy of nodal staging in colorectal cancer. This new concept of the systemic sentinel lymph node and a greater understanding of the interactions between systemic sentinel lymph nodes and standard sentinel lymph nodes may provide important information regarding the underlying mechanism of primary tumor lymphatic drainage. The enhanced permeability and retention effect can also play a role in the fluorescence of systemic sentinel lymph nodes, especially if these lymph nodes are inflamed. In this case, we can even imagine that this new technique will highlight more instances of lymph node-positive colorectal cancer. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/17976.

Sentinel Lymph Node Detection by Blue Dye Versus Indocyanine Green Fluorescence Imaging in Colon Cancer.

BACKGROUND/AIM: Nodal staging is used in colorectal cancer (CRC) to determine which patients should receive adjuvant chemotherapy. The aim of this study was to evaluate the role of indocyanine green fluorescence imaging (ICG-FI) in sentinel lymph node (SLN) detection compared to the standard technique. MATERIALS AND METHODS: Twenty patients with CRC admitted for elective colectomy were included (NCT01995591). Ex vivo SLN detection was performed using patent blue (PB) and free ICG injected around the tumor. RESULTS: Identification rates were 95% (19/20) for both techniques. Sensitivity was 43% for PB and 57% for ICG. Correlation between the techniques was 83%. FI was more sensitive in patients with body mass index (BMI) >25 kg/m(2) Serial section analysis did not allow for up-staging of patients. CONCLUSION: The use of ICG-FI is superior to the blue dye technique in patients with a BMI >25 kg/m(2) However, the sensitivity of ICG-FI in SLN detection remains low, with a high rate of false-negative results.

Near-Infrared Fluorescence Lymphatic Imaging to Reconsider Occlusion Pressure of Superficial Lymphatic Collectors in Upper Extremities of Healthy Volunteers.

BACKGROUND: There are very little scientific data on occlusion pressure for superficial lymphatic collectors. Given its importance in determining the transport capacity of lymphatic vessels, it is crucial to know its value. The novel method of near-infrared fluorescence lymphatic imaging (NIRFLI) can be used to visualize lymphatic flow in real time. The goal of this study was to see if this method could be used to measure the lymphatic occlusion pressure. METHODS: We observed and recorded lymph flow in the upper limb of healthy volunteers through a transparent cuff using near-infrared fluorescence lymphatic imaging. After obtaining a baseline of the lymph flow without pressure inside the cuff, the cuff was inflated by increments of 10 mm Hg starting at 30 mm Hg. A NIRFLI guided manual lymphatic drainage technique named "Fill & Flush Drainage Method" was performed during the measurement to promote lymph flow. Lymphatic occlusion pressure was determined by observing when lymph flow stopped under the cuff. RESULTS: We measured the lymphatic occlusion pressure on 30 healthy volunteers (11 men and 19 women). Mean lymphatic occlusion pressure in the upper limb was 86 mm Hg (CI ±3.7 mm Hg, α = 0.5%). No significant differences were found between age groups (p = 0.18), gender (p = 0.12), or limb side (p = 0.85). CONCLUSIONS: NIRFLI, a transparent sphygmomanometer cuff and the "Fill and Flush" manual lymphatic drainage method were used to measure the lymphatic occlusion pressure in 30 healthy humans. That combination of these techniques allows the visualization of the lymph flow in real time, while ensuring the continuous filling of the lymph collectors during the measurement session, reducing false negative observations. The measured occlusion pressures are much higher than previously described in the medical literature.

The α2,3-sialyltransferase encoded by myxoma virus is a virulence factor that contributes to immunosuppression.

Myxoma virus (MYXV) induces a lethal disease called Myxomatosis in European rabbits. MYXV is one of the rare viruses that encodes an α2,3-sialyltransferase through its M138L gene. In this study, we showed that although the absence of the enzyme was not associated with any in vitro deficit, the M138L deficient strains are highly attenuated in vivo. Indeed, while all rabbits infected with the parental and the revertant strains died within 9 days post-infection from severe myxomatosis, all but one rabbit inoculated with the M138L deficient strains survived the infection. In primary lesions, this resistance to the infection was associated with an increased ability of innate immune cells, mostly neutrophils, to migrate to the site of virus replication at 4 days post-infection. This was followed by the development of a better specific immune response against MYXV. Indeed, at day 9 post-infection, we observed an important proliferation of lymphocytes and an intense congestion of blood vessels in lymph nodes after M138L knockouts infection. Accordingly, in these rabbits, we observed an intense mononuclear cell infiltration throughout the dermis in primary lesions and higher titers of neutralizing antibodies. Finally, this adaptive immune response provided protection to these surviving rabbits against a challenge with the MYXV WT strain. Altogether, these results show that expression of the M138L gene contributes directly or indirectly to immune evasion by MYXV. In the future, these results could help us to better understand the pathogenesis of myxomatosis but also the importance of glycans in regulation of immune responses.

A translocation breakpoint disrupts the ASPM gene in a patient with primary microcephaly.

Primary microcephaly (microcephalia vera) is a developmental abnormality resulting in a small brain, with mental retardation. It is usually transmitted as an autosomal recessive trait, and six loci have been reported to date. We analyzed a translocation breakpoint previously reported in a patient with apparently sporadic primary microcephaly, at 1q31, where locus MCPH5 maps. The patient was lost to follow-up, and we sampled a maternal aunt who carried the familial translocation. FISH analyses showed that the insert of BAC clone RP11-32D17 spanned the breakpoint. The breakpoint was further located within a fragment of this insert corresponding to intron 17 of the ASPM gene, resulting in a predicted transcript truncated of more than half of its coding sequence. It is very likely that the proband carried a second ASPM mutation in trans, but he was not available for sampling and hence we could not confirm this hypothesis. Our observation adds to the mutation spectrum of ASPM in primary microcephaly, and is to our knowledge the second example of a constitutional, reciprocal translocation responsible for a bona fide autosomal recessive phenotype.