Evolutionary design of explainable algorithms for biomedical image segmentation.

An unresolved issue in contemporary biomedicine is the overwhelming number and diversity of complex images that require annotation, analysis and interpretation. Recent advances in Deep Learning have revolutionized the field of computer vision, creating algorithms that compete with human experts in image segmentation tasks. However, these frameworks require large human-annotated datasets for training and the resulting "black box" models are difficult to interpret. In this study, we introduce Kartezio, a modular Cartesian Genetic Programming-based computational strategy that generates fully transparent and easily interpretable image processing pipelines by iteratively assembling and parameterizing computer vision functions. The pipelines thus generated exhibit comparable precision to state-of-the-art Deep Learning approaches on instance segmentation tasks, while requiring drastically smaller training datasets. This Few-Shot Learning method confers tremendous flexibility, speed, and functionality to this approach. We then deploy Kartezio to solve a series of semantic and instance segmentation problems, and demonstrate its utility across diverse images ranging from multiplexed tissue histopathology images to high resolution microscopy images. While the flexibility, robustness and practical utility of Kartezio make this fully explicable evolutionary designer a potential game-changer in the field of biomedical image processing, Kartezio remains complementary and potentially auxiliary to mainstream Deep Learning approaches.

Efficacy and safety of crizotinib in ALK-positive systemic anaplastic large-cell lymphoma in children, adolescents, and adult patients: results of the French AcSé-crizotinib trial.

BACKGROUND: The French phase II AcSé-crizotinib trial aimed to evaluate the safety and efficacy of crizotinib in patients with ALK, ROS1, and MET-driven malignancies, including ALK-positive anaplastic large-cell lymphoma (ALK+ ALCL). METHODS: ALK+ ALCL patients 12 months or older with measurable disease and no standard care options available received crizotinib twice daily at 165 mg/m2 in children and adolescents and 250 mg in adults. The primary end-point was the response rate at 8 weeks. RESULTS: Twenty-eight patients were enroled between February 2014 and March 2018. Three patients who were not treated were excluded from the analysis. The median age was 19 years. The median previous line of chemotherapy was two. In the 24 patients with an evaluable response, the response rate at 8 weeks was 67% (95% CI: 47-82%). All patients discontinued crizotinib after a median treatment duration of 3.7 months: eight for progression, two for adverse events (AEs) related to prior treatments, and 15 by choice, including six for allogeneic stem-cell transplantation. The median follow-up was 45 months. Nine patients experienced an event: eight relapses (seven after crizotinib discontinuation and one after dose reduction), and one died in complete remission. The median duration of response was 43.3 months (95% CI: 8.3-not reached). The 3-year progression-free and overall survival rates were 40% (95% CI: 23-59%) and 63% (95% CI: 43-79%). Grade 3 or 4 treatment-related AEs occurred in 32% of patients. CONCLUSION: Crizotinib shows efficacy and an acceptable safety profile in ALK+ ALCL relapsed/refractory patients. However, a large proportion of patients experience a relapse after crizotinib discontinuation. Future studies will assess if prolonged ALK inhibitor exposure has curative potential without consolidation.

3D deconvolution of human skin immune architecture with Multiplex Annotated Tissue Imaging System.

Routine clinical assays, such as conventional immunohistochemistry, often fail to resolve the regional heterogeneity of complex inflammatory skin conditions. We introduce MANTIS (Multiplex Annotated Tissue Imaging System), a flexible analytic pipeline compatible with routine practice, specifically designed for spatially resolved immune phenotyping of the skin in experimental or clinical samples. On the basis of phenotype attribution matrices coupled to α-shape algorithms, MANTIS projects a representative digital immune landscape while enabling automated detection of major inflammatory clusters and concomitant single-cell data quantification of biomarkers. We observed that severe pathological lesions from systemic lupus erythematosus, Kawasaki syndrome, or COVID-19-associated skin manifestations share common quantitative immune features while displaying a nonrandom distribution of cells with the formation of disease-specific dermal immune structures. Given its accuracy and flexibility, MANTIS is designed to solve the spatial organization of complex immune environments to better apprehend the pathophysiology of skin manifestations.

Improving outcomes of childhood and young adult non-Hodgkin lymphoma: 25 years of research and collaboration within the framework of the European Intergroup for Childhood Non-Hodgkin Lymphoma.

The European Intergroup for Childhood Non-Hodgkin Lymphoma (EICNHL) was established 25 years ago with the goal to facilitate clinical trials and research collaborations in the field both within Europe and worldwide. Since its inception, much progress has been made whereby major improvements in outcomes have been achieved. In this Review, we describe the different diagnostic entities of non-Hodgkin lymphoma in children and young adults describing key features of each entity and outlining clinical achievements made in the context of the EICNHL framework. Furthermore, we provide an overview of advances in biopathology with an emphasis on the role of biological studies and how they have shaped available treatments. Finally, for each entity, we describe future goals, upcoming clinical trials, and highlight areas of research that require our focus going forward.

Epigenetically regulated PCDHB15 impairs aggressiveness of metastatic melanoma cells.

The protocadherin proteins are cell adhesion molecules at the crossroad of signaling pathways playing a major role in neuronal development. It is now understood that their role as signaling hubs is not only important for the normal physiology of cells but also for the regulation of hallmarks of cancerogenesis. Importantly, protocadherins form a cluster of genes that are regulated by DNA methylation. We have identified for the first time that PCDHB15 gene is DNA-hypermethylated on its unique exon in the metastatic melanoma-derived cell lines and patients' metastases compared to primary tumors. This DNA hypermethylation silences the gene, and treatment with the DNA demethylating agent 5-aza-2'-deoxycytidine reinduces its expression. We explored the role of PCDHB15 in melanoma aggressiveness and showed that overexpression impairs invasiveness and aggregation of metastatic melanoma cells in vitro and formation of lung metastasis in vivo. These findings highlight important modifications of the methylation of the PCDHß genes in melanoma and support a functional role of PCDHB15 silencing in melanoma aggressiveness.

DNA methylome combined with chromosome cluster-oriented analysis provides an early signature for cutaneous melanoma aggressiveness.

Aberrant DNA methylation is a well-known feature of tumours and has been associated with metastatic melanoma. However, since melanoma cells are highly heterogeneous, it has been challenging to use affected genes to predict tumour aggressiveness, metastatic evolution, and patients' outcomes. We hypothesized that common aggressive hypermethylation signatures should emerge early in tumorigenesis and should be shared in aggressive cells, independent of the physiological context under which this trait arises. We compared paired melanoma cell lines with the following properties: (i) each pair comprises one aggressive counterpart and its parental cell line and (ii) the aggressive cell lines were each obtained from different host and their environment (human, rat, and mouse), though starting from the same parent cell line. Next, we developed a multi-step genomic pipeline that combines the DNA methylome profile with a chromosome cluster-oriented analysis. A total of 229 differentially hypermethylated genes was commonly found in the aggressive cell lines. Genome localization analysis revealed hypermethylation peaks and clusters, identifying eight hypermethylated gene promoters for validation in tissues from melanoma patients. Five Cytosine-phosphate-Guanine (CpGs) identified in primary melanoma tissues were transformed into a DNA methylation score that can predict survival (log-rank test, p=0.0008). This strategy is potentially universally applicable to other diseases involving DNA methylation alterations.

Harmonization of programmed death-ligand 1 immunohistochemistry and mRNA expression scoring in metastatic melanoma: a multicentre analysis.

AIMS: The aim of this multicentre study was to harmonize programmed death-ligand 1 (PD-L1) immunohistochemistry (IHC) and melanoma scoring. To provide a reference for PD-L1 expression independently of the IHC protocol, PD-L1 mRNA expression was compared with IHC. METHODS AND RESULTS: Standardized PD-L1 assays (22C3, 28-8, SP142, SP263) and laboratory-developed tests (QR1, 22C3) were evaluated on three IHC platforms with a training set (seven cases). mRNA expression was determined by RNAscope (CD274/PD-L1 probe) and analysed with image analysis. PD-L1 IHC findings were scored by seven blinded pathologists using the tumour proportion score (TPS), the combined positive score (CPS), and the MELscore. This method was validated by three blinded pathologists on 40 metastatic melanomas. Concordances among various antibody/platforms were high across antibodies [intraclass correlation coefficient (ICC) >0.80 for the CPS], except for SP142. Two levels of immunostaining intensity were observed: high (QR1 and SP263) and low (28-8, 22C3, and SP142). Reproducibilities across pathologists were higher for QR1 and SP263 (ICC ≥0.87 and ICC ≥0.85 for the TPS and the CPS, respectively). QR1, SP263 and 28-8 showed the highest concordance with mRNA expression. We developed a standardized method for PD-L1 immunodetection and scoring, tested on 40 metastatic melanomas. Concordances among antibodies were excellent for all criteria, and concordances among pathologists were better for the MELscore than for other scores. CONCLUSION: Harmonization of PD-L1 staining and scoring in melanomas with good concordance is achievable with the PD-L1 IHC protocols applied to other cancers; this reproducible approach can simplify daily practice.

De novo generation of the NPM-ALK fusion recapitulates the pleiotropic phenotypes of ALK+ ALCL pathogenesis and reveals the ROR2 receptor as target for tumor cells.

BACKGROUND: Anaplastic large cell lymphoma positive for ALK (ALK+ ALCL) is a rare type of non-Hodgkin lymphoma. This lymphoma is caused by chromosomal translocations involving the anaplastic lymphoma kinase gene (ALK). In this study, we aimed to identify mechanisms of transformation and therapeutic targets by generating a model of ALK+ ALCL lymphomagenesis ab initio with the specific NPM-ALK fusion. METHODS: We performed CRISPR/Cas9-mediated genome editing of the NPM-ALK chromosomal translocation in primary human activated T lymphocytes. RESULTS: Both CD4+ and CD8+ NPM-ALK-edited T lymphocytes showed rapid and reproducible competitive advantage in culture and led to in vivo disease development with nodal and extra-nodal features. Murine tumors displayed the phenotypic diversity observed in ALK+ ALCL patients, including CD4+ and CD8+ lymphomas. Assessment of transcriptome data from models and patients revealed global activation of the WNT signaling pathway, including both canonical and non-canonical pathways, during ALK+ ALCL lymphomagenesis. Specifically, we found that the WNT signaling cell surface receptor ROR2 represented a robust and genuine marker of all ALK+ ALCL patient tumor samples. CONCLUSIONS: In this study, ab initio modeling of the ALK+ ALCL chromosomal translocation in mature T lymphocytes enabled the identification of new therapeutic targets. As ROR2 targeting approaches for other cancers are under development (including lung and ovarian tumors), our findings suggest that ALK+ ALCL cases with resistance to current therapies may also benefit from ROR2 targeting strategies.

Ultrarapid lytic granule release from CTLs activates Ca2+-dependent synaptic resistance pathways in melanoma cells.

Human cytotoxic T lymphocytes (CTLs) exhibit ultrarapid lytic granule secretion, but whether melanoma cells mobilize defense mechanisms with commensurate rapidity remains unknown. We used single-cell time-lapse microscopy to offer high spatiotemporal resolution analyses of subcellular events in melanoma cells upon CTL attack. Target cell perforation initiated an intracellular Ca2+ wave that propagated outward from the synapse within milliseconds and triggered lysosomal mobilization to the synapse, facilitating membrane repair and conferring resistance to CTL induced cytotoxicity. Inhibition of Ca2+ flux and silencing of synaptotagmin VII limited synaptic lysosomal exposure and enhanced cytotoxicity. Multiplexed immunohistochemistry of patient melanoma nodules combined with automated image analysis showed that melanoma cells facing CD8+ CTLs in the tumor periphery or peritumoral area exhibited significant lysosomal enrichment. Our results identified synaptic Ca2+ entry as the definitive trigger for lysosomal deployment to the synapse upon CTL attack and highlighted an unpredicted defensive topology of lysosome distribution in melanoma nodules.

Peripheral neuropathy and livedoid vasculopathy.

BACKGROUND: Livedoid vasculopathy (LV) is a chronic dermatosis associated with micro-thrombosis of the vessels of the dermis, leading to ischemic lesions and painful skin ulcerations of the lower limbs. This thrombosing occlusive vasculopathy, clearly distinct from 'classical vasculitis' (not related to alteration of vessel walls), may lead to peripheral neuropathy. OBJECTIVE: To clarify the main clinical, electrophysiological and pathological characteristics of peripheral neuropathy linked to LV. METHOD: We presented a series of personal cases of peripheral neuropathy due to LV. We also conducted a review of the literature (since the first description of LV in 1974) using multiple combinations of keywords from 'PubMed', 'Google Scholar' and 'ScienceDirect' databases according to the 'Preferred Reporting Items for Systematic reviews and Meta-Analyses' guidelines. RESULTS: We identified 16 patients (6 personal cases and 10 cases from the medical literature). Our personal cases were five females and one male, with a median age (at the onset of cutaneous signs of LV) of 38 (range 25-62). Several types of skin lesions of the lower limbs were observed. Median age at the onset of peripheral neuropathy symptoms was 48 years (range 29-66), with a main clinical and electrophysiological pattern of mononeuropathy multiplex. DISCUSSION: We observed a typical pattern of peripheral neuropathy, mostly mononeuropathy multiplex, whose pathophysiology might be related to occlusions of the small vessels of the nerves, as seen in the dermis. Moreover, LV may also be associated with other types of peripheral neuropathies (sometimes of autoimmune etiology) not directly related to the skin lesions. CONCLUSION: The 'ischemic form' of peripheral neuropathy linked to LV is mainly responsible for sensory disturbances (with multifocal distribution), sometimes for motor disturbances. This type of peripheral neuropathy has to be distinguished from 'classical vasculitic neuropathies' which are usually treated with antithrombotic therapies.

Impact of expert pathology review in skin adnexal carcinoma diagnosis: Analysis of 2573 patients from the French CARADERM network.

PURPOSE: To prospectively assess the impact of expert pathological review of skin adnexal carcinoma diagnosis in France. METHODS: From 2014 to 2019, 2573 samples from patients with newly diagnosed or suspected skin adnexal carcinomas were reviewed prospectively by expert pathologists through the national CARADERM (CAncers RAres DERMatologiques) network. Changes in diagnosis between referral and expert review were analysed regarding their potential impact on patient care or prognosis. RESULTS: The samples comprised 2205 newly diagnosed adnexal carcinomas, 129 benign adnexal tumours, 136 basal cell carcinomas, 74 squamous cell carcinomas, six cutaneous metastases and 13 other malignancies. There were 930 (42%) sweat gland carcinomas, of which porocarcinoma (261; 11.8%), microcystic adnexal carcinoma (125; 5.7%) and hidradenocarcinoma (109; 4.9%) were the most frequent subtypes; 778 (35%) hair follicle carcinomas, 238 (11%) sebaceous carcinomas and 212 (10%) extramammary Paget diseases/mammary-like anogenital gland adenocarcinomas. A diagnostic change between referral and expert review occurred in 503 (21.3%) patients, significantly higher for cases sent with a provisional diagnosis seeking an expert second opinion (45.7%) than for cases sent with a formal diagnosis (2.8%) (p < .0001). Sweat gland carcinomas were more prone to diagnostic discrepancies than other tumours (p < .0001), including 1.8% of patients with sweat gland carcinoma subtype misclassification with predicted clinical impact. Changes between benign and malignant conditions occurred in 117 samples (5% of patients). CONCLUSION: The study provides a unique description of the distribution of skin adnexal carcinomas and highlights the importance of expert review for these rare cancers. Optimal clinical management was impacted in a significant proportion of patients.

Recurrent FOXK1::GRHL and GPS2::GRHL fusions in trichogerminoma.

We report 21 cases of trichogerminoma harbouring previously undescribed FOXK1::GRHL1/2 or GPS2::GRHL1/2/3 in-frame fusion transcripts. Microscopic examination of a preliminary set of five cases revealed well-delimitated tumours located in the dermis with frequent extension to the subcutaneous tissue. Tumours presented a massive and nodular architecture and consisted of a proliferation of basaloid cells. A biphasic pattern sometime resulting in tumour cell nests ('cell balls') was present. Immunohistochemistry demonstrated the expression of cytokeratins (CKs) 15, 17, and PHLDA1. In addition, numerous CK20-positive Merkel cells were detected. RNA sequencing (RNA-seq) revealed a FOXK1::GRHL1 chimeric transcript in three cases and a FOXK1::GRHL2 fusion in two cases. In a second series for validation (n = 88), FOXK1::GRHL1/2 fusion transcripts were detected by RT-qPCR or FISH in an additional 12 trichogerminomas and not in any other follicular tumour entities or basal cell carcinoma cases (n = 66). Additional RNA-seq analysis in trichogerminoma cases without detected FOXK1::GRHL1/2 rearrangements revealed GPS2::GRHL1 fusion transcripts in two cases, GPS2::GRHL2 in one case, and GPS2::GRHL3 fusion transcript in one case. Therefore, our study strongly suggests that GRHL1/2/3 gene rearrangements might represent the oncogenic driver in trichogerminoma, a subset of follicular tumours characterized by immature features and numerous Merkel cells. © 2022 The Pathological Society of Great Britain and Ireland.

CD158k and PD-1 expressions define heterogeneous subtypes of Sezary syndrome.

Sezary syndrome (SS) is a rare leukemic form of cutaneous T-cell lymphoma. Diagnosis mainly depends on flow cytometry, but results are not specific enough to be unequivocal. The difficulty in defining a single marker that could characterize Sezary cells may be the consequence of different pathological subtypes. In this study, we used multivariate flow cytometry analyses. We chose to investigate the expression of classical CD3, CD4, CD7, and CD26 and the new association of 2 markers CD158k and PD-1. We performed lymphocyte computational phenotypic analyses during diagnosis and follow-up of patients with SS to define new SS classes and improve the sensitivity of the diagnosis and the follow-up flow cytometry method. Three classes of SS, defined by different immunophenotypic profiles, CD158k+ SS, CD158k-PD-1+ SS, CD158k and PD-1 double-negative SS, showed different CD8+ and B-cell environments. Such a study could help to diagnose and define biological markers of susceptibility/resistance to treatment, including immunotherapy.

Gene Expression Signature Associated with Clinical Outcome in ALK-Positive Anaplastic Large Cell Lymphoma.

Anaplastic large cell lymphomas associated with ALK translocation have a good outcome after CHOP treatment; however, the 2-year relapse rate remains at 30%. Microarray gene-expression profiling of 48 samples obtained at diagnosis was used to identify 47 genes that were differentially expressed between patients with early relapse/progression and no relapse. In the relapsing group, the most significant overrepresented genes were related to the regulation of the immune response and T-cell activation while those in the non-relapsing group were involved in the extracellular matrix. Fluidigm technology gave concordant results for 29 genes, of which FN1, FAM179A, and SLC40A1 had the strongest predictive power after logistic regression and two classification algorithms. In parallel with 39 samples, we used a Kallisto/Sleuth pipeline to analyze RNA sequencing data and identified 20 genes common to the 28 genes validated by Fluidigm technology-notably, the FAM179A and FN1 genes. Interestingly, FN1 also belongs to the gene signature predicting longer survival in diffuse large B-cell lymphomas treated with CHOP. Thus, our molecular signatures indicate that the FN1 gene, a matrix key regulator, might also be involved in the prognosis and the therapeutic response in anaplastic lymphomas.

Molecular diagnosis of T-cell lymphoma: a correlative study of PCR-based T-cell clonality assessment and targeted NGS.

Immunomorphological diagnosis of T-cell lymphoma (TCL) may be challenging, especially on needle biopsies. Multiplex polymerase chain reaction (PCR) assays to assess T-cell receptor (TCR) gene rearrangements are now widely used to detect T-cell clones and provide diagnostic support. However, PCR assays detect only 80% of TCL, and clonal lymphocyte populations may also appear in nonneoplastic conditions. More recently, targeted next-generation sequencing (t-NGS) technologies have been deployed to improve lymphoma classification. To the best of our knowledge, the comparison of these techniques' performance in TCL diagnosis has not been reported yet. In this study, 82 TCL samples and 25 nonneoplastic T-cell infiltrates were divided into 2 cohorts (test and validation) and analyzed with both multiplex PCR and t-NGS to investigate TCR gene rearrangements and somatic mutations, respectively. The detection of mutations appeared to be more specific (100.0%) than T-cell clonality assessment (41.7%-45.5%), whereas no differences were observed in terms of sensitivity (95.1%-97.4%). Furthermore, t-NGS provided a reliable basis for TCL diagnosis in samples with partially degraded DNA that was impossible to assess with PCR. Finally, although multiplex PCR assays appeared to be less specific than t-NGS, both techniques remain complementary, as PCR recovered some t-NGS negative cases.

Annular lipoatrophy of the ankle: Four new pediatric cases and a review of the literature.

Annular lipoatrophy of the ankle is a rare and unique acquired lipoatrophic panniculitis that mainly affects children. There is no consensus on treatment, and the long-term course is not well known. We present four new pediatric cases that contribute to the understanding of this rare disease.

[Impact of algorithms proposed by the Cutaneous Lymphoma French Study Group for diagnosis of cutaneous lymphoproliferations]. / Impact des algorithmes du groupe français d'étude des lymphomes cutanés (GFELC) sur le diagnostic anatomopathologique des lymphoproliférations cutanées.

After a first diagnosis proposition, management of cutaneous lymphomas requires a systematic review by an expert pathologist and each case is presented to a multidisciplinary meeting in the setting of the French Study Group of Cutaneous Lymphomas to propose an adequate treatment. A retrospective study of the 2760 cutaneous lymphoproliferations retrieved between 2010 and 2011 were analyzed and demonstrated the interest of diagnostic algorithms we built with the group. The objective of our study was to compare two cohorts from 2010-2011 and 2015-2017 regarding the proportion of cases sent for validation or expertise, the concordance and mismatch rates and potential diagnostic issues using our diagnostic algorithms. Between 2015-2017, 5640 skin lymphoproliferation cases were examined. It appeared that Pathologists were more confident and effective in finding the right diagnosis. Indeed, the rate of concordant diagnosis increased from 57% to 67%. Moreover, in comparison with the 2010-2011 concordant cases sent for expertise, 73.5% of concordant cases were sent for validation in 2015-2017. 14% of cases remained discordant, mainly sent for expertise. Furthermore, half of questionable cases (26.3%) were resolved after expertise, and 12.1% cases remained unsolved. These priority cases are important to be presented at multidisciplinary meeting. The analysis of discordant and doubtful cases unveiled recurrent diagnostic problems for which we proposed appropriate diagnostic algorithms including large B cell lymphomas, CD4+ T cell lymphoproliferations, epidermotropic CD8+ T-cell lymphoproliferations and the differential diagnosis of mycosis fongoïdes/Sezary syndrome versus inflammatory dermatitis.

Should treatment of ALK-positive anaplastic large cell lymphoma be stratified according to minimal residual disease?

In anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALK+ ALCL), positive minimal residual disease (MRD+) after the first chemotherapy course was proven of strong prognostic significance. We aimed to validate these results in 138 French patients. Eighty-seven patients had a detectable minimal disseminated disease at diagnosis (MDD+). Early MRD assessment was performed in 33 of 87 patients and was positive in 18 and negative in 15 (MRD-). Three-year progression-free survival was significantly correlated with the MDD/MRD status: 81.1% in MDD-, 69.6% in MDD+/MRD-, and 15.2% in MDD+/MRD+ patients. In conclusion, we confirmed on an independent cohort that the MDD/MRD status has strong prognosis significance in ALK+ ALCL.