Severe neuromuscular forms of glycogen storage disease type IV: Histological, clinical, biochemical, and molecular findings in a large French case series.

Glycogen storage disease type IV (GSD IV), also called Andersen disease, or amylopectinosis, is a highly heterogeneous autosomal recessive disorder caused by a glycogen branching enzyme (GBE, 1,4-alpha-glucan branching enzyme) deficiency secondary to pathogenic variants on GBE1 gene. The incidence is evaluated to 1:600 000 to 1:800 000 of live births. GBE deficiency leads to an excessive deposition of structurally abnormal, amylopectin-like glycogen in affected tissues (liver, skeletal muscle, heart, nervous system, etc.). Diagnosis is often guided by histological findings and confirmed by GBE activity deficiency and molecular studies. Severe neuromuscular forms of GSD IV are very rare and of disastrous prognosis. Identification and characterization of these forms are important for genetic counseling for further pregnancies. Here we describe clinical, histological, enzymatic, and molecular findings of 10 cases from 8 families, the largest case series reported so far, of severe neuromuscular forms of GSD IV along with a literature review. Main antenatal features are: fetal akinesia deformation sequence or arthrogryposis/joint contractures often associated with muscle atrophy, decreased fetal movement, cystic hygroma, and/or hydrops fetalis. If pregnancy is carried to term, the main clinical features observed at birth are severe hypotonia and/or muscle atrophy, with the need for mechanical ventilation, cardiomyopathy, retrognathism, and arthrogryposis. All our patients were stillborn or died within 1 month of life. In addition, we identified five novel GBE1 variants.

The Presence of the Autophagic Markers LC3B and Sequestosome 1/p62 in the Hydatidiform Mole.

Autophagy is implicated in normal pregnancy and various pathologic pregnancy conditions. Its presence in hydatidiform moles (HM) is unknown. We immunohistochemically studied 36 HM for LC3B and p62 to precisely determine their expression in the decidua, endometrium, and villi. Nineteen nonmolar pregnancies were also studied. LC3B was found in almost half of the villi and p62 was found in almost all villi. LC3B expression was significantly higher in complete HM than in partial HM. LC3B showed different expression patterns in trophoblast layers. LC3B and p62 expression was higher in molar than nonmolar pregnancies. Autophagic markers are present in HM and their expression differs between complete and partial moles.

WHO grading system for invasive pulmonary lung adenocarcinoma reveals distinct molecular signature: An analysis from the cancer genome atlas database.

Lung adenocarcinoma grading has gained interest in the past years. Recently a three-tier tumor grading was proposed showing that it is related to patients' prognosis. Nevertheless, the underlying molecular basis of this morphological grading remains partly unknown. The aim of our work is to take advantage of The Cancer Genome Atlas lung adenocarcinoma (TCGA_LUAD) cohort to describe the molecular data associated to tumor grading. We performed a study on publicly available data of the TCGA database first by assessing a tumor grade on downloadable tumor slides. Secondly we analyzed the molecular features of each tumor grade group. Our work was performed on a study group of 449 patients. We show that aneuploidy score was significantly different between grade 2 and grade 3 groups with different chromosomal imbalance (p < 0.001). SCGB1A1 mRNA expression was higher in grade 2 (p = 0.0179) whereas NUP155, CHFR, POLQ and CDC7 have a higher expression in grade 3 (p = 0.0189, 0.0427, 0.0427 and 0.427 respectively). GZMB and KRT80 have a higher methylation of DNA in grade 2 (p = 0.0201 and 0.0359 respectively). MT1G, CLEC12B and NDUFA7 have a higher methylation of DNA in grade 3 (p < 0.001, 0.0246 and 0.0359 respectively). We showed that the number of activated pathways is different between grade 2 and grade 3 patients (p = 0.004). We showed that differentially expressed genes by mRNA analysis and DNA methylation analysis involve several genes implied in chemoresistance. This could suggest that grade 3 lung adenocarcinoma might be more resistant to chemotherapy.

The immune microenvironment of the hydatidiform mole.

Gestational trophoblastic diseases (GTDs) are a heterogeneous group of lesions, the most frequent being the hydatidiform mole (HM). HMs are usually cured after surgical treatment or after chemotherapy in the case of a persistent trophoblastic activity. Immunotherapy could be an interesting alternative as a first-line or second-line treatment. However, only a few studies have explored the immune microenvironment of HMs. In the present retrospective study including 19 complete and 17 partial moles, we examined the composition of the immune cell microenvironment by immunohistochemistry using the following antibodies: CD4, CD8, CD56, PD-L1, S100, CD83, CD207, CD123, CD1a, CD11c, CD163, PAX5, and MUM1. In the decidual cells compartment, CD11c+ cells were the predominant population, followed by CD4+ cells, CD56+ NK cells, CD163+ macrophages, and CD8+ T lymphocytes.In the endometrial glands compartment, CD11c+ cells were the predominant population, followed by CD4+ cells, CD56+ NK cells, and CD8+ T lymphocytes. In the villi compartment, the predominant immune cells were CD4+ cells, followed by CD163+ macrophages and CD11c+ cells. Statistically significant differences were observed between partial and complete moles in all three compartments. The immune microenvironment of HMs is immunosuppressive, but it differs between complete and partial moles, the latter having a higher infiltrate of cells with phenotypes suggestive of immunosuppressive activities.

Fetal megacystis-microcolon: Genetic mutational spectrum and identification of PDCL3 as a novel candidate gene.

Megacystis-microcolon-intestinal-hypoperistalsis syndrome (MMIHS) is a severe congenital visceral myopathy characterized by an abdominal distension due to a large non-obstructed urinary bladder, a microcolon and intestinal hypo- or aperistalsis. Most of the patients described to date carry a sporadic heterozygous variant in ACTG2. More recently, recessive forms have been reported and mutations in MYH11, LMOD1, MYLK and MYL9 have been described at the molecular level. In the present report, we describe five patients carrying a recurrent heterozygous variant in ACTG2. Exome sequencing performed in four families allowed us to identify the genetic cause in three. In two families, we identified variants in MMIHS causal genes, respectively a nonsense homozygous variant in MYH11 and a previously described homozygous deletion in MYL9. Finally, we identified compound heterozygous variants in a novel candidate gene, PDCL3, c.[143_144del];[380G>A], p.[(Tyr48Ter)];[(Cys127Tyr)]. After cDNA analysis, a complete absence of PDLC3 expression was observed in affected individuals, indicating that both mutated transcripts were unstable and prone to mediated mRNA decay. PDCL3 encodes a protein involved in the folding of actin, a key step in thin filament formation. Presumably, loss-of-function of this protein affects the contractility of smooth muscle tissues, making PDCL3 an excellent candidate gene for autosomal recessive forms of MMIHS.

The Uterine Plexiform Lesions Revisited.

OBJECTIVES: Uterine lesions with plexiform morphology are uncommon lesions with debated histogenesis. Despite being an incidental and usually benign finding (plexiform tumorlet), some cases can pose diagnostic problems. Their paucity in the recent literature adds to these difficulties and often causes ambiguities. The objective of this study is to systematically review published cases to highlight the historical aspects of their recognition, reappraising their morphology, histogenesis, and differential diagnosis. METHODS: English literature is reviewed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, and 32 reports are analyzed. RESULTS: Most cases are reported in the fourth to sixth decades. In most cases (66.7%), plexiform lesions are incidental findings while 33.3% of cases have been the chief pathology. Size varies from 0.5 to 195 mm. Plexiform foci were solitary in 78.2% cases and multiple in 21.8%. In 67.8% of cases, the lesions are reported as myometrial, while 32.2% are arising from endometrial stroma. Immunohistochemistry shows smooth muscle and no sex cord marker expression. They are usually benign lesions, but worrisome features include plexiform morphology in disseminated peritoneal leiomyomatosis, intravenous leiomyomatosis, and diffuse uterine leiomyomatosis. CONCLUSIONS: Plexiform lesions represent a diverse pathology varying from epithelioid leiomyomas to epithelioid smooth muscle metaplasia of endometrial type of stroma.

Pathology of the Fallopian Tube: Tubal Involvement by Ovarian Tumors and Incidental Findings in the Nontumoral Setting.

The fallopian tube is thought to be the site of origin of most high-grade serous carcinomas (HGSCs). However, how often the tube is abnormal in the setting of other ovarian tumors is unknown. The aim of this study is to define the frequency of tubal abnormalities in the tumoral (n = 245) and nontumoral (n = 184) setting. We found that in ovarian tumors, 52.2% of the tubes were normal, while 39.2% were affected by the tumor. Abnormal tubes were found in 80% of HGSCs, in 21% of mucinous carcinomas, in 83.3% of seromucinous carcinomas, in 33.3% of endometrioid carcinomas, in 20% of clear-cell carcinomas, and in 10.5% of borderline tumors. Among normal tubes, almost 70% were histologically normal; transitional metaplasia was present in 17.4%, endometriosis in 8.1%, and adenofibroma in 2.2%, and 1.1% had an incidental serous intraepithelial tubal carcinoma. To conclude, the fallopian tube is abnormal in most serous carcinomas, and in a smaller number of endometrioid, clear-cell and mucinous carcinomas as well as borderline tumors. It is often abnormal in seromucinous tumors, but larger series are needed to study this rare subtype.

MLH1 promoter hypermethylation: are you absolutely sure about the absence of MLH1 germline mutation? About a new case.

Lynch syndrome accounts for 3-5% of colorectal cancers and is due to a germline mutation in one of the mismatch repair genes MLH1, MSH2, MSH6, and PMS2. Somatic hypermethylation of the MLH1 promoter is commonly associated to sporadic cases. Strategies have been developed to identify patients with Lynch Syndrome based on clinical findings, tumoral phenotype, family history and immunohistochemistry analysis. However, there still are some pitfalls in this strategy, possibly responsible for an underdiagnosis of Lynch syndrome. Here we report the case of a 37 years-old man presenting with two concomitant tumors located in the rectosigmoid and in the ileocecal angle. Both tumors were microsatellites instability-high (MSI-H) and showed a loss of MLH1 and PMS2 protein expression, but only one had MLH1 promoter hypermethylation. Constitutional analysis of mismatch repair genes could not be performed from a blood sample, because of the early death of the patient. However, tumoral tissue analyses revealed in both tumors a pathogenic variant in the MLH1 gene. Further analysis of the surrounding tumor-free tissue also showed the presence of this alteration of the MHL1 gene. Finally, the same pathogenic variant was present constitutionally in one of the siblings of the patient, confirming its hereditary nature. This new case of concomitant presence of MLH1 promoter hypermethylation and MLH1 germline mutation demonstrates that the presence of MLH1 promoter hypermethylation should not rule out the diagnosis of Lynch Syndrome.

Impact of delayed fixation and decalcification on PD-L1 expression: a comparison of two clones.

The bone is a frequent localization for lung non-small cell cancer metastasis; decalcification is required to permit tissue section. Pre-analytical conditions can influence the detection of immunohistochemical markers. The aim of our work is to evaluate PD-L1 expression in samples with delayed fixation and in decalcified tissue with chelating agent or acid at different time. Tumor-expressing PD-L1 and placental tissue were fixed at different times or decalcified with an acid decalcifier or EDTA for different durations. For 22C3 antibody, when tissues were decalcified with DC3, there was a significant decrease in the percentage of tumor cells or placental villi stained which after 4 h (p = 0.035 at 4 h). When EDTA is used for 22C3 antibody, there was a slight decrease in the percentage of stained tumor cells or villi but although there was a trend (p = 0.058 at 20 h), this was never statistically significant. For E1L3N antibody, when tissues were decalcified either with DC3 or EDTA, there was no significant decrease for the proportion of stained tumor cells or placental villi, neither for staining intensity for the first 24 h. The proportion of placental villi and tumor stained or intensity of staining was not significantly lower for any sample after delayed fixation also at 24 h for both PD-L1 clones. Delayed fixation does not affect the proportion of stained cell and intensity with PD-L1 immunohistochemistry. Decalcification also performed with EDTA lower the proportion and intensity of stained cells with PD-L1 immunohistochemistry.

Bardet-Biedl syndrome: Antenatal presentation of forty-five fetuses with biallelic pathogenic variants in known Bardet-Biedl syndrome genes.

Bardet-Biedl syndrome (BBS) is an emblematic ciliopathy associated with retinal dystrophy, obesity, postaxial polydactyly, learning disabilities, hypogonadism and renal dysfunction. Before birth, enlarged/cystic kidneys as well as polydactyly are the hallmark signs of BBS to consider in absence of familial history. However, these findings are not specific to BBS, raising the problem of differential diagnoses and prognosis. Molecular diagnosis during pregnancies remains a timely challenge for this heterogeneous disease (22 known genes). We report here the largest cohort of BBS fetuses to better characterize the antenatal presentation. Prenatal ultrasound (US) and/or autopsy data from 74 fetuses with putative BBS diagnosis were collected out of which molecular diagnosis was established in 51 cases, mainly in BBS genes (45 cases) following the classical gene distribution, but also in other ciliopathy genes (6 cases). Based on this, an updated diagnostic decision tree is proposed. No genotype/phenotype correlation could be established but postaxial polydactyly (82%) and renal cysts (78%) were the most prevalent symptoms. However, autopsy revealed polydactyly that was missed by prenatal US in 55% of the cases. Polydactyly must be carefully looked for in pregnancies with apparently isolated renal anomalies in fetuses.

EGFR, KRAS, BRAF and HER2 testing in metastatic lung adenocarcinoma: Value of testing on samples with poor specimen adequacy and analysis of discrepancies.

Molecular testing on metastatic lung adenocarcinoma or on non-small cell non-squamous lung carcinoma often relies on small specimen. In this group of patient with poor specimen adequacy, we analyzed the rate of EGFR, KRAS, BRAF and HER2 mutations compared to their rate in optimal specimen. We analyzed discrepancies in molecular testing results in patients with iterative analysis on several samples. We performed a retrospective study of 1538 samples consecutively analyzed. 263/665 (39,5%) biopsies and 37/708 (5,2%) surgical specimens were considered as samples with poor specimen adequacy (p<0,0001). A lower tumor cell content was associated with a lower rate of KRAS mutation: 15,8% in samples with <10% of tumor cells or <100 tumor cells versus 29,8% in samples with >10% tumor cell and >100 tumor cells (p=0,001). KRAS mutational rate was at 11,1% in cytology specimens, significantly lower than in biopsy or surgical specimens respectively at 28,2% and 28,5% (p=0,0002). Tumor cell content was not associated with mutational rate for EGFR, BRAF and HER2 mutations. DNA quantity was not associated with mutational rate for EGFR, KRAS, BRAF and HER2. A discrepancy in molecular testing was found in 16 patients. For 5 patients there was also a discrepancy for TTF-1 expression. On the 11 without TTF-1 discrepancy, specimen adequacy was not fulfilled in 10 cases at least for tumor content. Discrepancies were found in the case of low cellularity, poor cell content or testing on cytological specimens. Tumor cell content is a crucial parameter for molecular analysis rather than the type of specimen or the DNA quantity. Discrepancies in molecular testing results are rare but might suggest the presence of another tumor type, the emergence of another clone or a molecular testing in a sample with low cell content.

Confirmation that RIPK4 mutations cause not only Bartsocas-Papas syndrome but also CHAND syndrome.

CHAND syndrome is an autosomal recessive disorder characterized by curly hair, ankyloblepharon, and nail dysplasia. Only few patients were reported to date. A homozygous RIPK4 mutation was recently identified by homozygosity mapping and whole exome sequencing in three patients from an expanded consanguineous kindred with a clinical diagnosis of CHAND syndrome. RIPK4 was previously known to be implicated in Bartsocas-Papas syndrome, the autosomal recessive form of popliteal pterygium syndrome. We report here two cases of RIPK4 homozygous mutations in a fetus with severe Bartsocas-Papas syndrome and a patient with CHAND syndrome. The patient with CHAND syndrome harbored the same mutation as the one identified in the family previously reported. We thus confirm the implication of RIPK4 gene in CHAND syndrome in addition to Bartsocas-Papas syndrome and discuss genotype/phenotype correlations.

A case report of Muir-Torre syndrome in a woman with breast cancer and MSI-Low skin squamous cell carcinoma.

BACKGROUND: The tumor spectrum in the Lynch syndrome is well defined, comprising an increased risk of developing colonic and extracolonic malignancies. Muir-Torre syndrome is a variant with a higher risk of skin disease. Patients have been described carrying mutations in the mismatch repair genes and presenting tumors with unusual histology or affected organ not part of the Lynch syndrome spectrum. Hence, the real link between Lynch syndrome, or Muir-Torre syndrome, and these tumors remains difficult to assess. CASE PRESENTATION: We present the case of a 45-year-old-woman, diagnosed with breast cancer at 39 years of age and skin squamous cell carcinoma (SCC) at 41 years of age, without personal history of colorectal cancer. The microsatellite instability analysis performed on the skin SCC showed a low-level of microsatellite instability (MSI-Low). The immunohistochemical expression analysis of the four DNA mismatch repair proteins MLH1, MSH2, MSH6 and PMS2 showed a partial loss of the expression of MSH2 and MSH6 proteins. Germline deletion was found in MSH2 gene (c.1277-? _1661 + ?del), exon 8 to 10. Then, at 45 years of age, she presented hyperplastic polyps of the colon and a sebaceous adenoma. CONCLUSION: Squamous cell carcinomas have been described in Lynch syndrome and Muir-Torre syndrome in two studies and two case reports. This new case further supports a possible relationship between Lynch syndrome and squamous cell carcinoma.

Basic Molecular Pathology and Cytogenetics for Practicing Pathologists: Correlation With Morphology and With a Focus on Aspects of Diagnostic or Therapeutic Utility.

Morphology, as confronted in the everyday practice, often correlates with specific molecular features, which have important implications not only in pathogenesis and in diagnosis but also in prognosis and therapy. Thus, it is important that the classical pathology includes a sound knowledge of molecular aspects of disease. These molecular concepts are complex and not easily understood by all engaged in the routine practice of histopathology. Thus, the aim of this review is to present a summary of most of the necessary concepts for pathologists involving molecular pathology and genetics, beginning from basic definitions and mechanisms to major abnormalities and the methodology to detect them, correlating at the same time, the specific morphologic features associated with every abnormality.

Fetal anomalies associated with HNF1B mutations: report of 20 autopsy cases.

OBJECTIVES: To describe macroscopic and microscopic anomalies present in fetuses carrying hepatocyte nuclear factor-1 ß mutation, their frequency, and genotype/phenotype correlations. METHODS: Clinical data, ultrasound findings, genetic studies, and autopsy reports of 20 fetal autopsies were analyzed. Histology was reviewed by two pathologists. RESULTS: Macroscopic findings were typically unilateral or bilateral renal enlargement and cortical cysts. Renal lesions were associated with congenital anomalies of the kidney and urinary tract in 25% of cases. Microscopic renal anomalies were dominated by glomerulocystic kidney and renal dysplasia. Extra-renal manifestations such as pancreatic hypoplasia (75%) and genital anomalies (68%) were only detected at autopsy. In 40% of cases, there was heterozygous deletion of the whole gene. There were de novo mutations in 40%. CONCLUSION: This study underlines the importance of considering hepatocyte nuclear factor-1 ß mutations in fetuses with congenital anomalies of the kidney and urinary tract, especially when associated with pancreatic hypoplasia. No correlation between phenotype and genotype was found, highlighting high intra-familial variability in cases with inherited mutations. © 2016 John Wiley & Sons, Ltd.

Morphological and immunohistochemical analysis in ovaries and fallopian tubes of tamoxifen, letrozole and clomiphene-treated rats.

PURPOSE: Ovarian and tubal dysplasia may be precursors to ovarian cancer. The goal of this study was to check whether these histopathological lesions would be found after ovulation induction using tamoxifen, clomiphene citrate and letrozole. METHODS: Seventy-two rats were divided into four groups. In the first group, 24 rats received normal saline. The second group (16 rats) received clomiphene citrate for six cycles. The third group, divided into two sub-groups of eight rats each, were stimulated with tamoxifen for six cycles, with a dosage, respectively, of 0.4 and 0.8 mg/kg/day. In the last group, eight rats received letrozole 0.1 mg/kg/day and eight other rats received letrozole 0.5 mg/kg/day, for six cycles. Once the six cycles had been completed the rats were killed in order to remove ovaries and tubes for histopathological analysis (morphological, p53 and Ki67 immunohistochemical assessment). RESULTS: Histopathological lesions were found in both ovaries and tubes. The mean ovarian dysplasia score was significantly higher in the tamoxifen group whatever the dosage (p = 0.006 and 0.0002) and in the letrozole group with 0.5 mg/kg/day (p = 0.0002) compared with the control group. The mean tubal dysplasia score was significantly higher in all groups that received drug treatment compared with the control group, whatever the dosage used. The proliferation index (Ki67) was significantly higher in the tamoxifen and letrozole groups while no significant difference was found for apoptosis marker p53. CONCLUSIONS: Ovulation induction may induce histopathological abnormalities in ovaries and tubes with a different immunohistochemical profile in comparison with salpingo-oophorectomies for genetic risk.

Tubo-ovarian dysplasia in relationship with ovulation induction in rats.

OBJECTIVE: To assess tubo-ovarian dysplasia via morphologic and immunohistochemical study of rats exposed to ovulation stimulation protocols. DESIGN: Animal experimental study. SETTING: Academic research hospital. ANIMAL(S): 72 female Wistar rats divided into three groups. INTERVENTION(S): Stimulation protocols using follicle-stimulating hormone (FSH) or clomiphene citrate for 3, 6, or 12 cycles, after which the animals were killed. MAIN OUTCOME MEASURE(S): Ovarian and tubal dysplasia score and immunohistochemical assessment using p53 and Ki67. RESULT(S): The ovarian dysplasia score was statistically significantly higher after 12 stimulation cycles in the groups receiving FSH (group B) or clomiphene citrate (group C) compared with control (group A). The tubal dysplasia score was statistically significantly increased after only three stimulation cycles in groups B and C. The Ki67 proliferation marker was statistically significantly expressed in the ovaries from group C, and in the fallopian tubes from groups B and C. P53 was constantly low in all three groups. CONCLUSION(S): Ovulation stimulation may induce tubal and ovarian histopathologic and immunohistochemical abnormalities with a dose effect. The role of the fallopian tubes and their interaction with the ovaries require further study.

A mucoepidermoid carcinoma in a young man with intellectual disability: review of oral cancer in people with intellectual disability.

Oral tumors in patients with intellectual disabilities (ID) remain poorly documented, despite cancer incidence suggesting that malignancies are globally as frequent in this group as in the general population. A clinical case of a 36-year-old man with severe ID presenting with a mucoepidermoid carcinoma of intermediate grade in the right mandible is reported. Delayed diagnosis and problems managing complementary chemotherapy and radiotherapy are described. The literature review reported only 27 cases of malignant tumors in patients with ID. This finding indicates that oral tumors in patients with ID may be less frequent than in the general population, are usually diagnosed at an advanced stage, and may occur in patients who are younger than the general population. Diagnosis and treatment are difficult, implying a comprehensive knowledge of the underlying condition of each individual and the need for good communication skills to obtain patient cooperation, including an understanding of how the patient expresses pain.

Identification of mutations in TMEM5 and ISPD as a cause of severe cobblestone lissencephaly.

Cobblestone lissencephaly is a peculiar brain malformation with characteristic radiological anomalies. It is defined as cortical dysplasia that results when neuroglial overmigration into the arachnoid space forms an extracortical layer that produces agyria and/or a "cobblestone" brain surface and ventricular enlargement. Cobblestone lissencephaly is pathognomonic of a continuum of autosomal-recessive diseases characterized by cerebral, ocular, and muscular deficits. These include Walker-Warburg syndrome, muscle-eye-brain disease, and Fukuyama muscular dystrophy. Mutations in POMT1, POMT2, POMGNT1, LARGE, FKTN, and FKRP identified these diseases as alpha-dystroglycanopathies. Our exhaustive screening of these six genes, in a cohort of 90 fetal cases, led to the identification of a mutation in only 53% of the families, suggesting that other genes might also be involved. We therefore decided to perform a genome-wide study in two multiplex families. This allowed us to identify two additional genes: TMEM5 and ISPD. Because TMEM has a glycosyltransferase domain and ISPD has an isoprenoid synthase domain characteristic of nucleotide diP-sugar transferases, these two proteins are thought to be involved in the glycosylation of dystroglycan. Further screening of 40 families with cobblestone lissencephaly identified nonsense and frameshift mutations in another four unrelated cases for each gene, increasing the mutational rate to 64% in our cohort. All these cases displayed a severe phenotype of cobblestone lissencephaly A. TMEM5 mutations were frequently associated with gonadal dysgenesis and neural tube defects, and ISPD mutations were frequently associated with brain vascular anomalies.