Melanoma clonal heterogeneity leads to secondary resistance after adoptive cell therapy with tumor-infiltrating lymphocytes.

Adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TIL) is effective in melanoma patients, although long-term responses seem restricted to patients who have complete remissions. Many patients develop secondary resistance to TIL-ACT but the involved mechanisms are unclear. Here, we describe a case of secondary resistance to TIL-ACT likely due to intratumoral heterogeneity and selection of a resistant tumor cell clone by the transferred T cells. To our knowledge, this is the first case of clonal selection of a pre-existing non-dominant tumor cell clone and it demonstrates a mechanism involved in secondary resistance to TIL-ACT that could potentially change current clinical practice, because it advocates for T-cell collection from multiple tumor sites and analysis of tumor heterogeneity before the treatment with TIL-ACT.

Morphologic and molecular analysis of liver injury after SARS-CoV-2 vaccination reveals distinct characteristics.

BACKGROUND & AIMS: Liver injury after COVID-19 vaccination is very rare and shows clinical and histomorphological similarities with autoimmune hepatitis (AIH). Little is known about the pathophysiology of COVID-19 vaccine-induced liver injury (VILI) and its relationship to AIH. Therefore, we compared VILI with AIH. METHODS: Formalin-fixed and paraffin-embedded liver biopsy samples from patients with VILI (n = 6) and from patients with an initial diagnosis of AIH (n = 9) were included. Both cohorts were compared by histomorphological evaluation, whole-transcriptome and spatial transcriptome sequencing, multiplex immunofluorescence, and immune repertoire sequencing. RESULTS: Histomorphology was similar in both cohorts but showed more pronounced centrilobular necrosis in VILI. Gene expression profiling showed that mitochondrial metabolism and oxidative stress-related pathways were more and interferon response pathways were less enriched in VILI. Multiplex analysis revealed that inflammation in VILI was dominated by CD8+ effector T cells, similar to drug-induced autoimmune-like hepatitis. In contrast, AIH showed a dominance of CD4+ effector T cells and CD79a+ B and plasma cells. T-cell receptor (TCR) and B-cell receptor sequencing showed that T and B cell clones were more dominant in VILI than in AIH. In addition, many T cell clones detected in the liver were also found in the blood. Interestingly, analysis of TCR beta chain and Ig heavy chain variable-joining gene usage further showed that TRBV6-1, TRBV5-1, TRBV7-6, and IgHV1-24 genes are used differently in VILI than in AIH. CONCLUSIONS: Our analyses support that SARS-CoV-2 VILI is related to AIH but also shows distinct differences from AIH in histomorphology, pathway activation, cellular immune infiltrates, and TCR usage. Therefore, VILI may be a separate entity, which is distinct from AIH and more closely related to drug-induced autoimmune-like hepatitis. IMPACT AND IMPLICATIONS: Little is known about the pathophysiology of COVID-19 vaccine-induced liver injury (VILI). Our analysis shows that COVID-19 VILI shares some similarities with autoimmune hepatitis, but also has distinct differences such as increased activation of metabolic pathways, a more prominent CD8+ T cell infiltrate, and an oligoclonal T and B cell response. Our findings suggest that VILI is a distinct disease entity. Therefore, there is a good chance that many patients with COVID-19 VILI will recover completely and will not develop long-term autoimmune hepatitis.

Characterization of the Subcellular Distribution of Phospho-ß-catenin in Colorectal Cancer.

BACKGROUND/AIM: ß-Catenin is a multifunctional protein, which is localized to different subcellular compartments of the normal colon epithelium. The hyperactivation of Wnt pathway results in the nuclear accumulation of ß-catenin and induction of colorectal carcinogenesis. Although N-terminally hypo-phosphorylated ß-catenin (active ß-catenin) is known as the transcriptionally active form, phospho-S33/S37/T41-ß-catenin (phospho-ß-catenin) can also accumulate in the nucleus. In this study, we aimed to characterize the subcellular distribution of phospho-ß-catenin and the other forms of ß-catenin in normal colon epithelium and colorectal cancer (CRC). MATERIALS AND METHODS: Phosphorylated, hypo-phosphorylated, and the total pool of ß-catenin were evaluated in colon epithelium and CRC using immunohistochemistry, immunofluorescence staining, and western blotting. Tissue microarrays were used to determine the expression pattern of phospho-ß-catenin in CRC samples. RESULTS: Almost 11% (49/452) of CRCs expressed moderate to high levels of phospho-ß-catenin in the nucleus. In addition, hypo-phosphorylated and phosphorylated forms of ß-catenin localized to different subcellular regions in normal colon epithelium and CRC. Immunoblotting experiments suggested that truncated phospho-ß-catenin forms can be found in CRCs. CONCLUSION: Phospho-ß-catenin accumulates in the nucleus and different molecular weight ß-catenin proteins are present in colon cancer cells. To elaborate on the functional significance of nuclear phospho-ß-catenin, further studies should be performed.

Role of Reduced Bdnf Expression in Novel Apc Mutant Allele-induced Intestinal and Colonic Tumorigenesis in Mice.

BACKGROUND/AIM: Brain-derived neurotrophic factor (BDNF) is a growth factor of the neurotrophin family. Recent studies indicate that its expression is regulated by Wnt/ß-catenin signaling. In this study, we aimed to examine the effects of reduced Bdnf levels in an Apc mutant intestinal/colonic tumor mouse model. MATERIALS AND METHODS: We crossed Apc+/- and Bdnf+/- C57BL/6 mice. After genotyping the litters, Apc+/+ Bdnf+/+ (wild-type, wt), Apc+/- Bdnf+/+ (Apc mutant), Apc+/+ Bdnf+/- (Bdnf mutant), and Apc+/- Bdnf+/- (Apc/Bdnf double mutant) mice cohorts were generated. All mice were followed daily for 36 weeks and weighed once a week, and mice that died or reached a terminal stage before this period were also recorded and dissected. At the end of this period, all surviving mice were sacrificed, and tissue samples were collected. Polyp numbers in the small intestine and colon were counted. Microscopic slides were prepared for histopathological examination. Protein extraction was performed both for tumor and normal tissue analysis. RESULTS: A significant weight gain was observed in the Bdnf mutant and Apc/Bdnf double mutant cohorts compared to wt and Apc mutant controls. In Apc/Bdnf double mutant mice, the small intestinal polyp count was slightly decreased, and the colon polyp count increased significantly, and developed the disease phenotype significantly later than Apc mutant mice. CONCLUSION: Bdnf level has an important role in the Apc mutant intestinal and colonic tumorigenesis model. Modulation of Bdnf levels can be a potential therapeutic target in colorectal cancer.

Genetic and epigenetic analysis of hepatocellular adenomas with atypical morphological features.

BACKGROUND: Hepatocellular adenoma (HCA) is a rare liver tumour, which can have atypical morphological features such as cytological atypia, pseudoglandular architecture, and altered reticulin framework. Little is known about the genetic and epigenetic alterations of such HCAs and whether they show the alterations classically found in hepatocellular carcinoma (HCC) or in HCA without atypical morphology. METHODS: We analysed five HCAs with atypical morphological features and one HCA with transition to HCC. Every tumour was subtyped by immunohistochemistry, sequenced by a targeted NGS panel, and analysed on a DNA methylation microarray. RESULTS: Subtyping of the five HCAs with atypical features revealed two ß-catenin mutated HCA (b-HCA), two ß-catenin mutated inflammatory HCA (b-IHCA), and one sonic hedgehog activated HCA (shHCA). None of them showed mutations typically found in HCC, such as, e.g. TERT or TP53 mutations. The epigenomic pattern of HCAs with atypical morphological features clustered with reference data for HCAs without atypical morphological features but not with HCC. Similarly, phyloepigenetic trees using the DNA methylation data reproducibly showed that HCAs with morphological atypia are much more similar to nonmalignant samples than to malignant samples. Finally, atypical HCAs showed no relevant copy number variations (CNV). CONCLUSION: In our series, mutational, DNA methylation, as well as CNV analyses, supported a relationship of atypical HCAs with nonatypical HCAs rather than with HCC. Therefore, in cases with difficult differential diagnosis between HCC and HCA, it might be advisable to perform targeted sequencing and/or combined methylation/copy number profiling.

Severe Neuro-COVID is associated with peripheral immune signatures, autoimmunity and neurodegeneration: a prospective cross-sectional study.

Growing evidence links COVID-19 with acute and long-term neurological dysfunction. However, the pathophysiological mechanisms resulting in central nervous system involvement remain unclear, posing both diagnostic and therapeutic challenges. Here we show outcomes of a cross-sectional clinical study (NCT04472013) including clinical and imaging data and corresponding multidimensional characterization of immune mediators in the cerebrospinal fluid (CSF) and plasma of patients belonging to different Neuro-COVID severity classes. The most prominent signs of severe Neuro-COVID are blood-brain barrier (BBB) impairment, elevated microglia activation markers and a polyclonal B cell response targeting self-antigens and non-self-antigens. COVID-19 patients show decreased regional brain volumes associating with specific CSF parameters, however, COVID-19 patients characterized by plasma cytokine storm are presenting with a non-inflammatory CSF profile. Post-acute COVID-19 syndrome strongly associates with a distinctive set of CSF and plasma mediators. Collectively, we identify several potentially actionable targets to prevent or intervene with the neurological consequences of SARS-CoV-2 infection.

miR-579-3p Controls Hepatocellular Carcinoma Formation by Regulating the Phosphoinositide 3-Kinase-Protein Kinase B Pathway in Chronically Inflamed Liver.

Chronic liver inflammation causes continuous liver damage with progressive liver fibrosis and cirrhosis, which may eventually lead to hepatocellular carcinoma (HCC). Whereas the 10-year incidence for HCC in patients with cirrhosis is approximately 20%, many of these patients remain tumor free for their entire lives. Clarifying the mechanisms that define the various outcomes of chronic liver inflammation is a key aspect in HCC research. In addition to a wide variety of contributing factors, microRNAs (miRNAs) have also been shown to be engaged in promoting liver cancer. Therefore, we wanted to characterize miRNAs that are involved in the development of HCC, and we designed a longitudinal study with formalin-fixed and paraffin-embedded liver biopsy samples from several pathology institutes from Switzerland. We examined the miRNA expression by nCounterNanostring technology in matched nontumoral liver tissue from patients developing HCC (n = 23) before and after HCC formation in the same patient. Patients with cirrhosis (n = 26) remaining tumor free within a similar time frame served as a control cohort. Comparison of the two cohorts revealed that liver tissue from patients developing HCC displayed a down-regulation of miR-579-3p as an early step in HCC development, which was further confirmed in a validation cohort. Correlation with messenger RNA expression profiles further revealed that miR-579-3p directly attenuated phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) expression and consequently protein kinase B (AKT) and phosphorylated AKT. In vitro experiments and the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology confirmed that miR-579-3p controlled cell proliferation and cell migration of liver cancer cell lines. Conclusion: Liver tissues from patients developing HCC revealed changes in miRNA expression. miR-579-3p was identified as a novel tumor suppressor regulating phosphoinositide 3-kinase-AKT signaling at the early stages of HCC development.

Impact of COVID-19 on eating habits, sleeping behaviour and physical activity status of final-year medical students in Ankara, Turkey.

OBJECTIVE: The massive increase in COVID-19 cases led to the implementation of nationwide lockdown in Turkey. While enforced lockdown is an effective strategy to abate the transmission of the virus, it causes significant lifestyle changes in every part of the community. This study aims to assess the changes in eating habits, sleeping behaviour and physical activity status of final-year medical students (interns) whose education was suspended during the pandemic. DESIGN: In this cross-sectional epidemiological study, an online questionnaire was created. Interns were asked questions regarding socio-demographic status, eating habits, sleeping behaviour and physical activity status before and during COVID-19 pandemic. Changes in dietary habits and sleep quality were evaluated under the guidance of Turkey Dietary Guidelines, Determination of Nutritional Status Report, Turkey National Nutrition and Health Survey and Pittsburgh Sleep Quality Index. SETTING: Hacettepe University Faculty of Medicine - Ankara, Turkey. PARTICIPANTS: Among 536 students, a total of 340 students (63·4 %) answered the questionnaire. RESULTS: After the beginning of lockdown, an increase in BMI was observed in both female (P = 0·002) and male students (P = 0·013). Students having healthy dietary habits increased by 18·8 % and unhealthy dietary habits decreased by 3·2 % during lockdown (P < 0·001). Overall, 63·2 % of students reported a decrease in physical activity, and 33·6 % reported a reduction in sleep quality, whereas 38·6 % reported an improvement in sleep quality. There was no significant difference in sleep quality between genders (P = 0·075). CONCLUSIONS: This study indicated that medical school suspension and enforced lockdown led to significant changes in interns' eating behaviour, physical activity and sleeping habits.

Loss of CTNNB1 exon 3 in sclerosing angiomatoid nodular transformation of the spleen.

Sclerosing angiomatoid nodular transformation (SANT) is a rare vascular lesion of the spleen. Although several hypotheses have been suggested, the etiopathogenesis of SANT remains unknown. It is also unclear whether SANT is a reactive or a neoplastic lesion. Since CTNNB1 (ß-catenin gene) exon 3 mutations were frequently detected in some rare fibrovascular lesions, we aimed to investigate the presence of oncogenic CTNNB1 mutations in SANT cases. For this purpose, 7 cases of SANT with typical histopathological features were retrieved. First, the presence of CTNNB1 exon 3 alterations was examined with a recently described immunohistochemistry-based method. Then, the findings were confirmed with polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), and Sanger sequencing. In all cases, immunochemistry of ß-catenin gave a staining pattern that was suggestive of exon 3 alteration; however, no missense mutations were found in any case at the CTNNB1 exon 3 hotspot region. Subsequently, we screened for large interstitial deletions of CTNNB1 exon 3 which revealed short PCR products in three cases. Sequencing confirmed that these cases had large interstitial deletions, resulting in loss of the entire exon 3 of CTNNB1. In the remaining four cases, loss of exon 3 was documented at the cDNA level, although genomic deletion was not identified. These results demonstrate that loss of CTNNB1 exon 3 and stabilization of ß-catenin with activation of Wnt signaling pathway might have a significant role in the pathogenesis of SANT. Through this study, we provided important evidence for the neoplastic nature and pathogenesis of this disorder.

An immunohistochemical approach to detect oncogenic CTNNB1 mutations in primary neoplastic tissues.

The Wnt/ß-catenin signaling pathway is dysregulated in different types of neoplasms including colorectal cancer (CRC). Aberrant activation of this signaling pathway is a key early event in the development of colorectal neoplasms, and is mainly caused by loss of function mutations in Adenomatous Polyposis Coli (APC), and less frequently by ß-catenin stabilization mutations via missense or interstitial genomic deletions in CTNNB1. In this study, we have defined an immunohistochemical algorithm to dissect Wnt pathway alterations in formalin-fixed and paraffin-embedded neoplastic tissues. Basically, consecutive sections of tumor specimens were stained by immunohistochemistry with two different monoclonal antibodies against ß-catenin: one (anti-active ß-catenin antibody) recognizes hypo-phosphorylated ß-catenin and the other recognizes the total pool of ß-catenin. We validated the strategy in the HCT116 CRC cell line which has an in-frame deletion of ß-catenin serine 45, and then studied human tumor microarrays containing colon adenomas, CRCs, solid pseudopapillary neoplasms of the pancreas as well as the whole tissue sections of CRCs, desmoid fibromatosis, and pilomatrixoma of the skin. In some tumors, we found strong ß-catenin cytoplasmic and/or nuclear staining with the total ß-catenin antibody but no staining with the anti-active ß-catenin antibody. This was inferred to be an altered/mutant ß-catenin staining pattern. All six colon adenomas of the 126 total adenomas studied for the altered/mutant ß-catenin staining pattern had presumptively pathogenic point mutations or deletions in CTNNB1. Four of 10 CRCs with the alterated/mutant ß-catenin staining pattern studied in depth, from 181 total CRCs from tissue microarray, had pathogenic CTNNB1 mutations. The frequencies of CTNNB1 alterations in non-colonic tumors with altered/mutant ß-catenin staining ranged between 46 and 100%. Our results demonstrate that the immunohistochemical approach described here can detect oncogenic forms of ß-catenin in primary tissue samples and can also highlight other tumors with presumptive novel defects activating the Wnt/ß-catenin pathway.

A Case of Disseminated Hypopigmented Keratoses Improved with Oral Acitretin.

Disseminated hypopigmented keratosis is a distinct clinical entity and only few cases have been reported so far. Here, we present a 21-year-old man with almost 10-year history of hypopigmented, nonfollicular, keratotic lichenoid papules occurring on the extensor surfaces of the extremities, back and lumber region. Histopathological examination showed orthohyperkeratosis, irregular acanthosis, and sporadic papillomatosis with a normal amount of melanin and number of melanocytes. In addition, no marked inflammation or melanophages were seen. In order to exclude other possible causes, we performed laboratory tests and radiological examination which were all found to be normal. As the clinical and histopathological features of our patient were taken into account, it was considered to be compatible with the diagnosis of disseminated hypopigmented keratoses. So far, only topical therapies have been used with failure in the previously reported cases except one patient. Considering the extensive lesions, we treated the present patient with 5% salicylic acid in addition to oral acitretin and significant regression in all lesions was achieved, particularly on the keratosis.