Cancer Stem-Like Cells in a Case of an Inflammatory Myofibroblastic Tumor of the Lung.

Background: Inflammatory myofibroblast tumor (IMT) is a rare tumor with obscure etiopathogenesis in which different inflammatory cells and myofibroblastic spindle cells are seen histologically. Although the majority of these neoplasms have a benign clinical course, the malignant form has also been reported. The gold standard is surgical treatment for complete removal. Our report describes a 50-year-old woman who underwent surgery for IMT of the lung. The aim is to determine whether cancer stem cells may be present in IMT of the lung. Methods: In April 2018, the patient underwent surgery for tumor mass asportation through lateral thoracotomy. The histology of the tumor was consistent with IMT of the lung. The ALDEFLUOR assay, after tissue digestion, was used to identify and sort human lung cancer cells expressing high and low aldehyde dehydrogenase (ALDH) activity. SOX2, NANOG, OCT-4, and c-MYC positivity were additionally determined by immunohistochemistry. Results: The specimen contained 1.10% ALDHhigh cells among all viable lung cancer cells, which indicates the population of cancer stem cells is not negligible. Immunohistochemically assessed cell positivity for ALDH1A1, SOX2, NANOG, OCT-4, and c-MYC, which are considered as lung cancer stem-like cells markers. Conclusion: For the first time, we demonstrated the presence of cancer stem cells in a case of IMT of the lung. This finding may provide a base for considering new pathological and molecular aspects of this tumor. This perspective suggests further studies to understand the possibility of developing recurrence depending on the presence of cancer stem cells.

CD44+/EPCAM+ cells detect a subpopulation of ALDHhigh cells in human non-small cell lung cancer: A chance for targeting cancer stem cells?

OBJECTIVES: Several studies demonstrated that aldehyde dehydrogenase (ALDH) and CD44 are the most considered cancer stem cells (CSC) markers. However, a comparison between ALDH high cells and CD44+ cells have been previously described with no significant correlation. Indeed, the aim of the present research is to identify a superficial marker able to match with ALDH high cells population in freshly isolated human lung cancer cells. MATERIALS AND METHODS: This cross-sectional study analyzed the expression of ALDHhigh/low cells and the positivity for CD44 and epithelium cell adhesion molecule (EPCAM) antigens in surgical lung cancer tissues. The main approach was a cytofluorimetric analysis of ALDH expression and positivity for CD44/EPCAM on primary cell population obtained from 23 patients harboring NSCLC. RESULTS: There was a highly positive correlation between the expressions of ALDHhigh and CD44+/EPCAM+ cells, with a Pearson's correlation coefficient equal to 0.69 (95% CI 0.39-0.86; P = 0.0002), and Spearman's correlation coefficient equal to 0.52 (P = 0.0124). The average paired difference between the expression of ALDHhigh and CD44+/EPCAM+ cells was very close to 0, being 0.1% (SD 2.5%); there was no difference between these subpopulations in terms of means (95% CI = -1.0; 1.2%, P = 0.8464). These results highlight a strong similarity between ALDHhigh and CD44+/EPCAM+ cells. CONCLUSIONS: Our study is the first attempt which identifies a high correlation between the ALDHhigh and the CD44+/EPCAM+ cells, thus suggesting the possibility to use this superficial marker for future target treatments against lung cancer stem cells.

Correlating tumor-infiltrating lymphocytes and lung cancer stem cells: a cross-sectional study.

BACKGROUND: Lung cancer stem cells (LCSCs) are endowed with high aldehyde dehydrogenase (ALDH) expression and play roles in tumor proliferation, metastasis, and drug resistance. Their elusive nature may allow them to escape the immune response by tumor-infiltrating lymphocytes (TILs), which can positively affect the outcome in non-small cell lung cancer (NSCLC) patients. Despite independent investigations on both LCSCs and TILs, the relationship between the two has been very marginally considered. We analyzed whether these two cell types may be related as a prerequisite for novel diagnostic and therapeutic approaches. METHODS: In this cross-sectional study, NSCLC human surgical specimens from 12 patients were tested by ALDEFLUOR assay to identify ALDHhigh cells. Fluorescence-activated cell sorting (FACS) analyses for CD3+, CD4+, and CD8+ TILs were performed in combination with immunohistochemistry evaluation. RESULTS: Statistically positive correlations were found between ALDH+ and CD8+, and between ALDH+ and CD3+ cells populations; no correlation was found between ALDH+ and CD4+ cells. The expression of CD3+ and CD8+ by cells accounted for 40.1% and 58.7%, respectively, of the variability of ALDH+ cell expression by an R-squared index, which highlights the strong correlation between TILs and LCSCs. Immunohistochemistry revealed 6-25% positive cells. CONCLUSIONS: We report a correlation between cytotoxic TILs and LCSCs, which may contribute to the future development of targeted therapies focusing on the different roles of lymphocytes against lung cancer.

Isolation and Identification of Cancer Stem-Like Cells in Adenocarcinoma and Squamous Cell Carcinoma of the Lung: A Pilot Study.

Background: Lung cancer stem cells (CSCs) share many characteristics with normal stem cells, such as self-renewal and multipotentiality. High expression of aldehyde dehydrogenase (ALDH) has been detected in many tumors, particularly in the CSC compartment, and it plays an important role in tumor proliferation, metastasis, and drug resistance. CD44 is commonly used as a cell surface marker of cancer stem-like cells in epithelial tumors. The aim of this study was to isolate and analyze cancer stem-like cells from surgically removed specimens to compare lung adenocarcinoma (ADENO) and squamous (SQUAMO) cell carcinoma. Methods: The ALDEFLUOR assay was used to identify and sort ALDHhigh and ALDHlow human lung cancer cells following tissue digestion. Fluorescence-activated cell sorting analysis for CD44 was performed with tumor cells. Quantitative real-time PCR was performed to assess the expression of SOX2 and NANOG as stemness markers. ALDH1A1 expression was additionally determined by immunohistochemistry. Anchorage-independent ALDHhigh cell growth was also evaluated. ALDHhigh ADENO and SQUAMO cells were cultured to analyze spheroid formation. Results: All specimens contained 0.5-12.5% ALDHhigh cells with 3.8-18.9% CD44-positive cells. SOX2 and NANOG relative expression in ALDHhigh compared to ALDHlow cells in ADENO and SQUAMO was analyzed and compared between the histotypes. Immunohistochemistry confirmed the presence of ALDH1A1 in the sections. SOX2 and NANOG were expressed at higher levels in the ALDHhigh subpopulation than in the ALDHlow subpopulation only in ADENO cells, and the opposite result was seen in SQUAMO cells. In vitro functional assays demonstrated that ALDHhigh cells exhibited migration capacity with distinct behaviors between ALDHhigh spheres in ADENO vs. SQUAMO samples. Conclusions: Our results highlight the importance of a better characterization of cancer stem-like cells in ADENO and SQUAMO histotypes. This may suggest new differential approaches for prognostic and therapeutic purposes in patients with non-small-cell lung cancer.

Liver Angiopoietin-2 Is a Key Predictor of De Novo or Recurrent Hepatocellular Cancer After Hepatitis C Virus Direct-Acting Antivirals.

Recent reports suggested that direct acting antivirals (DAAs) might favor hepatocellular carcinoma (HCC). In study 1, we studied the proangiogenic liver microenvironment in 242 DAA-treated chronic hepatitis C patients with advanced fibrosis. Angiopoietin-2 (ANGPT2) expression was studied in tissue (cirrhotic and/or neoplastic) from recurrent, de novo, nonrecurrent HCC, or patients never developing HCC. Circulating ANGPT2,vascular endothelial growth factor (VEGF), and C-reactive protein (CRP) were also measured. In study 2, we searched for factors associated with de novo HCC in 257 patients with cirrhosis of different etiologies enrolled in a dedicated prospective study. Thorough biochemical, clinical, hemodynamic, endoscopic, elastographic, and echo-Doppler work-up was performed in both studies. In study 1, no patients without cirrhosis developed HCC. Of 183 patients with cirrhosis, 14 of 28 (50.0%) with previous HCC recurred whereas 21 of 155 (13.5%) developed de novo HCC. Patients with recurrent and de novo HCCs had significantly higher liver fibrosis (LF) scores, portal pressure, and systemic inflammation than nonrecurrent HCC or patients never developing HCC. In recurrent/de novo HCC patients, tumor and nontumor ANGPT2 showed an inverse relationship with portal vein velocity (PVv; r = -0.412, P = 0.037 and r = -0.409, P = 0.047 respectively) and a positive relationship with liver stiffness (r = 0.526, P = 0.007; r = 0.525, P = 0.003 respectively). Baseline circulating VEGF and cirrhotic liver ANGPT2 were significantly related (r = 0.414, P = 0.044). VEGF increased during DAAs, remaining stably elevated at 3-month follow-up, when it significantly related with serum ANGPT2 (r = 0.531, P = 0.005). ANGPT2 expression in the primary tumor or in cirrhotic tissue before DAAs was independently related with risk of HCC recurrence (odds ratio [OR], 1.137; 95% confidence interval [CI], 1.044-1.137; P = 0.003) or occurrence (OR, 1.604; 95% CI, 1.080-2.382; P = 0.019). In study 2, DAA treatment (OR, 4.770; 95% CI, 1.395-16.316; P = 0.013) and large varices (OR, 3.857; 95% CI, 1.127-13.203; P = 0.032) were independent predictors of de novo HCC. CONCLUSION: Our study indicates that DAA-mediated increase of VEGF favors HCC recurrence/occurrence in susceptible patients, that is, those with more severe fibrosis and splanchnic collateralization, who already have abnormal activation in liver tissues of neo-angiogenetic pathways, as shown by increased ANGPT2. (Hepatology 2018; 00:000-000).

Microenvironment inflammatory infiltrate drives growth speed and outcome of hepatocellular carcinoma: a prospective clinical study.

In HCC, tumor microenvironment, heavily influenced by the underlying chronic liver disease, etiology and stage of the tissue damage, affects tumor progression and determines the high heterogeneity of the tumor. Aim of this study was to identify the circulating and tissue components of the microenvironment immune-mediated response affecting the aggressiveness and the ensuing clinical outcome. We analyzed the baseline paired HCC and the surrounding tissue biopsies from a prospective cohort of 132 patients at the first diagnosis of HCC for immunolocalization of PD-1/PD-L1, FoxP3, E-cadherin, CLEC2 and for a panel of 82 microRNA associated with regulation of angiogenesis, cell proliferation, cell signaling, immune control and autophagy. Original microarray data were also explored. Serum samples were analyzed for a panel of 19 cytokines. Data were associated with biochemical data, histopathology and survival. Patients with a more aggressive disease and shorter survival, who we named fast-growing accordingly to the tumor doubling time, at presentation had significantly higher AFP levels, TGF-ß1 and Cyphra 21-1 levels. Transcriptomic analysis evidenced a significant downregulation of CLEC2 and upregulation of several metalloproteinases. A marked local upregulation of both PD-1 and PD-L1, a concomitant FoxP3-positive lymphocytic infiltrate, a loss of E-cadherin, gain of epithelial-mesenchymal transition (EMT) phenotype and extreme poor differentiation at histology were also present. Upregulated microRNA in fast-growing HCCs are associated with TGF-ß signaling, angiogenesis and inflammation. Our data show that fast HCCs are characterized not only by redundant neo-angiogenesis but also by unique features of distinctively immunosuppressed microenvironment, prominent EMT, and clear-cut activation of TGFß1 signaling in a general background of long-standing and permanent inflammatory state.

Annular lichenoid dermatitis of youth ... and beyond: a series of 6 cases.

Annular lichenoid dermatitis of youth (ALDY) is a clinico-pathologic entity described in children and young patients, clinically reminiscent of morphea, annular erythema, vitiligo or mycosis fungoides. We report on six patients presenting single or multiple lesions, distributed particularly on the flanks and abdomen, with clinical and histologic features consistent with ALDY. Two patients were young girls and four were adults males. Three patients received topical therapy and four showed complete resolution of the lesions after a 24-65 months follow-up. Analogously to the cases reported so far, immunohistochemistry showed a T cell infiltrate with a predominance of CD8+ lymphocytes, while T cell receptor rearrangement was absent in all cases. It seems appropriate to include annular lichenoid dermatitis of youth among the dermatoses with a lichenoid pattern. For the first time, we found that it can affect also adult patients, therefore we propose to rename the disease annular lichenoid dermatitis. The differential diagnosis with mycosis fungoides, especially in adult patients, is particularly crucial for their proper management and treatment.

Mismatch repair proteins expression and microsatellite instability in skin lesions with sebaceous differentiation: a study in different clinical subgroups with and without extracutaneous cancer.

Muir-Torre syndrome (MTS) is defined as the association of a sebaceous tumor or keratoacanthoma and an extracutaneous neoplasm, mainly from the gastrointestinal or genitourinary tracts. MTS is related to hereditary non-polyposis colorectal cancer (HNPCC), a syndrome with germline mutations in the mismatch repair (MMR) gene(s), leading to microsatellite instability (MSI). In this study, using immunohistochemistry and a microsatellite instability assay, we analyzed the incidence of MMR gene abnormalities in 79 sebaceous lesions from 70 patients, 26 of whom also had an extracutaneous visceral neoplasm. We were unable to investigate the family histories of our patients regarding other tumors in order to assess which of our cases met the Amsterdam criteria. Defective MMR protein expression (MMR-) was found in 18/70 (25.7%) patients, with an identical distribution between those having an isolated skin tumor (11/44, 25.0%) and those with an extracutaneous cancer (7/26, 25.4%). In the sporadic group, MMR negative lesions were significantly more frequent in extrafacial areas (P = 0.03). High concordance was found between MMR expression in sebaceous lesions and the extracutaneous neoplasm in the same patient (20/23, 86.9%), as well as between MMR expression and microsatellite status (18/20, 90%). In conclusion, this study confirms the value of immunohistochemistry to identify MMR defective tumors. However, since only a minority of sebaceous neoplasms in patients who also have an extracutaneous cancer display MMR defects, these techniques are of limited value for the identification of "clinically defined" MTS.

Comparative evaluation between external phased array coil at 3 T and endorectal coil at 1.5 T: preliminary results.

OBJECTIVE: The aim of this study was to compare the image quality and the diagnostic accuracy of endorectal coil 1.5 T MRI (erMRI) and phased-array coil 3 T MRI (3-T MRI) in the pretherapeutic staging of prostate cancer. METHODS: Twenty-nine consecutive patients, with pathological proven prostate cancer, have been examined in the same week with both erMRI and 3-T MRI. Two radiologists independently evaluated the image quality focusing on the following points: cancer tissue conspicuity, capsular infiltration and tumor involvement of seminal vesicles, neuro-vascular bundles, and apex. The radiologists assigned to each one of the above findings an image-quality score ranging from 1 to 5 (with 1 meaning "not visible," 2 "poorly visible," 3 "fairly visible," 4 "well visible with some artifacts," and 5 "clearly visible without artifacts".) Afterwards a comparative evaluation of the mean score obtained respectively by erMRI and 3 T MRI was done. Twenty-two of these 29 patients underwent radical prostatectomy. Assuming as gold standard the pathological report from the resected specimen, we compared the diagnostic accuracy of 3TMRI and erMRI in differentiating between tumors confined within the prostate gland (stage

Spitz nevus is relatively frequent in adults: a clinico-pathologic study of 247 cases related to patient's age.

Spitz nevus is a clinico-pathologic entity that can cause diagnostic concern, particularly in adults. Many studies have been performed to establish reliable histologic criteria, in the attempt to differentiate this lesion from melanoma. A series of 247 Spitz nevi, 6 of which were formerly classified as melanomas, were reviewed for clinical and histopathological parameters. Patients older than 20 comprised 66% of cases, with a predominance of women. The lower extremity was more affected in females of any age, whereas the trunk was more frequently involved in men over 40. Histopathologic examination showed the following differences among Spitz nevi related to age: acanthosis, parakeratosis, pagetoid infiltration, and Kamino bodies were more frequent in young people, whereas multinucleated melanocytes were more frequent in adults. The latter also had lesions that were less pigmented, with less maturation and more desmoplasia. At a mean follow-up of 94 months (range 52-172), recurrence at the site of biopsy or metastases were absent. In our study, a greater proportion of Spitz nevi occurred in adults than in previous series. Moreover, the relative incidence of Spitz nevus compared with melanoma in our population was higher than in other studies. Histopathologic criteria elaborated to diagnose Spitz nevus, applied to our cases, appeared reliable, allowing a correct diagnosis, even in adults.

Massive bone marrow crystal-storing histiocytosis in a patient with IgA-lambda multiple myeloma and extensive extramedullary disease. A case report.

We report a case of multiple myeloma (MM) displaying an unusual course, with metastatic spread in uncommon sites (gastroduodenal and upper respiratory tract, breast, skin and liver) and with a fatal outcome. In our patient this plasma cell neoplasm was associated with a rare condition named crystal-storing histiocytosis (CSH), resulting from the storage in reactive histiocytes of crystalline immunoglobulin inclusions. These crystal-forming paraprotein components are secreted by the neoplastic plasma cells and give rise to the crystalline material of the histiocytes only after their ingestion and degradation by the same histiocytes. In this disorder crystal-storing cells may be present in various tissues (in our case mainly in bone marrow), often with functional alterations of the involved organs. In our opinion the association of this "atypical" MM with CSH is to be considered an uncommon event.

Does mesoappendix infiltration predict a worse prognosis in incidental neuroendocrine tumors of the appendix? A clinicopathologic and immunohistochemical study of 15 cases.

We conducted a retrospective clinicopathologic and immunohistochemical study of the biologic significance of mesoappendix infiltration in 15 appendiceal neuroendocrine tumors selected from a series of 42 primary tumors. In all cases, the tumor was found incidentally and measured less than 2 cm (mean, 0.84 cm). In 13 cases, it was located in the tip of the appendix and in the midportion in 2. Histologically, none showed relationship with overlying mucosa. Necrosis was absent; mitotic figures were rare. The Ki-67 labeling index was low (1%-2%). In all cases, S-100 protein immunostaining disclosed positive elements with cytoplasmic dendritic processes closely intermingled with neuroendocrine neoplastic cells. All patients (8 males; 7 females; mean age, 38.2 years) underwent simple appendectomy. A right-sided hemicolectomy was performed subsequently in 1 case. After a mean follow-up of 52.6 months (range, 8-143 months), none had died of disease or had recurrent or metastatic disease. Our results confirm that appendiceal neuroendocrine tumors seem to have a different phenotype from those occurring in other gastrointestinal sites. Tumors less than 2 cm, even with mesoappendiceal infiltration, have an excellent prognosis, and simple appendectomy seems to be the appropriate therapeutic approach.