Papillary thyroid carcinoma tall cell variant shares accumulation of mitochondria, mitochondrial DNA mutations, and loss of oxidative phosphorylation complex I integrity with oncocytic tumors

open12siMDL was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC) ‘Bruna Martelli’ Fellowship.Papillary thyroid carcinoma tall cell variant (PTC-TCV), a form of PTC regarded as an aggressive subtype, shares several morphologic features with oncocytic tumors. Pathogenic homoplasmic/highly heteroplasmic somatic mitochondrial DNA (mtDNA) mutations, usually affecting oxidative phosphorylation (OXPHOS) complex I subunits, are hallmarks of oncocytic cells. To clarify the relationship between PTC-TCV and oncocytic thyroid tumors, 17 PTC-TCV and 16 PTC non-TCV controls (cPTC) were subjected to: (1) transmission electron microscopy (TEM) to assess mitochondria accumulation, (2) next-generation sequencing to analyze mtDNA and nuclear genes frequently mutated in thyroid carcinoma, and (3) immunohistochemistry (IHC) for prohibitin and complex I subunit NDUFS4 to evaluate OXPHOS integrity. TEM showed replacement of cytoplasm by mitochondria in PTC-TCV but not in cPTC cells. All 17 PTC-TCV had at least one mtDNA mutation, totaling 21 mutations; 3/16 cPTC (19%) had mtDNA mutations (p < 0.001). PTC-TCV mtDNA mutations were homoplasmic/highly heteroplasmic, 16/21 (76%) mapping within mtDNA-encoded complex I subunits. MtDNA mutations in cPTC were homoplasmic in 2 cases and at low heteroplasmy in the third case, 2/3 mapping to mtDNA-encoded complex I subunits; 2/3 cPTC with mtDNA mutations had small tall cell subpopulations. PTC-TCV showed strong prohibitin expression and cPTC low-level expression, consistent with massive and limited mitochondrial content, respectively. All 17 PTC-TCV showed NDUFS4 loss (partial or complete) and 3 of 16 cPTC (19%) had (partial) NDUFS4 loss, consistent with lack of complex I integrity in PTC-TCV (p < 0.001). IHC loss of NDUFS4 was associated with mtDNA mutations (p < 0.001). Four BRAF V600E mutated PTCs had loss of NDUSF4 expression limited to neoplastic cell subpopulations with tall cell features, indicating that PTCs first acquire BRAF V600E and then mtDNA mutations. Similar to oncocytic thyroid tumors, PTC-TCV is characterized by mtDNA mutations, massive accumulation of mitochondria, and loss of OXPHOS integrity. IHC loss of NDUFS-4 can be used as a surrogate marker for OXPHOS disruption and to reliably diagnose PTC-TCV.openTsybrovskyy, Oleksiy; De Luise, Monica; Biase, Dario; Caporali, Leonardo; Fiorini, Claudio; Gasparre, Giuseppe; Carelli, Valerio; Hackl, Dominik; Imamovic, Larisa; Haim, Silke; Sobrinho‐Simões, Manuel; Tallini, GiovanniTsybrovskyy, Oleksiy; De Luise, Monica; Biase, Dario; Caporali, Leonardo; Fiorini, Claudio; Gasparre, Giuseppe; Carelli, Valerio; Hackl, Dominik; Imamovic, Larisa; Haim, Silke; Sobrinho‐Simões, Manuel; Tallini, Giovann

Disseminated Histoplasmosis: A Challenging Differential Diagnostic Consideration for Suspected Malignant Lesions in the Digestive Tract

Histoplasmosis is well characterized as an endemic fungal disease restricted to certain areas of the USA. In Middle Europe, most patients present with acute pulmonary symptoms after travelling to endemic areas. Here, we want to illustrate the case of a 67-year-old man who presented with persistent oral ulcers, hoarseness, dysphagia, diarrhea, and weight loss to our Department of Otorhinolaryngology in December 2014. He was a retired construction worker and had a history of soil-disruptive activities in Africa and Middle and South America during employment. A positron emission tomography-computed tomography scan revealed prominent hypermetabolic lesions in the cecum and the lung, pointing towards a malignant disease. Surprisingly, histological examination of colonic and oral biopsies revealed abundant intracellular fungal elements, highly suspicious of Histoplasma capsulatum. Diagnosis was finally confirmed by panfungal polymerase chain reaction. Upon treatment with liposomal amphotericin followed by itraconazole, the severely ill patient showed an impressive clinical response. This case describes a disseminated manifestation of H. capsulatum years after the first exposure in an otherwise immunocompetent patient descending from a nonendemic area

Expression of cancer stem cell markers ALDH1 and CD44⁺/CD24^-/low in malignant pleural effusions of metastatic breast cancer patients.

<p>(A) Aldefluor assay measuring ALDH1 expression in individual pleura samples. (B) FACS dot plots of CD44⁺/CD24^-/low staining. First plots show settings for linage cocktail staining, positive cells were depleted. Both images A and B also include a representative plot of an unstained sample.</p

Genetic profiling of putative breast cancer stem cells from malignant pleural effusions

<div><p>A common symptom during late stage breast cancer disease is pleural effusion, which is related to poor prognosis. Malignant cells can be detected in pleural effusions indicating metastatic spread from the primary tumor site. Pleural effusions have been shown to be a useful source for studying metastasis and for isolating cells with putative cancer stem cell (CSC) properties. For the present study, pleural effusion aspirates from 17 metastatic breast cancer patients were processed to propagate CSCs <i>in vitro</i>. Patient-derived aspirates were cultured under sphere forming conditions and isolated primary cultures were further sorted for cancer stem cell subpopulations ALDH1⁺ and CD44⁺CD24^-/low. Additionally, sphere forming efficiency of CSC and non-CSC subpopulations was determined. In order to genetically characterize the different tumor subpopulations, DNA was isolated from pleural effusions before and after cell sorting, and compared with corresponding DNA copy number profiles from primary tumors or bone metastasis using low-coverage whole genome sequencing (SCNA-seq). In general, unsorted cells had a higher potential to form spheres when compared to CSC subpopulations. In most cases, cell sorting did not yield sufficient cells for copy number analysis. A total of five from nine analyzed unsorted pleura samples (55%) showed aberrant copy number profiles similar to the respective primary tumor. However, most sorted subpopulations showed a balanced profile indicating an insufficient amount of tumor cells and low sensitivity of the sequencing method. Finally, we were able to establish a long term cell culture from one pleural effusion sample, which was characterized in detail. In conclusion, we confirm that pleural effusions are a suitable source for enrichment of putative CSC. However, sequencing based molecular characterization is impeded due to insufficient sensitivity along with a high number of normal contaminating cells, which are masking genetic alterations of rare cancer (stem) cells.</p></div

Characterization of PL24.

<p>(A) Selected copy number plots of PL24. Depicted are segmented log2-ratio plots of chromosomes 1, 6, 17 and 18. (B) Representative picture of spheres in culture. (C) FACS dot plots of PL24 passage 20 left control with DEAB and right Aldefluor assay staining. (D) Immunofluorescent staining of FFPE PL24 spheres I ALDH1 (red) CK (green), II CD44 (red) CK (green), III Ki67 (red) CK (green), IV HEA (red) CK (green), V Vim (red) CK (green), VI Her2neu (green). All slides were counterstained with Dapi (blue). Measuring bar: 100 μm.</p

Sphere formation assay.

<p>Representative pictures from mammosphere assay. (A) PL11, left sphere from unsorted cells, middle sphere from CD44⁺/CD24^-/low subpopulation, right cells from CD44^-/CD24^- subpopulation. (B) PL13, left sphere from unsorted cells, middle sphere from ALDH1⁺ subpopulation, right cells from ALDH1^- subpopulation. Scale bar 50 μM. (C) Summary of sphere formation efficiency of all pleura samples with their different subpopulations.</p

Copy number profiles.

<p>Primary tumors of PL21 (A) and PL24 (E), the cultivated unsorted cells before cell sorting (B) (F), CD44-positive cell fraction of PL21 (C) and the ALDH1⁺ cell fraction of PL21 (D), respectively, after FACS cell sorting. PL24 long-term cultivation of unsorted spheroids (G). Depicted are segmented log2-ratio plots of the genome. The X-axis indicates the chromosome and Y-axis indicate the log2-ratios. Regions with log2 ratios > 2 indicate gain of chromosomal material and those regions with log2 ratios < 2 indicate loss of chromosomal material.</p