Thyroid hormones and platelet activation in COVID-19 patients.

PURPOSE: To retrospectively describe the association between thyroid hormones (TH) and platelet activation, as represented by mean platelet volume (MPV), in a cohort of patients hospitalized for COVID-19 with no known thyroid disease, and to correlate these data with the severity of COVID-19 and the occurrence of death/ARDS (Acute Respiratory Distress Syndrome). METHODS: 103 patients with real-time polymerase chain reaction (RT-PCR) testing-confirmed COVID-19 and hospitalized were enrolled. Serum samples were collected from patients upon admission before starting any treatment. Chi-squared test was used to determine the association between euthyroid sick syndrome (ESS) and COVID-19 severity. Multivariate logistic regression was performed to evaluate the best independent predictors of COVID-19 deaths/ARDS. RESULTS: 39/103 (37.9%) of patients were found to have ESS, and this condition was an independent predictor for the severity of COVID-19 (p = 0.003). Lower TSH and lower FT3/FT4 ratio correlated with higher MPV (p = 0,001 and p = 0.010), with an opposite trend with respect to what has been documented in non-COVID patients. Increasing MPV and lower FT3 significantly increased the risk, in COVID-19 patients, of an adverse outcome of death/ARDS. CONCLUSION: Increased platelet activation, as represented by increased MPV, has already been reported to correlate with COVID-19 severity, possibly as a consequence of cytokine release. We demonstrated, in a cohort of 103 patients with COVID-19, that MPV is inversely correlated to TH levels, in particular in the case of ESS, where downregulation of TH axis may occur in case of systemic cytokine inflammation and more severe outcomes (death/ARDS). That ESS itself may directly cause platelet activation, as demonstrated by higher MPV in these patients, is an interesting hypothesis which deserves further investigation.

Prognostic value of circulating tumor cells in nonmuscle invasive bladder cancer: a CellSearch analysis.

BACKGROUND: Circulating tumor cells (CTCs) provide prognostic information in patients with metastatic tumors. Recent studies have shown that CTCs are released in circulation in an early phase of cancer disease so that their presence is under investigation in the adjuvant setting. Few studies investigated the prognostic significance of CTCs enumeration in patients with metastatic and advanced bladder cancer. The current study has analyzed the presence of CTC in patients with nonmuscle-invasive bladder cancer (NMIBC). PATIENTS AND METHODS: Forty-four NMIBC patients were enrolled and included in a 24-month follow-up program. Blood drawings were carried out in all patients at the first diagnosis. CellSearch system (Veridex; LLC, Raritan, NJ) was used for CTCs enumeration. RESULTS: CTC were detectable in 8/44 patients (18%). Presence of CTC was found significantly associated to shorter time to first recurrence (6.5 versus 21.7 months, P < 0.001). Median time to progression was not reached, due to the short follow-up period. CTC presence was found associated to concomitant carcinoma in situ and higher T category. CONCLUSION: The detection of CTC in this setting of disease may allow to distinguish patients with high risk of recurrence from those with high risk of progression, as well as to early identify patients candidate for adjuvant treatment.

Circulating tumor cells (CTCs) in metastatic breast cancer (MBC): prognosis, drug resistance and phenotypic characterization.

BACKGROUND: the expression of ATP-binding cassette transporters on circulating tumor cells (CTCs) is predictive of response to chemotherapy in cancer patients. We tested the hypothesis that drug-resistant CTCs might have predictive value in metastatic breast cancer (MBC) and possibly retain stem-like properties. PATIENTS AND METHODS: CTCs obtained from 42 MBC patients were evaluated for multidrug-resistance-related proteins (MRPs), aldehyde dehydrogenase 1 (ALDH1), estrogen receptor α (ERα) and human epidermal growth factor receptor 2 (HER2/neu). Primary objective was to evaluate the prognostic and predictive value of CTCs profile. Secondary end points were the level of concordance in ERα and HER2/neu status between primary tumors and CTCs and the correlation in CTCs between ALDH1, drug resistance profile and number of MRPs. RESULTS: A difference in progression-free survival (PFS) was found between CTCs-positive and CTCs-negative patients. PFS was shorter in patients with a 'drug resistance' CTCs profile and in patients whose CTCs expressed two or more MRPs. No correlation was found between tumor characteristics and ALDH1. ALDH1 correlated to negative ERα and positive HER2/neu status in CTCs. The correlation between the number of MRPs expressed in CTCs and ALDH1 was statistically significant. CONCLUSION: in MBC, the presence of CTCs expressing MRPs and ALDH1 is predictive of response to chemotherapy.

Increased metastatic lymph node 64 and CYP17 expression are associated with high stage prostate cancer.

The metastatic lymph node 64 (MLN64), which is localized in the human chromosome 17, encodes a protein with strong homology with steroidogenic acute regulatory protein. Its overexpression in human breast carcinomas and MLNs led to the hypothesis that this protein could be involved in intraneoplastic steroidogenesis. In the present study, we investigated the expression of MLN64 in prostate cancer, another hormone-dependent tumor, and compared its expression with that of CYP17, the gene encoding for the key enzyme of androgen synthesis. We investigated by RT-PCR the expression of MLN64 and CYP17 in 60 prostatic tumors and compared their expression with the stage of disease and the appearance of relapses in a follow-up of 24 months. We found MLN64 and CYP17 expressed in all samples examined, with significantly higher expression in neoplastic tissues with respect to normal tissues (NTs). Moreover, only in neoplastic but not in NTs, a positive linear correlation was found between MLN64 and CYP17 gene expression. MLN64 and CYP17 expression seems to correlate with high stage, high Gleason score and short relapse-free time. These data, for the first time, demonstrate the presence of MLN64 and CYP17 expression in both normal and neoplastic prostatic tissues. The biological role of MLN64 in human prostate and, particularly, in neoplastic tissue is still unclear. Our findings concerning MLN64 and CYP17 gene expression and their significant positive correlation in human prostate cancer may suggest their possible role in intraneoplastic autonomous steroidogenesis.

Detection of epidermal growth factor receptor mRNA in peripheral blood: a new marker of circulating neoplastic cells in bladder cancer patients.

Despite the large number of studies performed in solid tumors, few attempts at molecular detection of urothelial cells in blood have been made. Specifically, only uroplakin II (UP-II) and cytokeratin 20 (CK-20) have been suggested as tumor markers in the blood of bladder cancer patients. Epidermal growth factor receptor (EGFR) mRNA expression was found in the blood of patients with some types of carcinoma; nevertheless, its expression has been never investigated in the blood of patients with urothelial tumors. We used a EGFR-based reverse transcription-PCR assay for the detection of tumoral cells in the blood of 27 patients with bladder cancer, in 30 healthy donors, and in 9 patients with cystitis. EGFR expression was compared with that of known markers of circulating epithelial cells, CK-19 and CK-20, and to a urothelial-specific marker, UP-II. Analysis by reverse transcription-PCR and Southern blot hybridization showed no evidence of EGFR and UP-II mRNA expression in any of the samples used as controls. Analysis of healthy donors showed mRNA expression for CK-19 and CK-20 in 6 of 30 and in 4 of 30 samples, respectively. All patients with cystitis resulted negative for EGFR expression, whereas 3 of 9, 2 of 9, and 3 of 9 were found expressing CK-19, CK-20, and UP-II, respectively. Among blood samples from tumoral patients, 74% had EGFR mRNA and 41% had positive signals for CK-19, whereas positivity for CK-20 and UP-II was found in 15% and 37% of patients, respectively. These results seem to indicate that EGFR mRNA in the blood may be a useful tumor marker in bladder cancer patients, as well as in other patients with epithelial tumors.

High levels of transforming growth factor-alpha (TGF-alpha) mRNA may predict local relapses in early stage urinary bladder cancer.

Elevated expression of transforming growth factor-alpha (TGF-alpha) gene has been previously reported in some types of human neoplasms, but its role in the pathogenesis of bladder cancer has still not been investigated. In the present study, we analysed 28 samples of early stage bladder tumours for the presence of TGF-alpha mRNA using reverse transcription-polymerase chain reaction (RT-PCR). We detected TGF-alpha mRNA in 71% (20/28) of these samples. When we related the expression levels of TGF-alpha with local relapses of patients during a follow-up of 2 years, we found that a high TGF-alpha expression level in bladder cancer was significantly associated with local relapses in patients with early stage tumours. The appearance of early relapses in tumours with high TGF-alpha expression levels may suggest the existence of an additional marker in the prediction of local relapses in patients with superficial disease.

Mutational analysis of the estrogen receptor ligand-binding domain: influence of ligand structure and stereochemistry on transactivation.

The mouse estrogen receptor (mER) exhibits ligand stereochemical specificity for indenestrol A (IA), a stilbestrol estrogen. IA has a chiral C3 methyl group, and the mER preferentially binds the S-enantiomer (IA-S), resulting in elevated biological activity when compared with the IA-R enantiomer. To elucidate the mechanisms for this stereochemical recognition, we have constructed a series of mERs with individual amino acid substitutions at Met521, His528, Met532, and Val537. The abilities of yeast-expressed wild-type and mutant mERs to transactivate an estrogen-responsive reporter gene construct were measured in the presence of diethylstilbestrol (DES) and IA enantiomers. The concentration of IA-S required to induce half-maximal transactivation by wild-type mER was 10-fold lower than IA-R, which is attributed to the 15-fold greater binding affinity for IA-S. Wild-type mER displayed similar dose-response curves for IA-R and demethyl IA, which lacks a C3 methyl group, demonstrating that the presence and correct orientation of the C3 methyl group on the IA compound is required for high-affinity ligand binding and transcriptional activity. Each mutant exhibited a reduced preference for IA-S enantiomer with respect to transactivation, suggesting that this region of the mER functions in ligand stereochemical recognition and activation. A mutation at Met532 diminished DES- and IA-S-induced transactivation by 7.5-fold and 40-fold respectively, with minimal change on their binding affinity. These data suggest that Met532 is required for transactivation induced by the potent agonist, IA-S, and the M532G mutation effectively uncouples IA-S ligand binding from transactivation. Use of these stereochemically different ligands in combination with mutagenesis of the mER demonstrates that ligand structure could influence transactivation by specifically altering the conformation of the mER AF-2 region.

Detection of basic fibroblast growth factor mRNA in urinary bladder cancer: correlation with local relapses.

Natural history of bladder cancer is characterized by high risk of disease progression even for patients with a clinical diagnosis of superficial disease; in these tumors, the occurrence of local relapse is known to be dependent on the angiogenesis rate. Basic fibroblast growth factor (bFGF), has been described to be elevated in urine and serum of patients with bladder cancer. We investigated the expression of bFGF at mRNA level in a panel of 32 transitional cell tumors of the urinary bladder and in normal bladder tissues used as controls. Expression of bFGF was found elevated in most tumors of high stage, where its presence was found correlated with the occurrence of early local relapses. Furthermore, bFGF was found highly expressed in the majority of tumors showing a high bcl-2 expression rate. Our data suggest that bFGF expression could contribute to the progression of disease; it may provide a prognostic indicator in the identification of patients with high risk for occurrence of local relapses.

Involvement of bcl-2 and bax gene expression in apoptosis and differentiation of the non-tumorigenic murine hematopoietic cell line, 32DC13(G).

32DCl3(G) is an interleukin-3 (IL-3) dependent, non-tumorigenic murine hematopoietic cell line which undergoes terminal differentiation into granulocytes when exposed to granulocyte-colony stimulating factor (G-CSF). This line therefore offers a convenient system to study the expression of genes involved in apoptosis and differentiation. In our experiments we have acquired evidence that during the differentiation pathway, likewise in apoptosis induced by IL-3 deprivation, detectable levels of bax mRNA appear, while bcl-2 expression decreases. These events are under the control of the p53 tumor-suppressor gene. In these cells, an overexpression of exogenous wild-type p53 leads to a decrease in bcl-2 mRNA and to the appearance of box mRNA, which instead is absent in the parental cells growing in IL-3 conditioned medium. Furthermore, results from experiments on p53 transfected cells demonstrate that excess wild-type p53 activity, on its own, fails to elicit apoptosis as long as IL-3 is present and does not induce differentiation if G-CSF is not added to the culture medium. We conclude that in apoptosis and differentiation of 32DCl3(G) the alterate ratio of bcl-2 and box gene expression, modulated by p53, is an early event dependent on IL-3 withdrawal and that the appearance of bax and the decrease of bcl-2 expression are necessary, but not sufficient for the acquisition of a completely mature granulocytic phenotype.

Follicle-stimulating hormone, testosterone, and hypoxia differentially regulate UDP-glucuronosyltransferase 1 isoforms expression in rat sertoli and peritubular myoid cells.

Uridine diphosphoglucuronosyltransferases (UGTs) are detoxifying enzymes responsible for the metabolism of endogenous and xenobiotics compounds. UGT isoforms are widely distributed in rat tissues showing a constitutive and inducible gene expression. However, little information is available concerning UGTs expression in testis. The UGT1A1, UGT1A2, and UGT1B1 mRNAs expression in whole rat testis, in Sertoli and peritubular myoid cells in basal conditions, and after hormonal and hypoxic stimulation were investigated by reverse transcriptase-polymerase chain reaction (RT-PCR). Constitutive expression of each UGT1 isoform was present in rat testis with higher levels of UGT1A2. UGT transcripts were also detected in Sertoli and peritubular myoid cells. After FSH stimulation, Sertoli cells showed an increase in UGT1B1 mRNA expression, whereas the levels of UGT1A1 and UGT1A2 resulted unmodified. The main effect induced by testosterone was a decrease of UGT1B1 mRNA expression in peritubular myoid cells, whereas in Sertoli cells an increase in UGT1A1 and UGT1B1 was observed. In hypoxic conditions, a reduction in UGTs mRNA levels was detected in both cell types. These findings suggest that rat UGT1 isoforms are regulated in testis by hormonal and environmental factors. Thus, it was speculated that alterations in UGTs expression and/or activity may be involved in the pathogenesis of testis injury.

Variable levels of bcl-2, bcl-x and bax mRNA in bladder cancer progression.

We investigated the expression of the anti-apoptotic genes bcl-2 and bcl-X and the pro-apoptotic gene bax in bladder tumors and normal samples from urinary bladder, using RT-PCR analysis. Bcl-2 mRNA was not detected in any of the normal samples, while it was found expressed in 66% of the low stage tumors and in 100% of the high stage tumors. Bax expression had an inverse progress, being present in 62% of the normal tissues examined, in 16% of the low stage tumors and in 14% of the high stage. Bcl-X gene expression was quite variable among all samples (37% in normal tissues, 50% in the low stage tumors and 14% in the high stage). bcl-X mRNA was only found in the isoform bcl-XL, with anti-apoptotic functions, whereas no sample expressed the isoform bcl-XS, which is known to suppress bcl-2 functions. Most samples expressing bcl-2 did not express bcl-X, and vice versa. These results, besides confirming the potential role of these genes in the pathogenesis of low stage bladder cancer strengthen the hypothesis concerning their possible interaction in the progression of disease.

Anti-estrogen activity in the yeast transcription system: estrogen receptor mediated agonist response.

The mouse estrogen receptor was expressed in yeast cells to study the mechanism of action of anti-estrogens. Tamoxifen and hydroxytamoxifen, estrogen antagonists in mammalian tissues, failed to antagonize estradiol-induced expression of a VitA2-ERE-CTC1-lacZ reporter gene construct and exhibited full agonist activity, while nafoxidine exhibited partial antagonism as well as partial agonism. ICI 164,384 is a potent anti-estrogen in both mouse and human estrogen receptor systems. Our previous studies in the mouse uterus indicated that rapid degradation of the estrogen receptor accounted for the loss of estrogen responsiveness. In yeast however, ICI 164,384 or an isomer ICI 182,780 were unable to antagonize estradiol at concentration of 200 microM. On the contrary, both ICI compounds exhibited partial agonist activity by stimulating beta-galactosidase activity to 50% that of estradiol. We examined the level of estrogen receptor in the yeast after treatment with estradiol, ICI 164,384 or vehicle by Western blot and found no ICI-induced reduction of estrogen receptor levels, but observed an increase in estrogen receptor following estradiol treatment. This indicates that the proteolytic activity responsible for degrading estrogen receptor in ICI 164,384-treated uteri or eukaryotic cells is not present in yeast. The agonist activity seen with ICI indicated that ICI-bound estrogen receptor is able to induce expression of an estrogen-responsive reporter gene. In support of this, estrogen receptor from ICI 164,384-treated yeast was able to bind an estrogen-responsive element in a gel-shift assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Two transcription activation functions in the amino terminus of the mouse estrogen receptor that are affected by the carboxy terminus.

To determine the characteristics of the N-terminal transactivation domain (AF-1) of the mouse estrogen receptor (ER), we constructed a number of deletion mutants. Wild-type and mutant receptors were expressed in yeast cells and assayed for their ability to transactivate an estrogen-responsive reporter plasmid (ERE-CYCl-LacZ) that contained a single estrogen response element of the vitellogenin A2 gene promoter. Deletion of the N-terminal 121 amino acids from the mouse ER resulted in a 50% reduction in transactivation activity compared with the full-length wild-type ER. Deletion of the first 150 amino acids resulted in loss of 90% transactivation activity. An ER deletion mutant lacking residues 121-154 retained full transcriptional activity, suggesting that this region plays a significant transacting role only when the first portion is deleted. A point mutation was introduced in the C-terminal region at Met-521 in order to study the possible interaction between the C-terminal ligand-binding domain and the N-terminal AF-1 region. This mutant ER, M521G, exhibited 150% of the transcriptional activity of the wild-type ER. An M521G mutant lacking the N-terminal 121 amino acids retained full transactivation activity, whereas, M521G lacking 150 amino acids resulted in only 10% of wild-type activity. These results suggest that residues 121-154 might interact with the C terminus to affect transcription. In summary, multiple N-terminal regions in the ER were identified that function in transactivation. Furthermore, a point mutation in the C-terminal portion of the ER may change the conformation of the ER ligand-binding domain, producing a more stable receptor/ligand complex that increases transcriptional activity. These data suggest that the N- and C-terminal portions of the ER interact in a cooperative manner to activate transcription from target genes.

[Familial papillary carcinoma of the thyroid: biogenetic identification and clinical assessment of 4 families]. / Il carcinoma papillifero della tiroide a carattere familiare: determinazioni biogenetiche e valutazioni cliniche su quattro gruppi familiari.

The Authors report 9 patients who were affected by familial papillary carcinoma of thyroid These patients were members of 4 families and they were selected in a general group of 97 patients affected by papillary cancer of the thyroid who underwent surgery from 1991 to 1998. The 9 patients were 1st degree relatives: two sisters, two sisters, two sisters and three brothers. The clinical course was similar in patients whether familiar or sporadic group, but average age in first was 10 yrs lower than in the latter group. Functional cervical dissection was needed only one time by lymphatic metastasis. Observed survival was 100% (follow up 92-16 months) and no specific complication was reported. Thyreoglobulin value was less than normal in every patients. Ret linkage analysis was always performed and no rearrangement was found; in 4 patients APC gene was detected but it was never seen. Case studies are consistent with an autosomal dominant trait that shows an high penetrance if associated with a permissive codominant trait. The authors believe that are necessary further studies on this occurrence. In papillary thyroid cancer familiarity was observed in 9.6%, than authors propose that relatives of thyroid papillary cancer should be underwent to screening.

Circulating tumor cells in metastatic colorectal cancer: do we need an alternative cutoff?

PURPOSE: To assess the prognostic and predictive value of circulating tumor cells (CTCs) in metastatic colorectal cancer (mCRC) irrespective of detection level. MATERIALS AND METHODS: We evaluated the prognostic and predictive significance of CTC count at baseline and under treatment in 119 mCRC subjects and compared the standard cutoff (≥3 CTCs/7.5 mL to ≥1 CTCs/7.5 mL). RESULTS: An overall comparison was made between patients with 0, 1-2 and ≥3 CTC (median PFS 8, 4 and 5 months, respectively). Two poor prognostic groups were found, including patients with ≥1 CTCs before and during treatment and patients with 0 CTC at baseline who converted to ≥1 CTCs (p = 0.014). CONCLUSIONS: The presence of at least 1 CTC at baseline count is predictive for poor prognosis in mCRC patients. Patients with 1-2 CTC should be switched from the favorable prognostic group--conventionally defined by the presence of <3 CTC--to the unfavorable, deserving a more careful monitoring.