Clinical relevance of gene mutations and rearrangements in advanced differentiated thyroid cancer.

BACKGROUND: Tumor genotyping is becoming crucial to optimize the clinical management of patients with advanced differentiated thyroid cancer (DTC); however, its implementation in clinical practice remains undefined. We herein report our single-center experience on molecular advanced DTC testing by next-generation sequencing approach, to better define how and when tumor genotyping can assist clinical decision making. MATERIALS AND METHODS: We retrospectively collected data on all adult patients with advanced DTC who received molecular profiling at the IRCSS Sant'Orsola-Malpighi Hospital from 2008 to 2022. The genetic alterations were correlated with radioactive iodide refractory (RAI-R), RAI uptake/disease status, and time to RAI resistance (TTRR) development. RESULTS: A significant correlation was found between RAI-R development and genetic alterations (P = 0.0001). About 48.7% of RAI-R cases were positive for TERT/TP53 mutations (as both a single event and comutations with other driver gene alterations, such as BRAF mutations, RAS mutations, or gene fusions), while the great majority of RAI-sensitive cases carried gene fusions (41.9%) or were wild type (WT; 41.9%). RAI uptake/disease status and time to TTRR were significantly associated with genetic alterations (P = 0.0001). In particular, DTC with TERT/TP53 mutations as a single event or as comutations displayed a shorter median TTRR of 35.4 months (range 15.0-55.8 months), in comparison to the other molecular subgroups. TERT/TP53 mutations as a single event or as comutations remained independently associated with RAI-R after Cox multivariate analysis (hazard ratio 4.14, 95% CI 1.51-11.32; P = 0.006). CONCLUSIONS: Routine testing for genetic alterations should be included as part of the clinical workup, for identifying both the subset of more aggressive tumors and the subset of tumors harboring actionable gene fusions, thus ensuring the appropriate management for all patients with advanced DTC.

Early and late virological monitoring of cytomegalovirus, Epstein-Barr virus, and human herpes virus 6 infections in small bowel/multivisceral transplant recipients.

BACKGROUND: Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) are the major causes of graft failure and posttransplantation mortality among small bowel and multivisceral transplantations (SB/MVT). Little is known about human herpes virus 6 (HHV-6) infections in transplant recipients. STUDY PURPOSE: The purposes of this study were to analyze the clinical relevance of CMV, EBV, and HHV-6 infections after small bowel transplantation and to establish whether routine monitoring for HHV-6 infection should be recommended for the prevention of severe complications in this population. METHODS: Ten adult patients were monitored based on CMV, EBV, and HHV6 DNA quantifications in blood and biopsy tissue samples. Three patients were monitored for at least 5 months (early period) and 7 patients were monitored for 1 to 5 years after transplantation (late period). RESULTS: In the early period, despite prophylaxis all 3 patients developed symptomatic CMV infections: 1 fever/diarrhea, 1 enteritis and rejection, as well as 1 fever and pneumonia. Only 1 patient developed EBV and HHV-6 infections. The average time of onset of CMV infection was 3 months after transplantation and only 24 days for HHV6 infection. In the late period, of the 7 SB/MVT recipients only 1 developed an EBV infection at 2 years after transplantation. No CMV or HHV-6 infections were identified in any patient. CONCLUSIONS: CMV infection is a major cause of organ disease and rejection in the early period after transplantation. EBV infection in adult recipients must be considered also in the late period, particularly in association with severe immunosuppression. Because HHV-6 infection occurs earlier than CMV/EBV, it may serve as an indicator for more intense virological surveillance.

Acute cellular rejection monitoring after intestinal transplant: utility of serologic markers and zoom videoendoscopy as support of conventional biopsy and clinical findings.

Acute cellular rejection (ACR) episodes in intestinal transplant recipients are diagnosed by histologic and clinical findings. We have applied zoom video endoscopy and the use of serologic markers granzyme B (GrB) and perforin (PrF) to monitor rejection together with conventional tools. Seven hundred eighty-two blood samples (obtained at the time of the biopsy) collected from 34 recipients for GrB/PrF upregulation were positive among 64.9% of ACRs during a 3-year follow-up. Considering only the first year results posttransplantation, it reached 73.1% of rejection events. Zoom videoendoscopy was used by our group in 29 recipients of isolated intestine (n = 24) or multivisceral transplantations (n = 5) to enable observation of villi and crypt areas. From more than 270 procedures, 84% of the zoom findings agreed with the histologic results, namely, a specificity of 95%. In fact, during ongoing ACR, villi were altered in 80% of cases. Both procedures were helpful to support conventional histologic findings and clinical symptoms of ACR in intestinal transplant recipients.

Two years' experience of acute rejection monitoring of intestinal transplant recipients by real-time PCR assessment of granzyme B and perforin up-regulation: considerations on diagnostic accuracy.

Granzyme B (GrB) and perforin are promising immunological markers to predict acute rejection of transplanted organs. Based on 2 years of experience with molecular monitoring on peripheral blood samples, we investigated the diagnostic accuracy of GrB/perforin gene up-regulation using real-time polymerase chain reaction (PCR) for prediction of acute cellular rejection (ACR) in intestinal transplantation recipients. Histology used as the reference standard. According to our definition of disease positivity (anything other than ACR score 0), GrB/perforin up-regulation showed 84% specificity but only 49% sensitivity. However, among the 26 false-negatives, 12 (46%) had an ACR score 1, which is indeterminate for rejection and no associated clinical manifestations; a further 10 (39%) had a score of 2 following rejection therapy (a confounder for GrB/perforin analysis). Thus only 4 (15%) false-negatives were actually associated with the onset of robust acute rejection. These data suggest that real-time PCR analysis for GrB/perforin up-regulation might play a role along with clinical criteria for detection of presymptomatic acute rejection episodes in intestinal recipients who require immediate endoscopy and pathological examination, especially during long-term follow-up.

Expression of connective tissue growth factor is a prognostic marker for patients with intrahepatic cholangiocarcinoma.

BACKGROUND AND AIMS: Connective tissue growth factor is a member of the 'CCN' protein family. Consistent with its profibrotic properties, it is over-expressed in several human epithelial malignancies. PATIENTS AND METHODS: We have retrospectively evaluated by immunohistochemistry the presence of connective tissue growth factor in archival tissues from 55 resected intrahepatic cholangiocarcinomas and compared its expression to the main pathological parameters, disease free and overall survival. RESULTS: Tumours were scored as high and low/absent expressers (> or =50%, 0-50% cells, respectively). Thirty-three of 55 cholangiocarcinomas (60%) were high and 22 (40%) low expressers. No significant correlation was found between connective tissue growth factor and tumour grade, tumour location, vascular and perineural invasion. Eighteen of 22 (82%) low/absent expressers and 12/33 (36%) high expressers had recurrence of disease (P=0.001). Low/absent expressers showed a poor disease free and overall survival compared with the higher expressers (P<0.001). Vascular invasion was related to tumour recurrence (P=0.025) and to decreased disease free survival (P<0.05). During proportional hazard regression analysis, only connective tissue growth factor was found to influence disease free survival (P=0.01). CONCLUSIONS: Expression of connective tissue growth factor is an independent prognostic indicator of both tumour recurrence and overall survival for intrahepatic cholangiocarcinoma patients regardless of tumour location, tumour grade, vascular and perineural invasion.

Potential of real-time PCR assessment of granzyme B and perforin up-regulation for rejection monitoring in intestinal transplant recipients.

Granzyme B (GrB) and perforin are promising markers to predict acute rejection episodes of transplanted organs. Having recently reported that immunohistochemical expression of GrB/perforin correlates with histologically assessed acute cellular rejection (ACR) episodes in intestinal transplantation recipients, herein we have additionally explored the potential of real-time polymerase chain reaction (PCR) assessment of GrB/perforin gene up-regulation in peripheral blood mononuclear cells. Both immunohistochemical evaluation of GrB/perforin expression and real-time PCR assessment of up-regulation, which was defined as a 2-fold increase with respect to "basal" levels during maintenance immunosuppressive protocols, were performed among a population of 23 intestinal transplant recipients under routine surveillance, in addition to histological analysis of ACR. The ACR scores showed direct relationships both with GrB/perforin immunohistochemistry (IHC) scores (P < .001) and with gene up-regulation by real-time PCR (P = .004). Furthermore, real-time PCR upregulation was associated with the IHC score (P < .001). A preliminary analysis of diagnostic accuracy-performed to gain information to plan future studies-indicated that when using histological assessment as the reference technique, our current definition of PCR up-regulation provided good specificity (84%) but insufficient sensitivity (44%) for a noninvasive prediction of ACR. The results of this pilot study suggested that real-time PCR analysis of GrB/perforin upregulation may help therapeutic decision making, and have the potential for detection of presymptomatic rejection. More extensive studies must investigate strategies to improve the sensitivity of the analyses of GrB/perforin up-regulation.

Granzyme B and perforin as predictive markers for acute rejection in human intestinal transplantation.

In human heart and kidney transplantations, granzyme B (GrB) and perforin have both been shown to be predictive markers for acute cellular rejection (ACR). We investigated the tissue expression and possible relationship of GrB and perforin to the clinical outcome, histopathology, and function of intestinal transplants. In 13 consecutive patients undergoing small intestine transplantation, histologic/immunohistochemical rejection monitoring was performed together with GrB and perforin immunostaining (score "0", 0%-10% positive lymphocytes; "1", 10%-25%; "2", 25%-50%; "3", >50%). Eleven patients are currently alive and well. All 11 had at least one episode of ACR: one patient had 6 episodes of severe ACR requiring retransplantation; the remaining 10 experienced only mild or moderate rejection. Both GrB and perforin were always co-expressed. A highly significant correlation was observed between GrB/perforin scores and histological severity of ACR (Pearson's coefficient, R < 0.0009). Interestingly, score 3 GrB/perforin immunostaining was recorded only in the context of severe ACR; all the histologically negative or "indeterminate" biopsies (n = 6) taken from a single affected patient showed GrB/perforin scores of 1 or 2. By contrast, none of the other tested histologically negative/"indeterminate" biopsies (n = 350), including those performed during graft stabilization, had raised GrB or perforin scores. We conclude that in intestinal transplantation recipients, a direct correlation seems to exist between histologically confirmed ACR and raised GrB/perforin immunohistochemical scores. Our findings suggest the need to investigate the possibility of predicting ACR by routine serum polymerase chain reaction (PCR) monitoring, which would reduce discomfort to patients.

Investigation of ErbB1 and ErbB2 expression for therapeutic targeting in primary liver tumours.

BACKGROUND: Molecular targets are needed for primary liver tumours. AIMS: ErbB1 and ErbB2 expression was analysed in neoplastic and surrounding tissue in surgical specimens from 52 hepatocellular carcinomas and 48 intrahepatic cholangiocarcinomas, randomly chosen from cases surgically treated in this institution. METHODS: ErbB1 and ErbB2 expression were evaluated immunohistochemically, the latter by Herceptest. Gene amplification of ErbB2 was tested by chromogenic in situ hybridisation. RESULTS: In normal/cirrhotic non-neoplastic tissue, the ErbB1 (but not ErbB2) antibody commonly stained normal hepatocytes and mature intrahepatic ducts. In neoplastic tissue, moderate/strong ErbB1 immunostaining occurred in 43/52 (85%) hepatocellular carcinomas and 39/48 (81%) intra-hepatic cholangiocarcinomas. With ErbB2 Herceptest, 0/52 (0%) hepatocellular carcinomas and 2/48 (4%) intra-hepatic cholangiocarcinomas had treatable scores of 2+/3+ (chromogenic in situ hybridisation confirmed gene amplification in the latter two cases only). Neither ErbB1 nor ErbB2 expression correlated with any of the main clinical-pathologic features or survival. CONCLUSIONS: Although not related to prognosis, ErbB1 could be a molecular target in a large percentage of patients with hepatocellular carcinoma or intrahepatic cholangiocarcinoma. Inclusion of anti-ErbB1 drugs such as ZD 1839 and c225 (and possibly also anti-ErbB2 drugs like Trastuzumab for a small subset of patients) in clinical trials is suggested.

Low p27 expression is an independent predictor of survival for patients with either hilar or peripheral intrahepatic cholangiocarcinoma.

PURPOSE AND EXPERIMENTAL DESIGN: The prognosis for intrahepatic cholangiocarcinoma (ICC) depends mainly on the feasibility of complete surgical resection. In the absence of demonstrated biological predictors of survival, we evaluated the prognostic value of the cyclin-dependent kinase inhibitor p27 by immunohistochemistry in a series of routine specimens from 47 ICC patients, 22 with the hilar and 25 with the peripheral subtype. Proliferation rate was also evaluated in the same cases by the MIB1 index. Tumors were scored as high, low, and negative p27 expressers (> or =50%, <50%, and no positive nuclei, respectively). RESULTS: High, low, and negative p27 expression was recorded in 18 (38%), 17 (36%), and 12 (26%) cases, respectively. No significant correlation was found between p27 expression and gender, age, tumor grade, tumor location, vascular or perineural invasion, or proliferative index. Tumors with low or absent p27 expression were associated with poor survival compared with the high-expresser group. Kaplan-Meier curves comparing different p27 expression levels with survival showed highly significant separation (P < 0.0001, log-rank test). With univariate Cox proportional hazard regression, only p27 score among all of the parameters was found to influence survival (P = 0.0003). CONCLUSION: We conclude that in ICC, low or absent p27 expression can predict poor survival, regardless of tumor location, pathological features, and tumor proliferation. Immunohistochemical detection of p27 on routine sections may provide the first biological prognostic marker for ICC, thus influencing the therapeutic strategies for these patients.

Her-2-neu expression and progression toward androgen independence in human prostate cancer.

BACKGROUND: Human prostate cancers are initially androgen dependent but ultimately become androgen independent. Overexpression of the Her-2-neu receptor tyrosine kinase has been associated with the progression to androgen independence in prostate cancer cells. We examined the expression of Her-2-neu in normal and cancerous prostate tissues to assess its role in the progression to androgen independence. METHODS: Prostate cancer tissue sections were obtained from 67 patients treated by surgery alone (UNT tumors), 34 patients treated with total androgen ablation therapy before surgery (TAA tumors), and 18 patients in whom total androgen ablation therapy failed and who developed bone metastases (androgen-independent [AI] disease). The sections were immunostained for Her-2-neu, androgen receptor (AR), prostate-specific antigen (PSA), and Ki-67 (a marker of cell proliferation) protein expression. Messenger RNA (mRNA) levels and gene amplification of Her-2-neu were examined by RNA in situ hybridization and fluorescent in situ hybridization(FISH), respectively, in a subset of 27 tumors (nine UNT, 11 TAA, and seven AI). All statistical tests were two-sided. RESULTS: Her-2-neu protein expression was statistically significantly higher in TAA tumors than in UNT tumors with the use of two different scoring methods (P =.008 and P =.002). The proportion of Her-2-neu-positive tumors increased from the UNT group (17 of 67) to the TAA group (20 of 34) to the AI group (14 of 18) (P<.001). When compared with UNT tumors, tumor cell proliferation was higher in AI tumors (P =.014) and lower in TAA tumors (P<.001). All tumors expressed AR and PSA proteins. Although Her-2-neu mRNA expression was high in TAA and AI tumors, no Her-2-neu gene amplification was detected by FISH in any of the tumor types. CONCLUSIONS: Her-2-neu expression appears to increase with progression to androgen independence. Thus, therapeutic targeting of this tyrosine kinase in prostate cancer may be warranted.

Acquired expression of p27 is a favorable prognostic indicator in patients with hepatocellular carcinoma.

The p27 cyclin-dependent kinase inhibitor is a negative regulator of cell-cycle progression. In many human epithelial malignancies, decreased expression of p27 correlates with high grade, early recurrence, and poor prognosis. To evaluate the prognostic significance of p27 in hepatocellular carcinoma (HCC), we studied 54 HCCs along with corresponding nontumoral tissue. Immunohistochemistry (IHC) and Western blot (WB) analysis before and after immunoprecipitation with Cdk2 were performed on paraffin-embedded tissues and protein homogenates, respectively, to compare localization and expression of the p27 protein and to determine the total and active (Cdk2-bound) fractions of p27. Correlations were analyzed between IHC-assessed levels of p27, survival, and major clinical and pathological variables. IHC revealed no p27 expression in the majority of hepatocytes from normal and cirrhotic liver, whereas 14 HCCs (26%) were high p27 expressers (>50% positive cells), 26 (48%) low expressers (<50% positive cells), and 14 (26%) negative. High IHC signals of p27 correlated with Cdk2-bound p27 as assessed by immunoprecipitation-WB; by contrast, WB alone displayed similar levels of p27 protein in all normal and tumoral samples. High IHC p27 expression correlated with prolonged survival (P = 0.027), whereas the presence of cirrhosis was associated with poor outcome (P = 0.029). We conclude that with respect to their nonneoplastic counterparts, a subset of HCCs acquire significant p27 expression and that high expression of p27 is a favorable independent prognostic parameter for HCC.

Stereotactic breast biopsy in a community hospital setting.

Our institution began using a stereotactic core needle breast biopsy system for mammographically detected breast lesions in November 1996. The system consists of a LORAD stereo imaging table and an 11-gauge vacuum-assisted mammotome (Biopsys Medical, Irvine, CA). All biopsies were performed with the combined efforts of a radiologist and a surgeon. Three hundred sixteen biopsies were attempted in 279 patients, with multiple biopsies in 31 patients. Indications included microcalcifications in 52 per cent of patients and a mass in 48 per cent of patients. Biopsy was unsuccessful in 20 patients (6.3%). Pathologic diagnoses included invasive ductal carcinoma (19 patients), invasive lobular carcinoma (2), ductal carcinoma in situ (17), atypical ductal hyperplasia (8), atypical lobular hyperplasia (1), fibroadenoma (40), lymph node (7), and benign (202). Invasive cancer, in situ carcinoma, or atypical hyperplasia was diagnosed in 46 (15.6%) lesions. Thirty-six patients had open biopsies. The core biopsy diagnosis was correct in 27 lesions, unable to be confirmed in six cases and changed in four cases, with three lesions upgraded and one case downgraded. The 11-gauge vacuum-assisted mammotome provides excellent accuracy for diagnosing mammographic abnormalities. A combined effort between radiology and surgical services is an effective way of using the stereotactic biopsy system.

High levels of BCL-2 messenger RNA detected by in situ hybridization in human hepatocellular and cholangiocellular carcinomas.

Immunocytochemistry has indicated that, in the liver, the bcl-2 gene is generally expressed in bile duct cells and tumors of biliary origin. Both in situ hybridization and immunocytochemistry were used to analyze the expression of bcl-2 messenger RNA (mRNA) and its protein product (Bcl-2) in the tissue of 50 pure primary liver tumor (PLT) specimens including 40 hepatocellular carcinoma (HCC) specimens and 10 cholangiocellular carcinoma (CC) specimens. The phenotype of the tumors expressing bcl-2 was confirmed by immunocytochemical assessment of the cytokeratin (CK) profile (CK8, CK18, CK7, and CK19). Whereas positive immunoreaction with the anti-Bcl-2 MoAb was revealed in only 8 (20%) of 40 HCC specimens and 1 (10%) of 10 CC specimens, high contents of bcl-2 mRNA were found in 26 (65%) of 40 HCC specimens and 9 (90%) of 10 CC specimens. Regarding the CK profile, only 25 (62%) of 40 HCC specimens showed pure hepatocytic lineage (CKs 8-18), whereas among the remaining 15 HCC specimens, positivity for either CK7 (12 specimens) or CK19 (5 specimens) was observed. All 10 CC specimens stained with CKs 8-18-19, and 8 of 10 stained with CK 7 as well. These results indicate that PLTs display a greater expression of bcl-2 mRNA than of the Bcl-2 protein. Furthermore, CK profile assessment confirmed that bcl-2 expression is not confined to liver tumors of biliary origin. In the absence of a well-demonstrated post-transcriptional control of the gene, the authors propose the detection of bcl-2 mRNA by in situ hybridization as a possible alternative method for assessing the expression of bcl-2 mRNA in PLT.

Unreliability of carotenoid electrochromism for the measure of electrical potential differences induced by ATP hydrolysis in bacterial chromatophores.

ATP hydrolysis induces the activation of the proton ATPase in chromatophores of Rhodobacter capsulatus supplemented with nigericine and 50 mM K+ (i.e. when delta pH < 0.2 units). The value of transmembrane electric potential (delta phi) driving this activation was measured using three different approaches: carotenoid electrochromism, uptake of SCN- and responses of the dye oxonol VI. The value of delta phi calculated from the SCN- uptake, on the basis of an internal volume determined experimentally, was about 140 mV, while that indicated by the electrochromic signal ranged between 35 and 70 mV. Only the value indicated by SCN- distribution is consistent with the energetic requirement for the activation of H(+)-ATPase.

DTIC therapy in patients with malignant intra-abdominal neuroendocrine tumors.

Malignant intra-abdominal neuroendocrine tumors are rare; consequently, a standard chemotherapeutic protocol for patients with unresectable disease has not been established. This prompted a review of our experience with dimethyltriazeno imidazole carboxamide (dacarbazine) (DTIC) treatment for these tumors. From 1976 to 1986, 14 patients were treated with DTIC for metastatic neuroendocrine tumors. There were seven men and seven women whose ages ranged from 19 to 76 years. Diagnoses included eight nonfunctioning islet-cell carcinomas, three retroperitoneal neuroendocrine tumors, two glucagonomas, and one ileal carcinoid. Before DTIC chemotherapy, four patients were treated with streptozotocin and 5-FU, and one was treated with cytoxan and methotrexate without response. Two patients who were initially treated with DTIC with no response were subsequently treated with streptozotocin and 5-FU without benefit. Standard treatment with DTIC consisted of monthly cycles of 250 mg/m2/day administered intravenously for 5 days. Seven patients had an objective response to DTIC with both improvement in quality of life and a decrease of more than 50% in tumor size on computerized tomography (CT) or liver scanning. Response duration ranged from 1 to 10 years. One patient with a glucagonoma was treated for two years and had no evidence of disease at laparotomy 7 years later. Four patients with nonfunctioning islet cell carcinoma had a positive response to DTIC, but three of these patients had tumor recurrence 3 to 6 years after treatment. Two patients with retroperitoneal neuroendocrine tumors had a positive response to DTIC treatment. One patient with a glucagonoma and one with a nonfunctioning islet-cell tumor had equivocal responses with transient clinical improvement but no objective changes in tumor size. Five patients did not respond; two were given DTIC therapy as a last resort and died 1 and 12 days later. Of the other three patients, two died 6 months and one 2 years after treatment. DTIC chemotherapy was effective in 50% of patients with intra-abdominal neuroendocrine tumors. Although DTIC therapy was associated with nausea, no major gastrointestinal, hematologic, or renal complications were noted. This favorable experience with DTIC chemotherapy for nonresectable intra-abdominal neuroendocrine tumors indicates that further clinical evaluation and use are warranted.

Use of a somatostatin analog (SMS 201-995) in the glucagonoma syndrome.

A long-acting somatostatin analog, SMS 201-995, is now available to treat the hormonal manifestations of islet cell tumors. We report its use in a patient with a metastatic glucagonoma refractory to conventional therapy. This patient, who was severely disabled by the rash of necrolytic migratory erythema and brittle diabetes mellitus, allowed us to evaluate the therapeutic efficacy of SMS 201-995 and to gain insight into the origin of the rash. SMS 201-995 was administered subcutaneously (.05 mg twice a day). The rash improved markedly within 48 hours and was completely resolved within 1 week of treatment. Insulin requirements decreased from 90 U/day to zero during the first week of treatment. Corresponding to improvement in clinical symptoms circulating glucagon levels showed a marked decrease. There was no substantial change in plasma or urinary levels of zinc or in plasma amino acid levels. When SMS 201-995 was stopped, the rash recurred within 36 hours and it improved within 48 hours of readministration. The rash and diabetes have remained well controlled during 8 months of therapy but no change in tumor size has been seen on CT scan. The rapid changes in the rash related to the administration of SMS 201-995 indicate that the pathogenesis of necrolytic migratory erythema is probably due to circulating hyperglucagonemia or some other hormonal substance produced by the tumor.