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Background and Aim: p.Cys282Tyr homozygosity is the prevalent genotype in (HFE)-related Hereditary Hemochromatosis with low penetrance and variable expression. However, liver cirrhosis and hepatocellular carcinoma remain the main causes of mortality in these patients. Detection of genetic modifiers identifying patients at risk for liver damage would be relevant for their clinical management. We evaluated proprotein convertase 7 (PCSK7) rs236918 as genetic marker of risk of liver fibrosis in an Italian cohort of p.Cys282Tyr homozygotes. Methods: Liver fibrosis was histologically assessed by Ishak score. We evaluated PCSK7 alleles and genotypes frequencies according to single or grouped staging scores: absent/mild fibrosis (stage: 0–2), moderate (stage: 3–4), and severe fibrosis/cirrhosis (stage: 5–6). Single nucleotide polymorphism genotyping was performed by restriction fragment length polymorphism or Taqman 5′-nuclease assays. Results: The rs236918 allele C frequency increased from stages 0–2 to 5–6 (7.1% vs 13.6%, vs 21.9%, P = 0.003). The wild-type genotype was significantly more frequent in the absent/mild fibrosis group (54.2%) compared with only 17% in patients with severe fibrosis/cirrhosis. At univariate proportional odds model, patients with GC + CC genotypes were 2.77 times (P = 0.0018) more likely to have worse liver staging scores than wild-type patients. In the adjusted analysis, odds ratio was 2.37 (P = 0.0218), and 2.56 (P = 0.0233) when the analysis was restricted to males. An exploratory mediation analysis suggested a direct effect of genotype on severe fibrosis/cirrhosis (odds ratio = 3.11, P = 0.0157), and a mild non-significant indirect effect mediated through iron accounting for 28%. Conclusions: These findings confirm that PCSK7 rs236918 C allele is a risk factor for cirrhosis development in Italian patients with HFE-Hemochromatosis.

Objectives: The International Registry of Lung Metastases was established in 1991 to assess the long-term results of pulmonary metastasectomy. Methods: The Registry has accrued 5206 cases of lung metastasectomy, from 18 departments of thoracic surgery in Europe ( n = 13), the United States ( n = 4) and Canada ( n = 1). Of these patients, 4572 (88%) underwent complete surgical resection. The primary tumor was epithelial in 2260 cases, sarcoma in 2173, germ cell in 363, and melanoma in 328. The disease-free interval was 0 to 11 months in 2199 cases, 12 to 35 months in 1857, and more than 36 months in 1620. Single metastases accounted for 2383 cases and multiple lesions for 2726. Mean follow-up was 46 months. Analysis was performed by Kaplan-Meier estimates of survival, relative risks of death, and multivariate Cox model. Results: The actuarial survival after complete metastasectomy was 36% at 5 years, 26% at 10 years, and 22% at 15 years (median 35 months); the corresponding values for incomplete resection were 13% at 5 years and 7% at 10 years (median 15 months). Among complete resections, the 5-year survival was 33% for patients with a disease-free interval of 0 to 11 months and 45% for those with a disease-free interval of more than 36 months; 43% for single lesions and 27% for four or more lesions. Multivariate analysis showed a better prognosis for patients with germ cell tumors, disease-free intervals of 36 months or more, and single metastases. Conclusions: These results confirm that lung metastasectomy is a safe and potentially curative procedure. Resectability, disease-free interval, and number of metastases enabled us to design a simple system of classification valid for different tumor types. (J Thorac Cardiovasc Surg 1997;113:37-49)

In non small cell lung cancer (NSCLC) patients undergoing surgery after induction chemotherapy, all mediastinal lymphnodes potentially involved by tumor should be resected whenever possible. Paratracheal bilateral lymphadenectomy for left sided tumors can be disabling, i.e. median sternotomy plus a thoracotomy to reach the subcarinal region. From the right side, an extensive ipsilateral dissection is feasible through a standard thoracotomy, but contralateral lymphnodes, especially in the left hilum and aortopulmonary window are considered inaccessible. A technical tip is shown to reach and dissect the left paratracheal and aortopulmonary window nodes through a simple right thoracotomy in right-lung cancer. The procedure has been carried out in 3 cases and proved to be technically feasible. The value of such a procedure as to staging accuracy, local disease control and survival should be evaluated in a clinical trial setting.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. Stereotactic body irradiation offers a non-invasive treatment modality for patients with early stage NSCLC who are not amenable to surgery or other invasive approaches because of their poor medical condition.Forty-three inoperable patients with NSCLC were treated with SBRT at our institution. A mean total dose of 30.5 Gy in 1-4 fractions was applied. The median follow-up duration was 14 months (range 6-36 months).The actuarial survival at two years was 53%: two patients died from cancer progression whereas a further 8 patients died from comorbidities. Acute toxicity was practically absent, with 7 (16.3%) patients suffering from grade 1 symptoms and two from (4.6%) grade II effects. At the time of this report, only 1 patient had grade II and 6 patients (13.9%) grade I chronic symptoms.Our results compare favourably with recently published studies and confirm that stereotactic radiotherapy has the potential to produce high local control rates with a low risk of lung toxicity in patients not amenable to curative resection. The low grade of side-effects is encouraging for shortening the treatment using a greater dose per fraction.

Background. Several inherited diseases cause hyperferritinemia with or without iron overload. Differential diagnosis is complex and requires an extensive work-up. Currently, a clinical-guided approach to genetic tests is performed based on gene-by-gene sequencing. Although reasonable, this approach is expensive and time-consuming and Next Generation Sequencing (NGS) technology may provide cheaper and quicker large-scale DNA sequencing. Methods. We analysed 36 patients with non-HFE-related hyperferritinemia. Liver iron concentration was measured in 33 by magnetic resonance. A panel of 25 iron related genes was designed using SureDesign software. Custom libraries were generated and then sequenced using Ion Torrent PGM. Results. We identified six novel mutations in SLC40A1, three novel and one known mutation in TFR2, one known mutation and a de-novo deletion in HJV, and a novel mutation in HAMP in ten patients. In silico analyses supported the pathogenic role of the mutations. Conclusions. Our results support the use of an NGS-based panel in selected patients with hyperferritinemia in a tertiary center for iron metabolism disorders. However, 26 out of 36 patients did not show genetic variants that can individually explain hyperferritinemia and/or iron overload suggesting the existence of other genetic defects or gene-gene and gene-environment interactions needing further studies.

Background and Aim: HFE-related Hemochromatosis (HH) is characterized by marked phenotype heterogeneity, probably due to the combined action of acquired and genetic factors. Among them, GNPAT rs11558492 was proposed as genetic modifier of iron status, but results are still controversial. To shed light on these discrepancies, we genotyped 298 Italian p.C282Y homozygotes and 169 healthy controls. Material and Methods: Allele and genotype frequencies were analysed and compared with those reported in Exome Variant Server (EVS). To explore the role of rs11558492 as a potential modifier of iron status, serum ferritin (SF), liver iron concentration (LIC) and iron removed (IR) were studied according to allele and genotype frequencies. In addition, the effect of the SNP on liver fibrosis was examined comparing patients with absent/mild-moderate fibrosis to those with severe fibrosis-cirrho-sis. Results: GNPAT rs11558492 minor allele (G) frequency (MAF) was 20.3% in HFE-HH, 17.2% in controls and 20.6% in EVS database. Genotype frequencies were 64% and 69.2% (AA), 31.2% and 27.2% (AG), 4.8% and 3.6% (GG) in HFE-HH and controls, respectively. No significant differences were found comparing genotype and allele frequencies even selecting subgroups of only-males with extreme phenotypes and low alcohol intake. SF, IR and LIC levels did not significantly differ according to rs11558492 genotypes. Also, MAF did not differ between patients with absent/mild fibrosis and severe fibrosis/cirrhosis. Conclusions: Our findings indicate that GNPAT rs11558492 is not a major modifier of iron status and is not associated with liver fibrosis in HFE-HH patients.

Hepcidin transcription is strongly down-regulated under hypoxic conditions, however whether hypoxia inhibits hepcidin directly or indirectly is still unknown. We investigated the time course of hypoxia-mediated hepcidin down-regulation in vivo in healthy volunteers exposed to hypobaric hypoxia at high altitude and, based on the hypothesis that circulating factors are implicated in hepcidin inhibition, we analyzed the effect of sera of these volunteers exposed to normoxia and hypoxia on hepcidin expression in Huh-7 cell lines. Hypoxia led to a significant hepcidin down-regulation in vivo that was almost complete within 72 h of exposure and followed erythropoietin induction. This delay in hepcidin down-regulation suggests the existence of soluble factor/s regulating hepcidin production. We then stimulated HuH-7 cells with normoxic and hypoxic sera to analyze the effects of sera on hepcidin regulation. Hypoxic sera had a significant inhibitory effect on hepcidin promoter activity assessed by a luciferase assay, although the amount of such decrease was not as relevant as that observed in vivo. Cellular mRNA analysis showed that a number of volunteers' sera inhibited hepcidin expression, concurrently with ID1 inhibition, suggesting that inhibitory factor(s) may act through the SMAD-pathway.

Although hyperferritinemia may be reflective of elevated total body iron stores, there are conditions in which ferritin levels are disproportionately elevated relative to iron status. Autosomal dominant forms of hyperferritinemia due to mutations in the L−ferritin IRE or in A helix of L−ferritin gene have been described, however cases of isolated hyperferritinemia still remain unsolved. We describe 12 Italian subjects with unexplained isolated hyperferritinemia (UIH). Four probands have affected siblings, but no affected parents or offspring. Sequencing analyses did not identify casual mutations in ferritin gene or IRE regions. These patients had normal levels of intracellular ferritin protein and mRNA in peripheral blood cells excluding pathological ferritin production at transcriptional and post-transcriptional level. In contrast with individuals with benign hyperferritinemia caused by mutations affecting the ferritin A helix, low rather than high glycosylation of serum ferritin was observed in our UIH subjects compared with controls. These findings suggest that subjects with UIH have a previously undescribed form of hyperferritinemia possibly attributable to increased cellular ferritin secretion and/or decreased serum ferritin clearance. The cause remains to be defined and we can only speculate the existence of mutations in gene/s not directly implicated in iron metabolism that could affect ferritin turnover including ferritin secretion.

BACKGROUND The effects of multimodality treatment on the survival of patients with esophageal carcinoma are unclear. The authors performed a prospective, Phase II study to assess the long-term results of chemotherapy plus radiotherapy (RT) on patients with esophageal squamous cell carcinoma. METHODS Of 106 consecutive patients who were recruited between 1985 and 1992, 101 patients were evaluable. Cisplatin (100 mg/m2 per day) on Day 1 and fluorouracil (1000 mg/m2 per day) on Days 1–4 were given for two cycles, with concomitant RT (30 grays [Gy] in 15 fractions) over 19 days. Patients with potentially resectable tumors were then assessed for curative surgery; the other patients received two more courses of chemotherapy and further RT (20 Gy in 10 fractions). RESULTS Of 40 patients who were candidates for surgery, 32 patients underwent surgery, and 24 patients had complete resection; 8 patients (25%) had no residual tumor in the specimen, and 12 patients (37%) had microscopic foci only. Surgical mortality was high (22%). Of 61 nonsurgical patients, 37 patients (61%) achieved complete clinical remission, and 14 patients (23%) achieved partial remission. The median survival for the entire series was 15 months (range, 1–136 months). The overall survival rate was 22% at 5 years and 12% at 10 years. At 10 years, freedom from disease progression was similar in the two groups (24%), whereas the median survival (22 months vs. 12 months) and the overall survival rates (17% vs. 9%) were better in nonsurgical patients compared with surgical patients, respectively, probably in relation to high surgical mortality. The larynx was preserved in 28% of 32 patients with cervical disease sites, with a 10-year disease free survival rate of 31%. Three deaths were attributed to nonsurgical treatments. CONCLUSIONS Careful multidisciplinary pretreatment evaluation can identify patients who are ineligible for surgery without compromising long-term results. For patients with inoperable disease, chemoradiotherapy can produce relatively good long-term results. The combined approach without surgery can permit laryngeal preservation in a useful fraction of patients. Cancer 2002;94:352–61. © 2002 American Cancer Society.

Hepcidin is a regulatory hormone that plays a major role in controlling body iron homeostasis. Circulating factors (holotransferrin, cytokines, erythroid regulators) might variably contribute to hepcidin modulation in different pathological conditions. There are few studies analysing the relationship between hepcidin transcript and related protein expression profiles in humans. Our aims were: a. to measure hepcidin expression at either hepatic, serum and urinary level in three paradigmatic iron overload conditions (hemochromatosis, thalassemia and dysmetabolic iron overload syndrome) and in controls; b. to measure mRNA hepcidin expression in two different hepatic cell lines (HepG2 and Huh-7) exposed to patients and controls sera to assess whether circulating factors could influence hepcidin transcription in different pathological conditions. Our findings suggest that hepcidin assays reflect hepatic hepcidin production, but also indicate that correlation is not ideal, likely due to methodological limits and to several post-trascriptional events. In vitro study showed that THAL sera down-regulated, HFE-HH and C-NAFLD sera up-regulated hepcidin synthesis. HAMP mRNA expression in Huh-7 cells exposed to sera form C-Donors, HFE-HH and THAL reproduced, at lower level, the results observed in HepG2, suggesting the important but not critical role of HFE in hepcidin regulation.

Iron is tightly connected to oxygen homeostasis and erythropoiesis. Our aim was to better understand how hypoxia regulates iron acquisition for erythropoiesis in humans, a topic relevant to common hypoxia-related disorders. Forty-seven healthy volunteers participated in the HIGHCARE project. Blood samples were collected at sea level and after acute and chronic exposure to high altitude (3400-5400 m above sea level). We investigated the modifications in hematocrit, serum iron indices, erythropoietin, markers of erythropoietic activity, interleukin-6, and serum hepcidin. Hepcidin decreased within 40 hours after acute hypoxia exposure (P < .05) at 3400 m, reaching the lowest level at 5400 m (80% reduction). Erythropoietin significantly increased (P < .001) within 16 hours after hypoxia exposure followed by a marked erythropoietic response supported by the increased iron supply. Growth differentiation factor-15 progressively increased during the study period. Serum ferritin showed a very rapid decrease, suggesting the existence of hypoxia-dependent mechanism(s) regulating storage iron mobilization. The strong correlation between serum ferritin and hepcidin at each point during the study indicates that iron itself or the kinetics of iron use in response to hypoxia may signal hepcidin down-regulation. The combined and significant changes in other variables probably contribute to the suppression of hepcidin in this setting.

Platinum microquantities were determined in plasma of patients affected by lung carcinoma during treatment with radiotherapy (RT) and concurrent low-dose continuous infusion of cis-dichlorodiammineplatinum(II) (CDDP). RT was given at 50 Gy in continuous course; CDDP was continuously infused at 4 mg/m(2) daily for 100 h/week for 5 weeks, and the infusions were separated by 68 h of rest. The percentage of free drug versus total drug in plasma was about 3%. It did not vary with therapy duration and was not significantly different from that found in 5-day continuous infusions at much higher daily doses. Nevertheless, maximal values of free Pt in plasma were very low and agreed with the low level of CDDP toxicity encountered on the present administration schedule.

Out of 52 consecutive patients resected for small cell lung carcinoma (SCLC) from 1976 to 1986, 19 were selected because they underwent nonradical surgery, 10 of them for locoregional spread and 9 for distant metastases. Of the former subset all received postoperative radiotherapy and 8 chemotherapy also. Three patients are alive and disease-free 37, 56 and 91 months after resection. Four patients had a distant recurrence, and 3 a locoregional failure. Patients of the latter subgroup received chemotherapy in 7 instances. None survived more than 16 months, distant metastases being the cause of death. In these patients NO status was associated with 13.3 months of mean survival, N1 with 8.5 months, and N2 with 6.7 months. Surgery and adjuvant treatments seem effective in achieving local control of SCLC despite nonradical resections. Tumor burden at locoregional sites does not preclude the possibility of long term survival.

From 1981 to 1986, 17 patients with resected small cell lung carcinoma (SCLC) staged as I or II according to the new TNM classification were recruited for a prospective study to evaluate the effctiveness of surgery and postoperative chemotherapy (plus locoregional radiotherapy only when a nonradical resection was accomplished) in the treatment of early stages of the disease. Six patients received full protocol chemotherapy (6 courses) and 8 a mean of 79.1% of the planned courses. Three patients received non adjuvant treatment. Locoregional radiotherapy for residual disease was administered in 2 cases. One patient died for myelosuppression due to chemotherapy and 10 for recurrences of cancer, all within the 20th postoperative month. Metastases accounted 80% of overall recurrences. Six patients were alive and tumor-free at 18, 22, 39, 44, 47 and 51 months from resection. Actuarial observed 3-year survival was 32%.