Incorporation of somatic BRAF mutation testing into an algorithm for the investigation of hereditary non-polyposis colorectal cancer.

Patients suspected on clinical grounds to have hereditary non-polyposis colorectal cancer (HNPCC) may be offered laboratory testing in order to confirm the diagnosis and to facilitate screening of pre-symptomatic family members. Tumours from an affected family member are usually pre-screened for microsatellite instability (MSI) and/or loss of immunohistochemical expression of mismatch repair (MMR) genes prior to germline MMR gene mutation testing. The efficiency of this triage process is compromised by the more frequent occurrence of sporadic colorectal cancer (CRC) showing high levels of MSI (MSI-H) due to epigenetic loss of MLH1 expression. Somatic BRAF mutations, most frequently V600E, have been described in a significant proportion of sporadic MSI-H CRC but not in HNPCC-associated cancers. BRAF mutation testing has therefore been proposed as a means to more definitively identify and exclude sporadic MSI-H CRC cases from germline MMR gene testing. However, the clinical validity and utility of this approach have not been previously evaluated in a familial cancer clinic setting. Testing for the V600E mutation was performed on MSI-H CRC samples from 68 individuals referred for laboratory investigation of suspected HNPCC. The V600E mutation was identified in 17 of 40 (42%) tumours showing loss of MLH1 protein expression by immunohistochemistry but in none of the 28 tumours that exhibited loss of MSH2 expression (P < 0.001). The assay was negative in all patients with an identified germline MMR gene mutation. Although biased by the fact that germline testing was not pursued beyond direct sequencing in many cases lacking a high clinical index of suspicion of HNPCC, BRAF V600E detection was therefore considered to be 100% specific and 48% sensitive in detecting sporadic MSI-H CRC amongst those cases showing loss of MLH1 protein expression, in a population of patients with MSI-H CRC and clinical features suggestive of HNPCC. Accordingly, we recommend the incorporation of BRAF V600E mutation testing into the laboratory algorithm for pre-screening patients with suspected HNPCC, whose CRCs show loss of expression of MLH1. In such tumours, the presence of a BRAF V600E mutation indicates the tumour is not related to HNPCC and that germline testing of MLH1 in that individual is not warranted. We also recommend that in families where the clinical suspicion of HNPCC is high, germline testing should not be performed on an individual whose CRC harbours a somatic BRAF mutation, as this may compromise identification of the familial mutation.

KIT immunohistochemistry and mutation status in gastrointestinal stromal tumours (GISTs) evaluated for treatment with imatinib.

AIMS: With the availability of effective but expensive treatment in the form of imatinib, accurate diagnosis of gastrointestinal stromal tumour (GIST) is extremely important. The aims of this study were: to describe the clinicopathological, immunohistochemical and molecular features of cases referred to a cancer centre with a possible diagnosis of GIST; to identify pitfalls in the performance and interpretation of KIT immunohistochemistry; to define the role of KIT mutation testing in making a diagnosis of GIST. METHODS AND RESULTS: Morphological review, KIT immunohistochemistry and mutation testing were performed on all cases referred with a diagnosis of GIST or where the diagnosis was under serious consideration on the basis of KIT immunopositivity with a view to treating with imatinib. Thirty-seven cases met the inclusion criteria. Of these, 26 were classified as GIST and 11 as non-GIST. Most GISTs showed strong diffuse membranous, cytoplasmic or paranuclear KIT immunopositivity. Some non-GISTs demonstrated patchy cytoplasmic KIT immunopositivity related to the immunohistochemical protocol used in the external laboratory, which led to erroneous diagnoses of GIST in nine (24%) cases. KIT mutations involving exons 11 or 9 were identified in 22 (88%) GISTs tested and none of the non-GISTs. CONCLUSIONS: An accurate diagnosis of GIST can be made on clinicopathological and immunohistochemical criteria without the need for mutational analysis in most cases, provided proper attention is paid to the immunohistochemical protocol used and, most importantly, control material. False-positive diagnoses of GIST potentially leading to inappropriate treatment with imatinib are more common than missed diagnoses.

Gastrointestinal stromal tumour treated with neoadjuvant imatinib.

This report describes a case of unresectable primary gastrointestinal stromal tumour (GIST) treated with imatinib on a neoadjuvant basis, before subsequent successful surgical resection. After six months of imatinib, computed tomography and positron emission tomography imaging demonstrated a significant size reduction and complete metabolic response to treatment, rendering the tumour resectable. Mutational analysis showed an activating KIT mutation in exon 11. The pathological appearance of the resected tumour was heterogeneous with extensive necrosis, cystic and myxoid change, extensive hypocellularity, and patchy foci of residual viable tumour. The implications for this management option of radiological, pathological, and molecular assessment are discussed.

Plasma granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor levels in critical illness including sepsis and septic shock: relation to disease severity, multiple organ dysfunction, and mortality.

OBJECTIVE: To define the circulating levels of granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) during critical illness and to determine their relationship to the severity of illness as measured by the Acute Physiology and Chronic Health Evaluation (APACHE) II score, the development of multiple organ dysfunction, or mortality. DESIGN: Prospective cohort study. SETTING: University hospital intensive care unit. PATIENTS: A total of 82 critically ill adult patients in four clinically defined groups, namely septic shock (n = 29), sepsis without shock (n = 17), shock without sepsis (n = 22), and nonseptic, nonshock controls (n = 14). INTERVENTIONS: None. MEASUREMENT AND MAIN RESULTS: During day 1 of septic shock, peak plasma levels of G-CSF, interleukin (IL)-6, and leukemia inhibitory factor (LIF), but not GM-CSF, were greater than in sepsis or shock alone (p < .001), and were correlated among themselves (rs = 0.44-0.77; p < .02) and with the APACHE II score (rs = 0.25-0.40; p = .03 to .18). G-CSF, IL-6, and UF, and sepsis, shock, septic shock, and APACHE II scores were strongly associated with organ dysfunction or 5-day mortality by univariate analysis. However, multiple logistic regression analysis showed that only septic shock remained significantly associated with organ dysfunction and only APACHE II scores and shock with 5-day mortality. Similarly, peak G-CSF, IL-6, and LIF were poorly predictive of 30-day mortality. CONCLUSIONS: Plasma levels of G-CSF, IL-6, and LIF are greatly elevated in critical illness, including septic shock, and are correlated with one another and with the severity of illness. However, they are not independently predictive of mortality, or the development of multiple organ dysfunction. GM-CSF was rarely elevated, suggesting different roles for G-CSF and GM-CSF in human septic shock.

Continuous plasmafiltration in sepsis syndrome. Plasmafiltration in Sepsis Study Group.

OBJECTIVE: To assess the effect of plasmafiltration (PF) on biochemical markers of inflammation, cytokines, organ dysfunction, and 14-day mortality in human sepsis. DESIGN: Multicenter, prospective, randomized, controlled clinical trial. SETTING: Seven university-affiliated intensive care units. PATIENTS: Thirty patients (22 adults, eight children) with new (<24 hrs) clinical evidence of infection and sepsis syndrome were enrolled. Fourteen of 30 (nine adults, five children) were randomized to PF. INTERVENTIONS: All patients received protocol-driven supportive intensive care, and those randomized to PF received continuous plasma exchange for 34 hrs using a hollow-fiber plasma filter. MEASUREMENTS AND MAIN RESULTS: Illness severity and risk of death were calculated with the Pediatric Risk of Mortality (children) and the Acute Physiology and Chronic Health Evaluation II (adults) scales. Plasma samples (0, 6, 24, and 48 hrs) were assayed for acute-phase proteins (albumin, globulin, C-reactive protein, alpha1-antitrypsin, haptoglobin), inflammatory mediators (complement fragment C3, thromboxane B2), and cytokines (interleukin-6, granulocyte colony-stimulating factor, leukemia inhibitory factor). Sieving coefficients were estimated from filtrate concentrations at 3 hrs. The two groups were matched for incidence of septic shock (13 of 14 vs. 11 of 16), refractory shock (three of 14 vs. six of 16), bacteremia (six of 14 vs. five of 16), severity of illness, and calculated risk of death (0.68 vs. 0.64). There was no difference in mortality. Eight of 14 PF patients (57%) and eight of 16 controls (50%) survived for 14 days (p = .73, Fisher's exact test). Multiple logistic regression revealed age (odds ratio, 16.4:1; 95% confidence interval, 2.12-infinity) and shock (10.6:1; 1.32-infinity) as significant predictors of death; plasmafiltration was associated with a nonsignificant reduction in the risk of death (odds ratio, 1.78:1; 95% confidence interval, 0.20-18.1). The mean (SD) number of organs failing in the first 7 days in the PF group was 2.57 (0.94) vs. 2.94 (0.85) in controls (p = .37, Mann-Whitney U test). Both groups had similarly elevated plasma concentrations of all inflammatory mediators except complement fragment C3 at study entry. Leukemia inhibitory factor was detectable in four patients only. PF did not influence mean concentrations of interleukin-6, granulocyte colony-stimulating factor, thromboxane B2, total white cell count, neutrophil count, or platelet count, but it was associated with significant reductions of alpha1-antitrypsin, haptoglobin, C-reactive protein, and complement fragment C3 in the first 6 hrs (p < .05). The sieving coefficients for all inflammatory mediators approached unity. CONCLUSIONS: PF caused a significant attenuation of the acute-phase response in sepsis. There was no significant difference in mortality, but there was a trend toward fewer organs failing in the PF group that suggests that this procedure might be beneficial.

Differential alterations in plasma colony-stimulating factor concentrations in meningococcaemia.

To determine whether circulating levels of any of the colony-stimulating factors (CSF) might contribute to the host response in severe sepsis, plasma concentrations of granulocyte CSF (G-CSF), granulocyte-macrophage CSF (GM-CSF), and macrophage CSF (M-CSF) were measured by immunoassays in 20 subjects with meningococcaemia, a bloodstream infection caused by Neisseria meningitidis, that has proven to be a valuable model to study the responses of other inflammatory mediators during sepsis and septic shock in humans. Plasma G-CSF concentrations were transiently elevated in most subjects during the early phase of meningococcaemia, and were higher in subjects with septic shock (mean +/- s.d. = 165 +/- 142 ng/ml, n = 9) compared with those who remained normotensive (mean +/- s.d. = 7 +/- 2 ng/ml, n = 10) (P < 0.05). Peak plasma G-CSF concentrations > 10 ng/ml were associated with the development of septic shock (P < 0.01), disseminated intravascular coagulation (P < 0.01), fulminant infection (P < 0.05), and a fatal outcome (P < 0.01). Plasma GM-CSF concentrations > 1 ng/ml were briefly present in subjects with life-threatening septic shock (1-15 ng/ml, n = 5), and were strongly associated with fulminant meningococcaemia (P < 0.01). Plasma M-CSF concentrations were marginally elevated in all subjects, but were not associated with complications related to or arising from sepsis-induced organ injury. This study demonstrates that plasma levels of G-CSF, GM-CSF and M-CSF show very different responses during meningococcaemia, changes which presumably reflect the different roles played by these mediators in sepsis and, potentially, in septic shock.

Leukemia inhibitory factor protects against experimental lethal Escherichia coli septic shock in mice.

Leukemia inhibitory factor (LIF) has recently been associated with septic shock in humans. In this study we sought to determine, in mice, the role of LIF in septic shock. During sublethal endotoxemia, serum LIF levels, as determined by radio-receptor competition assay, peaked at 2 h and were low (3 ng/ml), whereas in lethal Escherichia coli septic shock serum LIF levels rose progressively (> 30 ng/ml) in the premorbid phase coincident with the development of tissue injury. Single i.v. injections of high doses (up to 50 micrograms per mouse) of recombinant murine LIF had no obvious acute detrimental effects, whereas continued i.p. administration (30 micrograms per mouse per day) for 3-4 days induced a fatal catabolic state without evidence of preceding hemodynamic collapse or shock. Simultaneous or subsequent administration of high doses of LIF had no effect on mortality from sublethal and lethal E. coli septic shock, whereas prior administration conferred significant protection against lethality (P << 0.001 by log-rank test), an effect that was dose and interval dependent. This protective effect resembled endotoxin tolerance and was characterized by suppression of E. coli-induced serum tumor necrosis factor concentration (P < 0.05), reduction in the number of viable bacteria (P < 0.05), and prevention of sepsis-induced tissue injury. These observations suggest that systemic LIF production is part of the host response to both endotoxin and sepsis-induced tissue injury.

Leukemia inhibitory factor: association with intraamniotic infection.

OBJECTIVE: Our purpose was to investigate whether leukemia inhibitory factor is associated with intraamniotic infection. STUDY DESIGN: A comparative clinical study of amniotic fluid leukemia inhibitory factor concentrations was performed. RESULTS: Leukemia inhibitory factor was undetectable (< 1 ng/ml) by radioreceptor assay during normal pregnancy at midtrimester and at term. Among women in labor those with intraamniotic infection had higher leukemia factor concentrations than those without infection before term (p < 0.001) and at term (p < 0.005). The leukemia inhibitory factor concentrations correlated with the amniotic fluid white blood cell counts (r = 0.47) (p < 0.001). In cultured human gestational tissue explants, leukemia inhibitory factor release was significantly enhanced by endotoxin, interleukin-1 alpha, and tumor necrosis factor-alpha however, leukemia inhibitory factor did not enhance the release of prostaglandin E2 by these tissues. CONCLUSIONS: Amniotic fluid leukemia inhibitory factor concentrations were elevated during intraamniotic infection and gestational tissues released leukemia inhibitory factor in response to bacterial products and inflammatory mediators.

Circulating leukemia inhibitory factor levels correlate with disease severity in meningococcemia.

Circulating concentrations of the proinflammatory cytokine leukemia inhibitory factor (LIF) were prospectively determined by radioreceptor competition assay (sensitivity, 1 ng/mL) in 33 subjects with meningococcemia. LIF was detected in the plasma of 13 subjects and was associated with development of septic shock (P < .01), disseminated intravascular coagulation (P < .05), multiorgan failure (P < .05), and death (P < .01). Plasma LIF concentrations were highest (1-1772 ng/mL) at hospital admission and became undetectable within 36 h, and the peak levels correlated inversely with systolic blood pressure (r, -.70, P < .001), peripheral blood leukocyte count (r, -.58, P < .01), and prodromal interval (r, -.60, P < .001). Plasma LIF concentrations > 400 ng/mL were present only in subjects with fatal fulminant infection. LIF concentrations in plasma collected within 12 h of hospital admission correlated with disease severity in patients with meningococcemia. It is likely that LIF participates in the host response to infection, and it may contribute to the pathogenesis of septic shock.

Increased levels of leukemia inhibitory factor in synovial fluid from patients with rheumatoid arthritis and other inflammatory arthritides.

OBJECTIVE: To examine synovial fluid (SF) from patients with arthritis, for the presence of the cytokine leukemia inhibitory factor (LIF). METHODS: SF from 152 subjects was examined for LIF, using a radioreceptor competition assay. RESULTS: LIF was present at concentrations of 1-43 ng/ml in the SF of 23% of patients with rheumatoid arthritis (RA) or other inflammatory or infectious arthritides but in only 1 of 29 patients with osteoarthritis (P < 0.01). In the RA patients, the SF LIF concentration correlated significantly with the peripheral blood white blood cell count (WBC) (P < 0.05) and the SF WBC count (P < 0.01), but not with other clinical or radiologic parameters of disease activity or progression. CONCLUSION: LIF is implicated as a potential mediator of the local or systemic inflammatory response or the joint destruction seen in inflammatory arthritis.

Induction of leukemia inhibitory factor in human synovial fibroblasts by IL-1 and tumor necrosis factor-alpha.

The cytokines, IL-1 alpha and TNF-alpha, induced a dose-dependent production of leukemia inhibitory factor (LIF) in cultured human synovial fibroblast-like cells, as measured by a radioreceptor competition assay. Significant levels of LIF were first detected in cell supernatants between 2 and 4 h after cytokine addition. Increases were also observed in LIF mRNA steady-state expression. Evidence is presented for down-regulation of the IL-1-induced LIF activity by an endogenous cyclo-oxygenase product(s); the glucocorticoid, dexamethasone, lowered the IL-1-induced LIF activity and mRNA expression. A synergistic effect was noted between the actions of IL-1 alpha and TNF-alpha, and between IL-1 and transforming growth factor-beta. IFN-gamma could not induce LIF formation in the synovial cells but inhibited the stimulatory effect of IL-1. These results suggest that cytokine-stimulated synovial fibroblasts may be a major source of intraarticular LIF production in the joints of patients with inflammatory arthritis. Synoviocyte-derived LIF may activate monocyte/macrophages in the lesions and may contribute to the bone changes and certain systemic manifestations in patients with inflammatory joint disease.

Prostatic embryonal rhabdomyosarcoma in adults. A clinicopathologic review.

Embryonal rhabdomyosarcoma of the prostate is a rare, highly malignant tumor that occurs predominantly in male infants and children, in whom it is the most common prostatic sarcoma. Six cases occurring in adults have been published, and the authors report three additional cases. The natural history is characterized by rapid growth, with the typical formation of large pelvic or abdominal masses, often leading renal failure due to bilateral ureteric obstruction. The tumor eventually disseminates widely, mainly to the lungs, bone, liver, and serosal surfaces, and unlike most other sarcomas, regional lymph node metastases are common. Combined modality therapy has resulted in marked improvement in survival rates and reduced surgical morbidity for children with these tumors. However, in adults the prognosis remains poor, with all patients dying of disseminated disease within 16 months of histologic diagnosis (mean survival, 8 months).

Clearance and fate of leukemia-inhibitory factor (LIF) after injection into mice.

Leukemia-inhibitory factor (LIF) elicits effects on a broad range of cell types, including cells of the monocytic and megakaryocytic series, embryonal stem cells, hepatocytes, adipocytes, and osteoblasts. Native and recombinant LIF, injected intravenously into adult mice, had an initial half-life of 6-8 min and a more prolonged second clearance phase. Clearance of 125I-LIF from the circulation was paralleled by a rapid accumulation in the kidneys, liver, lungs, and spleen and a more gradual accumulation in the thyroid gland. Labeling of the renal glomerular tufts, parenchymal hepatocytes, splenic red pulp, alveolar pneumocytes, and thyroid follicular cells as well as of megakaryocytes and osteoblasts in the bone cavities, placental trophoblasts, and cells of the choroid plexus was demonstrable autoradiographically. The appearance of a large amount of nonprecipitable 125I in the urine suggested that the kidneys were the major route of LIF clearance from the body.

Rare earth deposits in a deceased movie projectionist. A new case of rare earth pneumoconiosis?

The subject described in this case report, a movie projectionist, had approximately 25 years of occupational exposure to carbon arc lamp fumes. The carbon arc deposits were visible in histological sections as small granules within macrophages of the tracheobronchial lymph nodes and hepatic Kupffer's cells. Electron microprobe analysis by energy dispersive analysis of x-rays showed the granules to be composed of the rare earth elements cerium, lanthanum and neodymium, which are the major constituents of carbon arc rods. Tissue concentrations, as determined by inductively coupled plasma spectroscopy, were approximately 250-2000 times those of unexposed controls, and there was evidence of their redistribution throughout the reticuloendothelial system. There were no respiratory symptoms, or radiographic or histological pulmonary changes attributable to the progressive accumulation of the rare earth elements, and as such the patient cannot be considered to have suffered from pneumoconiosis. Twenty-one published cases of rare earth pneumoconiosis, mainly in photoengravers exposed to carbon arc fumes, are reviewed and suggest that rare earth oxides are not innocuous dusts. With the increasingly widespread use of rare earth elements there is a likelhood that further occupational groups may have significant but unrecognised exposure.

'Corkscrew-like' bacteria associated with gastritis.

Gastric spiral bacteria morphologically unlike Campylobacter pylori have recently been described in patients with gastritis. We report an additional case. These organisms are as yet unnamed, remain uncultured and are morphologically similar to organisms present in the gastric mucosa of various mammalian species. Their possible role in the pathogenesis of gastritis is discussed.

Bladder granulomata after bladder surgery.

Bladder granulomata were found in 17 (3%) of 539 patients who had bladder biopsy or resection. The finding of granulomata in 13.6% of patients who had at least two surgical procedures, but never in the first biopsy specimen of any patient was highly significant statistically (P much less than 0.001). The occurrence of granulomata exclusively in patients with bladder carcinoma was related to the significantly greater number of biopsies performed in these patients and not to carcinoma, per se. There were two types of granulomata: necrotizing, palisading granulomata (NPG) resembling rheumatoid granulomata; and foreign-body-type granulomata (FBG). They often occurred together in the same specimen, and transitions from FBG to NPG were evident histologically. The granulomata apparently healed by fibrous scarring. Energy-dispersive analysis of x-rays (EDAX) did not reveal any inorganic foreign material, but showed sulphur in some granulomata, possibly released from necrotic stroma and urothelium. Clinical and morphologic evidence is presented indicating that the granulomata arose as a local reaction to tissue necrosis caused by surgery and/or cautery.