Improving molecular testing and personalized medicine in non-small-cell lung cancer in Ontario.

BACKGROUND: Although molecular testing has become standard in managing advanced nonsquamous non-small-cell lung cancer (nsclc), most patients undergo minimally invasive procedures, and the diagnostic tumour specimens available for testing are usually limited. A knowledge translation initiative to educate diagnostic specialists about sampling techniques and laboratory processes was undertaken to improve the uptake and application of molecular testing in advanced lung cancer. METHODS: A multidisciplinary panel of physician experts including pathologists, respirologists, interventional thoracic radiologists, thoracic surgeons, medical oncologists, and radiation oncologists developed a specialty-specific education program, adapting international clinical guidelines to the local Ontario context. Expert recommendations from the program are reported here. RESULTS: Panel experts agreed that specialists procuring samples for lung cancer diagnosis should choose biopsy techniques that maximize tumour cellularity, and that conservation strategies to maximize tissue for molecular testing should be used in tissue processing. The timeliness of molecular reporting can be improved by pathologist-initiated reflex testing upon confirmation of nonsquamous nsclc and by prompt transportation of specimens to designated molecular diagnostic centres. To coordinate timely molecular testing and optimal treatment, collaboration and communication between all clinicians involved in diagnosing patients with advanced lung cancer are mandatory. CONCLUSIONS: Knowledge transfer to diagnostic lung cancer specialists could potentially improve molecular testing and treatment for advanced lung cancer patients.

BRCA1, BRCA2 and breast cancer: a concise clinical review.

Less than 5% of breast cancers are hereditary, but over 90% of hereditary breast cancers are caused by a mutation of either BRCA1 or BRCA2. The mutation may be inherited from either the maternal or the paternal side of the family. Clinicians should consider specific criteria in the family history to determine when a patient may benefit from counselling and appropriate testing. Testing is generally offered only to patients who are at high risk and is currently estimated to have a sensitivity of about 85%. Test protocols are primarily oriented to detecting frameshift and nonsense mutations that cause premature protein truncations. Missense mutations also occur, but they are less common and sometimes not clearly of clinical significance. Laboratory results need to be correlated with the clinical picture, and genetic counselling is a critical component in maximizing the benefits of testing. In the future, application of more refined clinical criteria, as well as expected improvements in laboratory techniques, will undoubtedly lead to significantly better outcomes and options in surveillance and management for hereditary breast and ovarian cancer syndromes caused by mutations of BRCA1 and BRCA2.

CD34-positive acute promyelocytic leukemia is associated with leukocytosis, microgranular/hypogranular morphology, expression of CD2 and bcr3 isoform.

Acute promyelocytic leukemia (APL) has a favorable prognosis. Current therapy includes chemotherapy used in combination with all-trans-retinoic acid (ATRA). Although the differentiating effects of ATRA on promyelocytes have been well established, in vitro studies have shown that less-differentiated APL blasts (CD34(+)) demonstrate a variable responsiveness to ATRA. To assess the clinical relevance of this finding, we analyzed a cohort of 38 patients with t(15;17) and/or PML-RARalpha APL to determine the incidence and laboratory features of CD34(+) APL. Thirty-two percent (12/38) of cases were CD34(+). There was a difference in WBC at presentation between CD34(+) and CD34(-) cases (34.6 +/- 9.2, mean +/- standard error vs. 5.4 +/- 2.0, P = 0.009). Patients with CD34(+) APL demonstrated a micro/hypogranular phenotype (75%) (P = 0.001), co-expression of CD2(+) (83%) (P = 0.001), and the bcr3 isoform (100%) (P = 0.017). In contrast, CD34(-) cases demonstrated hypergranular morphology (65%), CD2(+) (15%), and the bcr1 isoform (50%). A high presenting WBC count (\G10 x 10(9)/L) was associated with an inferior overall survival (Log rank = 0.0047). Patients with CD34(+) APL demonstrated an incidence of early mortality of 50%. Despite a marked correlation between CD34 positivity and increased WBC count, overall survival of CD34(+) and CD34(-) cases did not differ significantly in our small cohort. Immunophenotypic analysis for CD34 expression should be included in future large APL trials to determine if detection of CD34(+) blasts represents an independent adverse prognostic factor.

Wolf-Hirschorn syndrome resulting from partial monosomy 4p/trisomy 9p.

An infant girl was referred for a genetic consultation because of facial appearance suggestive of Wolf-Hirschorn syndrome (WHS), growth retardation and generalized hypotonia. She had an unbalanced karyotype 46,XX,der(4)t(4;9)(p15.2;p22)mat resulting in the deletion of the critical region for WHS and duplication of the critical region for the 9p duplication syndrome. The mother and the grandmother of proposita were the carriers of an apparently balanced translocation 46,XX,t(4;9)(p15.2;p22). The infant's phenotype was characteristic of WHS syndrome rather than that of duplication 9p phenotype. This is probably the first description of WHS phenotype resulting from a familial 4;9 translocation.

Familial Evans syndrome: a report of an affected sibship.

PURPOSE: This report describes the clinical course of three siblings, all of whom had Evans syndrome in childhood. PATIENTS: The coexistence of autoimmune hemolytic anemia and thrombocytopenia, in the absence of a known underlying cause, led to the diagnosis of Evans syndrome in a 4-month-old girl and subsequently in her two brothers when they were 4 and 13 years old. RESULTS: The 4-month-old girl had a life-threatening relapsing course unresponsive to corticosteroids, intravenous gamma-globulin, thymectomy, and cyclophosphamide. She eventually responded to splenectomy. Her two brothers had milder disease that responded to corticosteroids. Cytogenetic analyses revealed the presence of a familial Y;15 translocation in all three children and their father. CONCLUSION: There are few reported cases of familial Evans syndrome, and they are usually associated with an inherited congenital abnormality. We report the unusual finding of three siblings with the disease and no known congenital abnormality.

Serum antibodies to Balamuthia mandrillaris, a free-living amoeba recently demonstrated to cause granulomatous amoebic encephalitis.

Free-living amoebae cause three well-defined disease entities: a rapidly fatal primary meningoencephalitis, a chronic granulomatous amoebic encephalitis (GAE), and a chronic amoebic keratitis. GAE occurs in immunocompromised persons. Recently, another type of free-living amoeba, Balamuthia mandrillaris, has been shown to cause GAE. The finding that this amoeba has caused infection in some healthy children has raised the possibility that humans may lack immunity to B. mandrillaris. Human serum was examined for the presence of surface antibodies specific for this amoeba by immunofluorescence. Sera from adults contained titers of 1/64-1/256 of anti-B. mandrillaris antibodies (IgM and IgG classes), which did not cross-react with other amoebae. Cord blood contained very low antibody levels, but levels similar to those in adults were seen in serum of 1- to 5-year-old children.

Morphometric analysis of bovine lymphomas classified according to the National Cancer Institute Working Formulation.

The National Cancer Institute Working Formulation (NCI-WF) for the subjective classification of human non-Hodgkin's lymphoma is readily applicable to the classification of bovine lymphomas. Forty-nine cases of bovine lymphoma were analysed morphometrically to see if nuclear size and cleavage were distributed continuously or discretely between different NCI-WF tumour cell types. The mean nuclear area (+/- standard error of the mean, SE) was significantly greater (P < 10(-6)) in cells from the different types of diffuse large-cell lymphoma than in cells from the different types of small-cell lymphoma (42.91 +/- 1.21 micron 2 vs 19.33 +/- 1.08 micron 2, respectively); there was no overlap between the two groups. The mean nuclear are (+/- SE) of cells from diffuse large-cell lymphomas was significantly greater (P < 10(-4)) than that of cells from small non-cleaved lymphomas (42.74 +/- 1.72 micron 2 vs 27.54 +/- 1.08 micron 2, respectively), and there was again no overlap between the two groups; these two cell types are difficult to distinguish by any criteria other than size. Additionally, the cell-to-cell variability in nuclear area within a given tumour was significantly greater (P < 0.001) for the diffuse large-cell type than for the small non-cleaved cell type. The mean nuclear form factor (+/- SE) and mean nuclear contour indices (+/- SE) of the diffuse large cleaved cell type (0.53 +/- 0.02 and 5.08 +/- 0.11, respectively) were significantly different (P < 0.01 to 10(-6)) from the same parameters in the diffuse large-cell type (0.82 +/- 0.01 and 3.94 +/- 0.04, respectively). Some of the major criteria of the NCI-WF used subjectively to discriminate between bovine lymphoma cell types were supported by morphometric measurements. The magnitude of the differences in nuclear morphological characteristics between bovine lymphoma cell types was such that there was no overlap.

Morphology of acute promyelocytic leukemia with cytogenetic or molecular evidence for the diagnosis: characterization of additional microgranular variants.

Early diagnosis of t(15;17) acute promyelocytic leukemia (APL) is essential because of the associated disseminated intravascular coagulation and the unique response of the disease to all-trans retinoic acid (ATRA) therapy. Early diagnosis depends primarily on morphological recognition. The French-American-British (FAB) classification, however, does not describe all morphological variations that occur in APL. In 25 cases with evidence of APL confirmed by cytogenetic and/or molecular analysis, we found a heterogeneous morphological group. The most common form of APL was heterogeneous and consisted of various combinations of cells in which hypergranular cells and some cells with multiple Auer rods were obvious. In some cases, one cell predominated. This led to the description of five subcategories. These included the classical FAB M3 with hypergranular cells and multiple Auer rods; the FAB variant with hypogranular bilobed cells; the basophilic cell type of McKenna et al. [Br. J. Haematol 50:201, 1982]; and two additional subtypes, one consisting of differentiated promyelocytes and a few blast cells (M2-like), and the other consisting largely of blast cells and a few early promyelocytes (M1-like). Immunophenotyping revealed a pattern of CD33 and/or CD13 positivity, and CD14 and HLA-DR negativity in 96% of cases. CD2 was positive in the FAB variant and in the subtype with basophilic cells, but negative with other subtypes. Three out of five cases with basophilic cell predominance [McKenna et al.: Br J Haematol 50:201, 1982], and one out of two M2-like cases, responded to ATRA therapy. Awareness of the heterogeneity and the atypical morphologic subtypes found in t(15;17) APL will contribute to improved recognition and early institution of ATRA therapy.

Accidental infant death and stroller-prams.

A three-month-old boy and an eight-month-old boy died from accidental positional asphyxia and hanging, respectively, after being placed to sleep unsupervised in stroller-prams. Both infants had moved down towards the fronts of the stroller-prams. The younger infant fell out when the footplate collapsed and he was found hanging from a metal bar on the side. The older infant had partly slipped through the front and was suspended with his head and arms within the stroller-pram and with his face pushed firmly into the mattress by a horizontal metal bar. Stroller-prams are a potentially dangerous sleeping environment unless infants are closely supervised, gaps in the front of stroller-prams closed and upright footplates stabilised.

Nonrandom chromosomal abnormalities in bovine lymphoma.

Despite detailed knowledge of the genetic map of the bovine leukemia virus (BLV), the mechanism whereby BLV infection results in transformation and B-lineage restriction of tumors is poorly understood. The aim of this study was to gain new insight into pathogenetic mechanisms of BLV-induced tumorigenesis by determining the karyotypes of BLV-associated lymphomas in cattle. Metaphases in cells from lymphoid tumors from 20 mature dairy cows were banded and analyzed after short-term, unstimulated culture. Nineteen out of twenty cases exhibited clonal abnormalities, 17 cases were hyperdiploid, and 16 cases had extremely complex chromosomal changes. Recurrent chromosomal anomalies were identified and there was clear evidence for the evolution of increasing chromosomal instability in 12 cases. The most common abnormalities were the acquisition of additional small chromosomes (23-29); trisomy of chromosomes 5 and 7, and Robertsonian translocations and isochromosome rearrangements involving chromosomes 10, 12, 23, and 26. Monosomy X, trisomy X, and translocations involving the X chromosome were also detected. Chromosomes 2, 3, 4, 6, 8, 9, 11, 13, 14, 19, and 21 were infrequently involved in either structural or numerical changes. Structural rearrangements of chromosomes 10, 12, 23, and 26 may reflect primary abnormalities occurring relatively early in transformation, whereas trisomy 5 may be an extremely common secondary abnormality. While comparison of these findings with the current bovine gene map raises intriguing possibilities for pathogenetic mechanisms, further studies are needed before hypothetical mechanisms linking chromosomal abnormalities with BLV-induced transformation can be made.

Gene transfer into hematopoietic stem cells: long-term maintenance of in vitro activated progenitors without marrow ablation.

Adoptive transfer of genetically modified somatic cells will play an increasingly important role in the management of a wide spectrum of human diseases. Among the most appealing somatic cells as potential gene transfer vehicles are hematopoietic cells, because of their wide distribution and their well-characterized capacities for proliferation, differentiation, and self-renewal. Genes can be readily transferred into short-lived and lineage-restricted hematopoietic cells, but there remains a need to develop reliable methods for gene transfer into hematopoietic stem cells in large animals. In this work, we used a gene transfer approach in which hematopoietic cells in long-term marrow cultures were exposed to the replication-defective retrovirus N2, bearing the reporter gene neo, on multiple occasions during 21 days of culture. Genetically marked cultured autologous cells were infused into 18 canine recipients in the absence of marrow-ablative conditioning. neo was detected by Southern blotting and/or the polymerase chain reaction in the marrow, blood, marrow-derived granulocyte/macrophage and erythroid progenitors, and cultured T cells in dogs after infusion. In most dogs, the proportion of long-term marrow culture cells contributing to hematopoiesis rose during the first 3 months after infusion and peaked within the first 6. The maximal levels attained were between 10% and 30% G418-resistant (neo-positive) granulocyte/macrophage progenitors. At 12 months, five dogs maintained greater than 10% G418-resistant progenitors, and for two of them this level exceeded 20%. Two dogs had greater than 5% G418-resistant hematopoietic progenitors at 24 months after infusion. Our data suggest that very primitive hematopoietic progenitors are maintained in long-term marrow cultures, where they can be triggered into entering the cell cycle. In vivo, these activated cells likely continue normal programs of proliferation, differentiation, and self-renewal. Their progeny can be maintained at clinically relevant levels for up to 2 years without the requirement that endogenous hematopoiesis be suppressed through chemo- or radiotherapy prior to adoptive transfer. Long-term marrow culture cells may thus be ideal targets for gene therapy involving adoptive transfer of transduced hematopoietic cells.

Preparation and transfusion of canine platelet concentrates.

A protocol was developed for preparation of platelet concentrates (PC) to support thrombocytopenic dogs. Four clinically normal dogs with platelet counts that ranged from 200 to 330 x 10(9) platelets/L were used as donors. One unit (450 ml) of blood was collected by venipuncture into a double blood bag. Whole blood (WB) was centrifuged for 4 minutes at 1,000 x g (braking time = 2 minutes, 30 seconds) to prepare platelet-rich plasma (PRP). The PRP was expressed into the satellite bag and was centrifuged for 10 minutes at 2,000 x g (braking time = 2 minutes, 36 seconds). The platelet-poor plasma was expressed, leaving 40 to 70 ml of plasma and the pelleted platelets in the satellite bag. The resulting PC was left undisturbed for 60 minutes to promote disaggregation, and the platelets were then resuspended by gentle manual agitation. Forty-eight PC were prepared. Mean (+/- SD) platelet yield from WB to PRP was 78 (+/- 13)% (range, 35 to 97%); yield from PRP to PC was 94 (+/- 6)% (range, 75 to 100%); and overall yield (PC from WB) was 74 (+/- 13)% (range, 36 to 91%). Mean PC platelet count was 8.0 (+/- 3.0) x 10(10) platelets/PC (range, 2.3 to 13.4 x 10(10) platelets/PC). The WBC content was 0.1 to 2.3 x 10(9) platelets/PC, representing 3 to 74% of WBC in the WB. Hematocrit was 0.1 to 26.2%. Results of bacterial and fungal culturing were negative. The PC were irradiated (18 Gy) and transfused to 5 cross-matched dogs undergoing bone marrow transplantation that developed profound thrombocytopenia of up to 8 weeks' duration.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical and pathological findings in dogs following supralethal total body irradiation with and without infusion of autologous long-term marrow culture cells.

We developed a canine model for autologous bone marrow transplantation (AuBMT) with long-term marrow culture (LTMC) cells. Marrow was harvested from nine normal dogs. Harvests from dogs 2-7 were placed into 21 day LTMC. Cells in LTMC from dogs 4-7 were labelled with the neomycin phosphotransferase gene neo. Dogs were given 60Co total body irradiation (TBI) and then infused with LTMC cells: dog 1 received 500 cGy TBI and 2.08 x 10(8)/kg uncultured marrow cells. Dogs 2-7 received 600-800 cGy TBI and 0.07-0.45 x 10(8)/kg LTMC cells. Dogs 8 and 9 received 600 and 800 cGy TBI, respectively, but no infusion of marrow or LTMC cells. For all dogs, profound myelosuppression developed during week 1 and pyrexia developed during week 2. Enrofloxacin was given from one day before TBI until a peripheral neutrophil count > 1.0 x 10(9)/L was achieved, which eliminated Escherichia coli from feces. Dogs 1, 2 and 5-9 also received gentamicin and/or combination beta-lactam antibiotics. Numerous platelet transfusions were needed to control hemorrhages in all dogs except dog 1. Dog 1 achieved neutrophils > 1.0 x 10(9)/L on day 15, while dogs 2 and 5-9 achieved this count on days 33-48. Dogs 3 and 4 died on days 17 and 18, respectively, of beta-hemolytic streptococcal sepsis and hemorrhage, with no evidence of hematopoiesis at necropsy. The marker gene, neo, was documented in lymphoid and myeloid cells of dogs 5-7 up to 21 months post-AuBMT. Our studies indicate that dogs can recover following supralethal TBI and can survive the delayed engraftment associated with AuBMT using LTMC cells, if they receive intensive platelet and antimicrobial therapy. Used prophylactically for such therapy, enrofloxacin achieved selective intestinal decontamination, but did not prevent sepsis when used as the sole antimicrobial agent during myelosuppression. Furthermore, our studies indicate that infused LTMC cells, at the above doses, can contribute to hematopoietic recovery, but are not essential for recovery following TBI, and do not shorten the period of prolonged profound myelosuppression induced by TBI.

The use of an implantable central venous (Hickman) catheter for long-term venous access in dogs undergoing bone marrow transplantation.

Methods were developed for the insertion and maintenance of long-term central venous catheters in dogs in order to provide reliable venous access during bone marrow transplantation. Single-lumen, 9.6 Fr Hickman catheters with a VitaCuff were used. The catheter was inserted into the jugular vein via a surgical cut-down, and tunnelled subcutaneously to exit over the thoracic spine. Fluoroscopic guidance was necessary to ensure proper positioning of the catheter tip in the right atrium. The catheter was secured at the venous entrance site with a grommet and at the cutaneous exit site with a finger-cuff suture. The exit site was bandaged; dressings were changed daily. Five dogs were studied. Catheter insertion and maintenance techniques were developed using two dogs. For the other three dogs, which developed 7 wk of profound myelosuppression induced by total body irradiation, the catheters were used for blood sampling and infusions of antibiotics, fluids, and blood products. For these three dogs there were 261 total catheter-days. Complete catheter obstruction did not occur. Partial obstruction (inability to withdraw blood) occurred for 13 days with one catheter. The tip of this catheter was in the cranial vena cava. One irradiated dog had a staphylococcal exit site infection for several days after catheter insertion, which resolved with antibiotic therapy. Infections of the subcutaneous tunnel, and catheter associated bacteremia, were not identified. Infectious and hemorrhagic complications of myelosuppression were less severe than in six other dogs where intermittent venipuncture was used for vascular access during radiation induced myelosuppression. In conclusion, long-term central venous catheterization is feasible in dogs during profound myelosuppression and markedly facilitates patient management.

Effects of different hepatic pathologies on disposition of alfentanil in anaesthetized patients.

We have studied the influence of different hepatic pathologies on the disposition of alfentanil in 23 unpremedicated patients (six healthy control subjects, six patients with liver dysfunction of alcoholic aetiology and 11 patients with non-alcohol related liver disease). All patients received a bolus of alfentanil 500 micrograms i.v. as supplement to 67% nitrous oxide and isoflurane in oxygen anaesthesia. Plasma drug concentrations were measured in venous blood samples at intervals up to 24 h by radio-immunoassay and protein binding was determined by equilibrium dialysis. Kinetic estimates were determined using non-compartmental analysis. Patients with non-alcoholic liver disease had lesser plasma clearance (114.8 (range 66.8-213.5) ml min-1) than the alcoholic group (158.8 (100.0-220.7) ml min-1) or controls (187.4 (125.2-269.5) ml min-1). In all three groups, there was considerable intersubject variability, with a bimodal distribution in the non-alcoholic group. This group also had a smaller apparent volume of distribution at steady state. Mean residence time was prolonged in the alcoholic group compared with controls (284.9 (217.8-362.2) min vs 226.8 (201.2-250) min). Protein binding was decreased in the alcoholic group compared with controls (84.9 (SD 4.2)% vs 89.3 (2.1)%); this was attributable to a lesser plasma alpha 1-acid glycoprotein concentration (0.55 (0.18) g litre-1 vs 0.89 (0.21) g litre-1). Free drug clearance was reduced in both liver dysfunction groups compared with controls.

Autologous transplantation of canine long-term marrow culture cells genetically marked by retroviral vectors.

Retroviral infection of bone marrow cells in long-term marrow cultures (LTMCs) offers several theoretical advantages over other methods for gene transfer into hematopoietic stem cells. To investigate the feasibility of this approach in a large animal model system, we subjected LTMCs from nine dogs to multiple infections with retrovirus containing the neomycin phosphotransferase gene (neo) during 21 days of culture. Feeder layers, cocultivation, polycations, and selection were not used. The in vitro gene transfer efficiency was 70% as determined by polymerase chain reaction amplification of neo sequences in colony-forming unit granulocyte-macrophage (CFU-GM) obtained from day-21 LTMCs. Day-21 LTMC cells were infused into autologous recipients with (four dogs) and without (three dogs) marrow-ablative conditioning. At 3 months posttransplant, up to 10% of marrow cells contained the neo gene. This percentage declined to 0.1% to 1% at 10 to 21 months posttransplant. Neo was also detected in individual CFU-GM, burst-forming unit-erythroid (BFU-E), and CFU-Mix progenitors derived from marrow up to 21 months postinfusion and in cultures of peripheral blood-derived T cells up to 19 months postinfusion. There was no difference in the percentage of neo-marked cells present when dogs that received marrow ablative conditioning were compared with dogs receiving no conditioning. Detection of neo-marked marrow cells almost 2 years after autologous transplantation in a large mammalian species shows that retroviral infection of marrow cells in LTMCs is a potentially nontoxic and efficient protocol for gene transfer. Further, our results suggest that marrow conditioning and in vivo selection pressure to retain transplanted cells may not be absolute requirements for the retention of genetically marked cells in vivo.

Two unusual tumours of the gastrointestinal tract in a patient with tuberous sclerosis.

A 16 year old girl with an established diagnosis of tuberous sclerosis presented with a 1 year history of swelling of the left cheek. A 2 cm diameter tumour was excised which showed histological features of a solid variant of a minor salivary gland basal cell adenoma. One year later during laparotomy and excision of multiple renal angiomyolipomas, a 5 cm diameter subserosal tumour was found at the hepatic flexure of the colon. Examination of biopsy material revealed a leiomyoma. This case is presented to demonstrate two tumours that have not to the authors' knowledge been previously described in a young patient with tuberous sclerosis. Although the association may be coincidental, these tumours could represent two rare associations of tuberous sclerosis.