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.

Long-term culture of canine marrow: cytogenetic evaluation of purging of lymphoma and leukemia.

We established and maintained long-term cultures of marrow from normal dogs and dogs with lymphoma or leukemia by single inoculations of mononuclear cell suspensions. Media containing only horse sera (as opposed to horse and fetal calf sera) and catalase (for antioxidative effect) supported improved culture viability, as indicated by increased recovery of progenitor cells (granulocyte-macrophage colony-forming units, CFU-GM) and the release of abundant erythroid cells in the cultures for up to 3 weeks. CFU-GM were maintained for at least 3-4 weeks of culture. Culture appearance, cell counts, and assays of CFU-GM were used to compare the culture kinetics of tumor-involved marrow to normal marrow specimens. Cultures of marrow with extensive tumor involvement tended to be less viable, apparently due to a relative lack of competent progenitors. To investigate whether canine long-term marrow culture provided a purging effect similar to the loss of tumor cells noted in human long-term cultures of marrow from patients with chronic myelogenous leukemia (CML) or acute myelogenous leukemia (AML), we established long-term marrow cultures from 28 dogs with histologically confirmed untreated lymphoma or leukemia. Eleven of these dogs had cytogenetically marked tumor cells in the marrow at the initiation of culture. In six dogs with lymphoma and one dog with acute monocytic leukemia (AMoL) French-American-British classification (FAB) M4 leukemia, we could detect no cytogenetic evidence for persistence of the tumor clones in individually plucked or pooled CFU-GM grown from 3-week-old long-term cultures. In one case of AML (FAB M2), 80% of CFU-GM recovered from long-term cultures at 4 weeks still contained an extra metacentric marker chromosome associated with the continued presence of the leukemic clone in the cultures. Our documentation of a purging effect for some tumors supports the use of this canine model system in the investigation of autologous marrow transplantation with long-term cultured cells for humans with lymphoma and leukemia.

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.

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.

A case of hartmannellid amebic meningoencephalitis in Zambia.

A case of amebic meningoencephalitis recognized in an adult Zambian is described. This is the first authenticated case from Africa. The morphologic features of the organism, its ability to form cysts in tissue, and the granulomatous tissue response denote that the ameba is an hartmannellid rather than a Naegleria. Free-living amebas of the family Hartmannellidae have not been incriminated before as a cause of primary amebic meningoencephalitis in man. To our knowledge this is the only case where such an ameba was responsible for fulminating meningoencephalitis. The presence of the amebas in a cellulocutaneous abdominal lesion suggests hematogenous dissemination.

Sensitivity to amphotericin B of a Naegleria sp. isolated from a case of primary amoebic meningoencephalitis.

An amoeba of the genus Naegleria causing fatal meningoencephalitis in a human subject has been investigated for its sensitivity to several drugs. Penicillin, sulphadiazine, chloramphenicol, oxytetracycline hydrochloride, streptomycin, methotrexate, emetine, quinine, and metronidazole had no effect on the organism in vitro at levels in excess of those likely to be attained therapeutically in the brain. Amphotericin B was highly amoebicidal in vitro and protected mice from infection with the organism. Used in high dosage by the intraventricular as well as the intravenous route, this drug might be successful in the treatment of further cases of primary amoebic meningoencephalitis.

Cytogenetic studies: an essential part of the paediatric necropsy.

Chromosome studies were attempted on 97% of necropsies carried out in the Department of Histopathology of the Adelaide Children's Hospital over the four-year period ending May 1981. Results were obtained from 89% of necropsies of which 7.5% had major chromosome abnormalities. The chromosome results are analysed according to the category of the necropsy and to primary cause of death. It is recommended that cytogenetic studies be performed on all stillbirths and infants dying at less than 28 days of age except in cases of isolated CNS malformation, sudden infant death syndrome (SIDS), trauma, or known single gene defects.

Two-year experience of management of bleeding esophageal varices with a coordinated treatment program based on injection sclerotherapy.

The results of 61 consecutive patients treated for bleeding esophageal varices with a coordinated multidisciplinary protocol are described. The primary form of treatment after vigorous resuscitation was fiberoptic endoscopic injection sclerotherapy under general anesthetic. Thirteen patients failed to be controlled by injection, and eight were able to be treated by percutaneous transhepatic embolization. Those patients who were unable to undergo embolization or whose bleeding did not stop after embolization were controlled by surgery. The overall mortality rate with the 2-year limit was 29 patients (47%); however, only 18 deaths (29%) were related to the hospital admission for bleeding. Only one patient died of continued variceal bleeding. All of the other deaths were from later liver failure or unrelated disease. The results of the study confirmed the high mortality rate in patients with severe liver disease (Child's grade C) undergoing surgical control of bleeding, and it was shown that when control was obtained with injection sclerotherapy and embolization, the 1-year survival rate of a similar group of patients may be as high as 32%.

A fatal case of meningoencephalitis due to a free-living amoeba of uncertain identity--probably acanthamoeba sp.

There are 2 main types of meningoencephalitis caused by free-living amoebae. The first is a well-defined acutely fatal disease resembling fulminating bacterial meningitis. It is caused by the single species Naegleria fowleri. The second is a more poorly defined disease that runs a subacute or chronic course and is characterized by focal granulomatous lesions in the brain. The causative organisms are probably Acanthamoeba sp. in most cases, but it is possible that other genera may be involved. The first case of the subacute form of the disease to be recognized in Australia is described. A 2 1/2-yr-old, previously well girl presented with ataxia and lower motor neurone paralyses. The cerebrospinal fluid was pleocytic and she was thought to be suffering from a relatively minor viral brain-stem encephalitis. Her symptoms persisted in a peculiarly fluctuating way for 30 d when she suddenly collapsed and died from an intracranial haemorrhage. Necropsy showed focal granulomatous lesions associated with necrotizing vasculitis in the basal regions of the brain. The lesions contained well preserved free-living amoebae which were morphologically different from N. fowleri and most closely resembled Acanthamoeba sp. The ultrastructure of the organisms was particularly well preserved and is described in some detail. Immunohistological studies also excluded N. fowleri but were inconclusive for Acanthamoeba or other genera of free-living amoebae. Difficulties with the diagnosis and treatment of this disease are discussed and some practical suggestions are made.

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.

The cytology, histology and prevalence of cell types in canine lymphoma classified according to the National Cancer Institute Working Formulation.

No significance has been shown yet between the cytological subtypes of canine lymphoma and clinical behaviour. This paper describes and illustrates the cytological and histological criteria for application of the National Cancer Institute Working Formulation classification system, a scheme with demonstrated prognostic capability for human non-Hodgkin's lymphomas, to a series of 285 canine lymphomas. The Working Formulation can be used without difficulty for canine lymphomas. Low grade follicular tumors were found to be much less common, and high grade, aggressive tumors much more common than these cell types in humans. Low grade tumors tend to have low mitotic rates and high grade tumors tend to have high mitotic rates. There may be an association between hypercalcemia and lymphoblastic cell type. A review of available literature data for canine lymphomas suggests that prognostic extrapolation of clinical behaviour based on human lymphoma data may be possible. These results suggest that there may be strong similarities of morphology and behaviour between human non-Hodgkin's lymphomas and canine lymphomas.

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.

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)

Laboratory abnormalities in patients with cancer.

In this problem-oriented review of abnormalities associated with cancer, we have emphasized distinctive diagnostic points related to pathogenesis for each condition and outlined how the approach to management is determined by pathogenesis. For abnormalities of the complete blood count, it is important to distinguish between abnormalities directly related to marrow malignancy and abnormalities associated with extramarrow malignancy. Hemopoietic tumors consist of developmentally deficient blood cells produced by a clonal population of malignant stem cells. Tumors infiltrating marrow cause overcrowding in the limited marrow microenviroment. Extramarrow malignancies cause blood abnormalities, but the potential for normal marrow function is present. Abnormalities of blood cells secondary to therapy are usually clearly identified by consideration of clinical history. The initial differential diagnosis for hypercalcemia is malignancy. An aggressive diagnostic approach may be needed to identify the neoplasm, and therapy should incorporate measures to prevent renal failure. Hypoproteinemia and hyperproteinemia may be caused by neoplasia. Monoclonal gammopathies should be identified and may be associated with hyperviscosity syndrome. Hypoglycemia in the adult animal is most frequently caused by insulin-secreting tumors, but it has also been associated with hepatic and other tumors. Increased blood urea nitrogen, creatinine, lipase, amylase, and liver enzyme activities may also be caused by malignancy. Inadequate urine concentrating ability may be caused by hypercalcemia or malignancy-associated renal insufficiency. Hematuria in older animals is suggestive of urinary tract neoplasia. Exfoliated tumor cells may be identified in the urine sediment of these patients.

Clinical management of the cancer patient. Taking a biopsy.

Good biopsy protocol depends on excellent interaction with diagnostic pathologists. This article reviews the essentials of diagnostic appraisal, specimen preparation, biopsy interpretation, proper reporting, and implementation of biopsy results in case management. The emphasis is on biopsy of malignancy.