Hybrid capture-based genomic profiling of circulating tumor DNA from patients with estrogen receptor-positive metastatic breast cancer.

BACKGROUND: Genomic changes that occur in breast cancer during the course of disease have been informed by sequencing of primary and metastatic tumor tissue. For patients with relapsed and metastatic disease, evolution of the breast cancer genome highlights the importance of using a recent sample for genomic profiling to guide clinical decision-making. Obtaining a metastatic tissue biopsy can be challenging, and analysis of circulating tumor DNA (ctDNA) from blood may provide a minimally invasive alternative. PATIENTS AND METHODS: Hybrid capture-based genomic profiling was carried out on ctDNA from 254 female patients with estrogen receptor-positive breast cancer. Peripheral blood samples were submitted by clinicians in the course of routine clinical care between May 2016 and March 2017. Sequencing of 62 genes was carried out to a median unique coverage depth of 7503×. Genomic alterations (GAs) in ctDNA were evaluated and compared with matched tissue samples and genomic datasets of tissue from breast cancer. RESULTS: At least 1 GA was reported in 78% of samples. Frequently altered genes were TP53 (38%), ESR1 (31%) and PIK3CA (31%). Temporally matched ctDNA and tissue samples were available for 14 patients; 89% of mutations detected in tissue were also detected in ctDNA. Diverse ESR1 GAs including mutation, rearrangement and amplification, were observed. Multiple concurrent ESR1 GAs were observed in 40% of ESR1-altered cases, suggesting polyclonal origin; ESR1 compound mutations were also observed in two cases. ESR1-altered cases harbored co-occurring GAs in PIK3CA (35%), FGFR1 (16%), ERBB2 (8%), BRCA1/2 (5%), and AKT1 (4%). CONCLUSIONS: GAs relevant to relapsed/metastatic breast cancer management were identified, including diverse ESR1 GAs. Genomic profiling of ctDNA demonstrated sensitive detection of mutations found in tissue. Detection of amplifications was associated with ctDNA fraction. Genomic profiling of ctDNA may provide a complementary and possibly alternative approach to tissue-based genomic testing for patients with estrogen receptor-positive metastatic breast cancer.

Characterization of the novel HLA-B*18:79 allele.

Characterization of the novel HLA B*18:79 allele is described.

Characterization of a novel allele, HLA-A*03:132.

A*03:132 differs from A*03:01:01:01 at nucleotide 853 (codon 261) in exon 4.

Characterization of four novel HLA alleles, including two in the same haplotype.

Characterization of the novel HLA alleles A*02:330, A*11:108, B*40:175, and B*40:176 is described.

Characterization of a novel allele, HLA-B*15:228.

B*15:228 differs from B*15:01:01:01 at three nucleotides in exon 4.

Characterization of three novel HLA alleles with silent nucleotide substitutions.

Characterization of the novel HLA alleles B*35:11:03, B*42:01:03, and B*51:01:35 is described.

Case Report of a Diagnosed Septic Hip Joint Treated with a 2-Stage Hip Arthroplasty.

Introduction: Identifying and determining appropriate treatment of adult hip septic arthritis (SA) can be quite challenging. Although rare, the annual incidence of this diagnosis is approximately 8 cases per 100,000 patients. The timing of patient symptoms is wide spread. The presentation may be acute, subacute, or even chronic, and moreover, the disease process may be masked by an underlying etiology. Once diagnosed, SA requires rapid and aggressive treatment. Case Report: A 67-year-old patient presented with left hip pain. Physical examination shifted the differential diagnosis from osteoarthritis to a possible septic joint. Elevated inflammatory markers were revealed. Joint aspiration was obtained, which demonstrated rare Group G streptococcus. Two-stage hip arthroplasty was performed. Intra-operative cultures still reveal no growth of bacteria, and the patient is progressing well. Conclusion: Adult septic hip arthritis is a rare diagnosis. Hence, a proper history, physical examination, infectious laboratory workup is important. The treatment of the condition is based on the duration of symptoms and the physician's clinical gestalt.

Isolation of lymphocytopathic retroviruses from San Francisco patients with AIDS.

Infectious retroviruses have been detected in 22 of 45 randomly selected patients with acquired immune deficiency syndrome (AIDS) and in other individuals from San Francisco. The AIDS-associated retroviruses (ARV) studied in detail had a type D morphology, Mg2+-dependent reverse transcriptase, and cytopathic effects on lymphocytes. The viruses can be propagated in an established adult human T cell line, HUT-78. They cross-react with antiserum to the lymphadenopathy-associated retrovirus isolated from AIDS patients in France. Antibodies to ARV were found in all 86 AIDS patients and in a high percentage of 88 other homosexual men in San Francisco. This observation indicates the widespread presence of these lymphocytopathic retroviruses and their close association with AIDS.

Interferon alfa-2a and 13-cis-retinoic acid in renal cell carcinoma: antitumor activity in a phase II trial and interactions in vitro.

PURPOSE: A phase II trial of interferon alfa-2a (IFN) and 13-cis-retinoic acid (CRA) was conducted in patients with renal cell carcinoma (RCC). In vitro studies were performed to investigate potential mechanisms of interaction. PATIENTS AND METHODS: Forty-four patients were treated. IFN was given daily at 3 MU and escalated to 6 and 9 MU if tolerated. The dose of CRA was 1 mg/kg/d. The effects of combining CRA and IFN on the proliferation of five RCC cell lines were examined, and retinoid sensitivity was correlated to the expression of retinoic acid receptors. RESULTS: Thirteen (30%) of 43 assessable patients achieved a major response (three complete and 10 partial). Responding sites included bone metastases and renal primary tumors. Seven responding patients remain progression-free at 10+ to 19+ months. The response proportion was higher than in our prior experience with IFN, which was 10% in 149 patients. Eleven of 12 renal cancer cell lines were resistant to CRA alone; one, SK-RC-06, showed 90% inhibition of cell growth. CRA augmented the antiproliferative effect of IFN in several IFN-sensitive cell lines, but not in IFN-resistant lines. Northern blot analysis showed that expression of retinoic acid receptor-beta (RAR-beta) was repressed and not induced by retinoic acid in retinoic acid-insensitive RCC lines. However, RAR-beta expression was induced by retinoic acid in SK-RC-06 cells. CONCLUSION: IFN and CRA showed antitumor activity in patients with advanced RCC, and the proportion and nature of response suggested CRA added therapeutic benefit to IFN. A phase III randomized trial of IFN plus CRA versus IFN alone and a phase II trial of single-agent CRA have been initiated.

Enhanced AKR leukemogenesis by the dual tropic viruses. II. Effect on cell-mediated immune responses.

We examined the relationship of the leukemia-accelerating properties of a dual-tropic virus (DTV-70) (when injected into the thymus of 14-day-old AKR mice) to its ability to impair T cell functions. Splenic lymphocytes from virus-infected AKR mice were found to have reduced T cell mitogenic responses; moreover, these cells suppressed phytohemagglutinin stimulation of cells from normal, uninfected AKR mice. The response to the B cell mitogen lipopolysaccharide was slightly enhanced at 15 days following DTV-70 infection and was unaffected at later ages. AKR mice infected with DTV-70 showed reduced ability to develop delayed-type hypersensitivity reaction and interleukin 2 production. In contrast, spleen cells from the virus-infected mice responded normally to allogeneic stimulation in mixed lymphocyte culture and mounted an almost normal graft versus host reaction. The data suggest that DTV-70 impairs certain T cell functions that could interfere with immune surveillance and thus permit progression of preleukemic cells into overt leukemia. These T cell functions are suppressed normally by 6 months of age, perhaps by spontaneously arising DTV.

Enhanced AKR leukemogenesis by the dual tropic viruses. I. The time and site of origin of potential leukemic cells.

The occurrence of potential leukemia cells (PLC) among bone marrow, spleen, and thymus of AKR mice during the preleukemic period was tested by an in vivo transplantation bioassay. The presence of PLC in 30- and 75-day-old AKR mice was demonstrated mostly among bone marrow cells, less in spleen, and was lacking in thymus. Occurrence of PLC in young AKR mice was shown to be thymus independent. However, progression of PLC from young donors (14-80 days old) into overt leukemia following transplantation into F1 recipients was shown to be dependent on specific host conditions including an intact thymus and an Fv-1nn allele. In contrast, PLC from 7-9-month-old AKR mice or frank leukemic cells when transplanted grew in any intact or thymectomized histocompatible host, thereby indicating their autonomous growth state. Infection of 2-week-old AKR mice with the dual-tropic virus DTV-70 induced characteristic changes in the thymus and accelerated leukemia development. DTV-70 inoculation into 14-day-old AKR mice did not change the spontaneous PLC distribution pattern in the tested host organs within 30 days postinfection, nor did it change PLC-specific host requirements for further progression into leukemic cells; however, it enhanced PLC transition to autonomous leukemic cells. The preferential cell tropism of DTV-70 for target cells (prothymocytes) among bone marrow and young spleen cells rather than for thymocytes was also demonstrated in an in vitro-in vivo test. The dual tropic virus may act as a promoter on preexisting PLC (present mostly among bone marrow cells) by enhancing their ability to progress into autonomous leukemic cells.

Expression of retinoic acid receptor beta in human renal cell carcinomas correlates with sensitivity to the antiproliferative effects of 13-cis-retinoic acid.

The differentiation and growth suppressive effects of retinoic acid are mediated through retinoic acid nuclear receptors (RARs and RXRs), which are ligand-activated transcription factors. Recent data suggest that both altered and regulated expression of RARs are linked to retinoic acid response in a cell context-dependent manner. This study examined the antiproliferative effects of 13-cis-retinoic acid (cRA) on 12 renal cancer cell lines and correlated these findings with the basal and induced expression of RAR-alpha, -beta and -gamma. Eleven of 12 renal cancers that were either resistant to or only minimally inhibited by cRA did not basally express RAR-beta as determined by Northern blot analysis. In these cells, cRA treatment did not induce RAR-beta expression. In contrast, 1 of 12 cell lines (SK-RC-06) was >90% inhibited by cRA and basally expressed RARbeta. Furthermore, RAR-beta mRNA in SK-RC-06 cells was up-regulated by cRA treatment. Amplification of cDNA using PCR and RAR-beta isoform-specific primer pairs revealed that only SK-RC-06 cells expressed the RAR-beta1 isoform. Expression of RAR-alpha transcripts was abundant in all 12 cell lines examined, whereas low levels of RAR-gamma transcripts were detectable in 6 of 10 renal cancers. Expression of RAR-alpha and RAR-gamma was not affected by cRA. These data showing that the majority of renal cancer cell lines are resistant to cRA suggest that: (a) resistance to the antiproliferative action of cRA correlates with repressed RAR-beta mRNA expression; and (b) the antiproliferative effects of cRA in renal cancer cells are mediated through RAR-beta1.

Application of electrospray mass spectrometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry for molecular weight assignment of peptides in complex mixtures.

Electrospray mass spectrometry (ES/MS) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF/MS) were used to provide mass spectra from seven elapid snake venoms. Spectral interpretation was much simpler for MALDI/TOF/MS. ES/MS proved more useful for the provision of molecular weight data for very closely related peptides, but suppression of higher molecular weight compounds was seen to occur during flow injection analysis. MALDI/TOF/MS proved useful for providing a complete picture of the venom, but the low resolution led to obscuring of major ions, and the mass accuracy was poorer for known peptides. Suppression also occurred during MALDI/TOF/MS but could be overcome using alternative matrices because the spectra were very dependent on the choice of matrix. ES/MS and MALDI/TOF/MS provide complementary and confirmatory information such that for the anal sis of complex peptide mixtures (snake venoms), the use of both techniques is desirable.

Hypertrophic osteoarthropathy in association with pulmonary metastasis from osteogenic sacroma.

Hypertrophic osteoarthropathy in children is rare, especially when it is associated with lung metastsis. The patient reported herein had metastic osteogenic sarcoma and failed to respond to chemotherapy or thoractomy. There are several theories as to the pathogenesis of osteoarthropathy, but none is totally convincing. Vagotomy or transection of intercostal nerves has been advocated to relieve the symptoms of osteoarthropathy.

Craniofrontonasal dysostosis with deafness and axillary pterygia.

Craniofrontonasal dysostosis (CFND) is an inherited disorder previously referred to as craniofrontonasal dysplasia. However, there is no evidence of tissue dysplasia and, therefore, the term dysostosis has been substituted. The disorder is characterized by frontonasal dysostosis, coronal craniostenosis, and the variable presence of other skeletal defects, including short webbed neck, sloping shoulders, polydactyly, syndactyly, and broad first toes. Here we report an affected mother and daughter who also have limited hip abduction. In addition, the mother had an axillary pterygia, congenital footplate fixation of the left ear, and right sensorineural hearing loss; these manifestations have not been reported previously in CFND and expand the phenotype of this syndrome. Both patients had marked restriction of shoulder abduction, and the mother had limited forearm pronation; these manifestations have been reported in only one other patient with CFND. Awareness of the possibility of these abnormalities may allow for early intervention by physical therapy and hearing aides in infants and young children with these manifestations as a component of CFND.

Reduction of radiation exposure during radiography for scoliosis.

To reduce the radiation exposure received by young scoliosis patients during treatment, six changes in technique were instituted: (1) a posteroanterior projection, (2) specially designed leaded acrylic filters, (3) a high-speed screen-film system, (4) a specially designed cassette-holder and grid, (5) a breast-shield, and (6) additional filtration in the x-ray tube the thyroid, breast, and abdominal areas were made on an Alderson phantom. They revealed an eightfold reduction in abdominal exposure for both the posteroanterior and the lateral radiographys. There was a twentyfold reduction in exposure to the thyroid for the posteroanterior radiography from 100 to less than five milliroentgens and for the lateral radiograph there was a 100-fold reduction from 618 to six milliroentgens. For the breasts there was a sixty-ninefold reduction from 344 to less than five milliroentgens for the posteroanterior radiography and a fifty-fivefold reduction from 277 to less than five milliroentgens for the lateral radiograph. These reductions in exposure were obtained without significant loss in the quality of the radiographs and in most instances with an improvement in the over-all quality of the radiograph due to the more uniform exposure.

A physiologically based toxicokinetic model for lake trout (Salvelinus namaycush).

A physiologically based toxicokinetic (PB-TK) model for fish, incorporating chemical exchange at the gill and accumulation in five tissue compartments, was parameterized and evaluated for lake trout (Salvelinus namaycush). Individual-based model parameterization was used to examine the effect of natural variability in physiological, morphological, and physico-chemical parameters on model predictions. The PB-TK model was used to predict uptake of organic chemicals across the gill and accumulation in blood and tissues in lake trout. To evaluate the accuracy of the model, a total of 13 adult lake trout were exposed to waterborne 1,1,2,2-tetrachloroethane (TCE), pentachloroethane (PCE), and hexachloroethane (HCE), concurrently, for periods of 6, 12, 24 or 48 h. The measured and predicted concentrations of TCE, PCE and HCE in expired water, dorsal aortic blood and tissues were generally within a factor of two, and in most instances much closer. Variability noted in model predictions, based on the individual-based model parameterization used in this study, reproduced variability observed in measured concentrations. The inference is made that parameters influencing variability in measured blood and tissue concentrations of xenobiotics are included and accurately represented in the model. This model contributes to a better understanding of the fundamental processes that regulate the uptake and disposition of xenobiotic chemicals in the lake trout. This information is crucial to developing a better understanding of the dynamic relationships between contaminant exposure and hazard to the lake trout.

The use of computerised tomography in dislocation of the hip and femoral neck anteversion in children.

Computerised tomography is useful in the diagnosis of abnormalities of the hip in children, particularly in assessing the size and shape of the acetabulum, the position and congruity of the femoral head relative to the acetabulum, and the degree of femoral anteversion or retroversion. It is most useful when limited hip movement and previous operations preclude adequate clinical examination and assessment by routine radiographic techniques. It is not recommended for routine use in screening congenital dislocation of the hip or in diagnosis or follow-up of Perthes' disease or slipped capital femoral epiphysis.

Branchial elimination of superhydrophobic organic compounds by rainbow trout (Oncorhynchus mykiss).

The branchial elimination of pentachloroethane and four congeneric polychlorinated biphenyls by rainbow trout was measured using a fish respirometer-metabolism chamber and an adsorption resin column. Branchial elimination was characterized by calculating a set of apparent in vivo blood:water partition coefficients (P(BW)). Linear regression was performed on the logarithms of P(BW) estimates and the log K(OW) value for each compound to give the fitted equation: log P(BW)=0.76 x log K(OW)-1.0 (r(2)=0.98). The linear nature of this relationship provides support for existing models of chemical flux at fish gills and suggests that a near equilibrium condition was established between chemical in venous blood entering the gills, including dissolved and bound forms, and dissolved chemical in expired branchial water. In vivo P(BW) estimates were combined with P(BW) values determined in vitro for a set of lower log K(OW) compounds (Bertelson et al., Environ. Toxicol. Chem. 17 (1998) 1447-1455) to give the fitted relationship: log P(BW)=0.73 x log K(OW)-0.88 (r(2)=0.98). The slope of this equation is consistent with the suggestion that chemical binding to non-lipid organic material contributes substantially to blood:water chemical partitioning. An equation based on the composition of trout blood (water content and the total amount of organic material) was then derived to predict blood:water partitioning for compounds with log K(OW) values ranging from 0 to 8: log P(BW)=log[(10(0.73 log K(ow)) x 0.16)+0.84].

Comparative evaluation of culture- and BAX polymerase chain reaction-based detection methods for Listeria spp. and Listeria monocytogenes in environmental and raw fish samples.

Two commercial polymerase chain reaction (PCR)-based Listeria detection systems, the BAX for Screening/Listeria monocytogenes and the BAX for Screening/Genus Listeria, and a culture-based detection system, the Biosynth L. monocytogenes Detection System (LMDS), were evaluated for their ability to detect L. monocytogenes and Listeria spp. in raw ingredients and the processing environment. For detection of L. monocytogenes from raw fish, enrichment was performed in Listeria enrichment broth (LEB), followed by plating on both Oxford agar and LMDS L. monocytogenes plating medium (LMPM). Detection of Listeria and L. monocytogenes from environmental samples was performed using LMDS enrichment medium, followed by plating on both Oxford agar and LMPM. A total of 512 environmental samples and 315 raw fish were taken from two smoked fish processing facilities and screened using these molecular and cultural Listeria detection methods. The BAX for Screening/L monocytogenes was used to screen raw fish and was 84.8% sensitive and 100% specific. The BAX for Screening/Genus Listeria was evaluated on environmental samples and had 94.7% sensitivity and 97.4% specificity. In conjunction with enrichment in LEB, LMPM had a sensitivity and specificity for detection of L. monocytogenes from raw fish of 97.8 and 100%, respectively. Use of LMDS enrichment medium followed by plating on LMPM allowed for sensitivity and specificity rates of 94.8 and 100%, respectively, for detection of L. monocytogenes from environmental samples. We conclude that both the BAX systems and the use of LMPM allow for reliable and rapid detection of Listeria spp. and L. monocytogenes. While the BAX systems provide screening results in about 3 days, the use of LMPM allows for L. monocytogenes isolation in 4 to 5 days.