Deciphering radiological stable disease to immune checkpoint inhibitors.

BACKGROUND: 'Stable disease (SD)' as per RECIST is a common but ambiguous outcome in patients receiving immune checkpoint inhibitors (ICIs). This study aimed to characterize SD and identify the subset of patients with SD who are benefiting from treatment. Understanding SD would facilitate drug development and improve precision in correlative research. PATIENTS AND METHODS: A systematic review was carried out to characterize SD in ICI trials. SD and objective response were compared to proliferation index using The Cancer Genome Atlas gene expression data. To identify a subgroup of SD with outcomes mirroring responders, we examined a discovery cohort of non-small-cell lung cancer (NSCLC). Serial cutpoints of two variables, % best overall response and progression-free survival (PFS), were tested to define a subgroup of patients with SD with similar survival as responders. Results were then tested in external validation cohorts. RESULTS: Among trials of ICIs (59 studies, 14 280 patients), SD ranged from 16% to 42% in different tumor types and was associated with disease-specific proliferation index (ρ = -0.75, P = 0.03), a proxy of tumor kinetics, rather than relative response to ICIs. In a discovery cohort of NSCLC [1220 patients, 313 (26%) with SD to ICIs], PFS ranged widely in SD (0.2-49 months, median 4.9 months). The subset with PFS >6 months and no tumor growth mirrored partial response (PR) minor (overall survival hazard ratio 1.0) and was proposed as the definition of SD responder. This definition was confirmed in two validation cohorts from trials of NSCLC treated with durvalumab and found to apply in tumor types treated with immunotherapy in which depth and duration of benefit were correlated. CONCLUSIONS: RECIST-defined SD to immunotherapy is common, heterogeneous, and may largely reflect tumor growth rate rather than ICI response. In patients with NSCLC and SD to ICIs, PFS >6 months and no tumor growth may be considered 'SD responders'. This definition may improve the efficiency of and insight derivable from clinical and translational research.

Dissecting the clinicopathologic, genomic, and immunophenotypic correlates of KRASG12D-mutated non-small-cell lung cancer.

BACKGROUND: Allele-specific KRAS inhibitors are an emerging class of cancer therapies. KRAS-mutant (KRASMUT) non-small-cell lung cancers (NSCLCs) exhibit heterogeneous outcomes, driven by differences in underlying biology shaped by co-mutations. In contrast to KRASG12C NSCLC, KRASG12D NSCLC is associated with low/never-smoking status and is largely uncharacterized. PATIENTS AND METHODS: Clinicopathologic and genomic information were collected from patients with NSCLCs harboring a KRAS mutation at the Dana-Farber Cancer Institute (DFCI), Memorial Sloan Kettering Cancer Center, MD Anderson Cancer Center, and Imperial College of London. Multiplexed immunofluorescence for CK7, programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), Foxp3, and CD8 was carried out on a subset of samples with available tissue at the DFCI. Clinical outcomes to PD-(L)1 inhibition ± chemotherapy were analyzed according to KRAS mutation subtype. RESULTS: Of 2327 patients with KRAS-mutated (KRASMUT) NSCLC, 15% (n = 354) harbored KRASG12D. Compared to KRASnon-G12D NSCLC, KRASG12D NSCLC had a lower pack-year (py) smoking history (median 22.5 py versus 30.0 py, P < 0.0001) and was enriched in never smokers (22% versus 5%, P < 0.0001). KRASG12D had lower PD-L1 tumor proportion score (TPS) (median 1% versus 5%, P < 0.01) and lower tumor mutation burden (TMB) compared to KRASnon-G12D (median 8.4 versus 9.9 mt/Mb, P < 0.0001). Of the samples which underwent multiplexed immunofluorescence, KRASG12D had lower intratumoral and total CD8+PD1+ T cells (P < 0.05). Among 850 patients with advanced KRASMUT NSCLC who received PD-(L)1-based therapies, KRASG12D was associated with a worse objective response rate (ORR) (15.8% versus 28.4%, P = 0.03), progression-free survival (PFS) [hazard ratio (HR) 1.51, 95% confidence interval (CI) 1.45-2.00, P = 0.003], and overall survival (OS; HR 1.45, 1.05-1.99, P = 0.02) to PD-(L)1 inhibition alone but not to chemo-immunotherapy combinations [ORR 30.6% versus 35.7%, P = 0.51; PFS HR 1.28 (95%CI 0.92-1.77), P = 0.13; OS HR 1.36 (95%CI 0.95-1.96), P = 0.09] compared to KRASnon-G12D. CONCLUSIONS: KRASG12D lung cancers harbor distinct clinical, genomic, and immunologic features compared to other KRAS-mutated lung cancers and worse outcomes to PD-(L)1 blockade. Drug development for KRASG12D lung cancers will have to take these differences into account.

Clinical definition of acquired resistance to immunotherapy in patients with metastatic non-small-cell lung cancer.

Acquired resistance (AR) to programmed cell death protein 1/programmed death-ligand 1 [PD-(L)1] blockade is frequent in non-small-cell lung cancer (NSCLC), occurring in a majority of initial responders. Patients with AR may have unique properties of persistent antitumor immunity that could be re-harnessed by investigational immunotherapies. The absence of a consistent clinical definition of AR to PD-(L)1 blockade and lack of uniform criteria for ensuing enrollment in clinical trials remains a major barrier to progress; such clinical definitions have advanced biologic and therapeutic discovery. We examine the considerations and potential controversies in developing a patient-level definition of AR in NSCLC treated with PD-(L)1 blockade. Taking into account the specifics of NSCLC biology and corresponding treatment strategies, we propose a practical, clinical definition of AR to PD-(L)1 blockade for use in clinical reports and prospective clinical trials. Patients should meet the following criteria: received treatment that includes PD-(L)1 blockade; experienced objective response on PD-(L)1 blockade (inclusion of a subset of stable disease will require future investigation); have progressive disease occurring within 6 months of last anti-PD-(L)1 antibody treatment or rechallenge with anti-PD-(L)1 antibody in patients not exposed to anti-PD-(L)1 in 6 months.

Clinical and molecular correlates of PD-L1 expression in patients with lung adenocarcinomas.

BACKGROUND: Programmed death-ligand 1 (PD-L1) expression is the only FDA-approved biomarker for immune checkpoint inhibitors (ICIs) in patients with lung adenocarcinoma, but sensitivity is modest. Understanding the impact of molecular phenotype, clinical characteristics, and tumor features on PD-L1 expression is largely unknown and may improve prediction of response to ICI. PATIENTS AND METHODS: We evaluated patients with lung adenocarcinoma for whom PD-L1 testing and targeted next-generation sequencing (using MSK-IMPACT) was performed on the same tissue sample. Clinical and molecular features were compared across PD-L1 subgroups to examine how molecular phenotype associated with tumor PD-L1 expression. In patients treated with anti-PD-(L)1 blockade, we assessed how these interactions impacted efficacy. RESULTS: A total of 1586 patients with lung adenocarcinoma had paired PD-L1 testing and targeted next-generation sequencing. PD-L1 negativity was more common in primary compared to metastatic samples (P < 0.001). The distribution of PD-L1 expression (lymph nodes enriched for PD-L1 high; bones predominantly PD-L1 negative) and predictiveness of PD-L1 expression on ICI response varied by organ. Mutations in KRAS, TP53, and MET significantly associated with PD-L1 high expression (each P < 0.001, Q < 0.001) and EGFR and STK11 mutations associated with PD-L1 negativity (P < 0.001, Q = 0.01; P = 0.001, Q < 0.001, respectively). WNT pathway alterations also associated with PD-L1 negativity (P = 0.005). EGFR and STK11 mutants abrogated the predictive value of PD-L1 expression on ICI response. CONCLUSION: PD-L1 expression and association with ICI response vary across tissue sample sites. Specific molecular features are associated with differential expression of PD-L1 and may impact the predictive capacity of PD-L1 for response to ICIs.

COVID-19 in patients with lung cancer.

BACKGROUND: Patients with lung cancers may have disproportionately severe coronavirus disease 2019 (COVID-19) outcomes. Understanding the patient-specific and cancer-specific features that impact the severity of COVID-19 may inform optimal cancer care during this pandemic. PATIENTS AND METHODS: We examined consecutive patients with lung cancer and confirmed diagnosis of COVID-19 (n = 102) at a single center from 12 March 2020 to 6 May 2020. Thresholds of severity were defined a priori as hospitalization, intensive care unit/intubation/do not intubate ([ICU/intubation/DNI] a composite metric of severe disease), or death. Recovery was defined as >14 days from COVID-19 test and >3 days since symptom resolution. Human leukocyte antigen (HLA) alleles were inferred from MSK-IMPACT (n = 46) and compared with controls with lung cancer and no known non-COVID-19 (n = 5166). RESULTS: COVID-19 was severe in patients with lung cancer (62% hospitalized, 25% died). Although severe, COVID-19 accounted for a minority of overall lung cancer deaths during the pandemic (11% overall). Determinants of COVID-19 severity were largely patient-specific features, including smoking status and chronic obstructive pulmonary disease [odds ratio for severe COVID-19 2.9, 95% confidence interval 1.07-9.44 comparing the median (23.5 pack-years) to never-smoker and 3.87, 95% confidence interval 1.35-9.68, respectively]. Cancer-specific features, including prior thoracic surgery/radiation and recent systemic therapies did not impact severity. Human leukocyte antigen supertypes were generally similar in mild or severe cases of COVID-19 compared with non-COVID-19 controls. Most patients recovered from COVID-19, including 25% patients initially requiring intubation. Among hospitalized patients, hydroxychloroquine did not improve COVID-19 outcomes. CONCLUSION: COVID-19 is associated with high burden of severity in patients with lung cancer. Patient-specific features, rather than cancer-specific features or treatments, are the greatest determinants of severity.

Severe immune-related adverse events are common with sequential PD-(L)1 blockade and osimertinib.

BACKGROUND: Concurrent programmed death-ligand-1 (PD-(L)1) plus osimertinib is associated with severe immune related adverse events (irAE) in epidermal growth factor receptor (EGFR)-mutant non-small-cell lung cancer (NSCLC). Now that PD-(L)1 inhibitors are routinely used as adjuvant and first-line treatments, sequential PD-(L)1 inhibition followed by osimertinib use may become more frequent and have unforeseen serious toxicity. METHODS: We identified patients with EGFR-mutant NSCLC who were treated with PD-(L)1 blockade and EGFR- tyrosine kinase inhibitors (TKIs), irrespective of drug or sequence of administration (total n = 126). Patient records were reviewed to identify severe (NCI-CTCAE v5.0 grades 3-4) toxicity. RESULTS: Fifteen percent [6 of 41, 95% confidence interval (CI) 7% to 29%] of all patients treated with sequential PD-(L)1 blockade followed later by osimertinib developed a severe irAE. Severe irAEs were most common among those who began osimertinib within 3 months of prior PD-(L)1 blockade (5 of 21, 24%, 95% CI 10% to 45%), as compared with >3-12 months (1 of 8, 13%, 95% CI 0% to 50%), >12 months (0 of 12, 0%, 95% CI 0% to 28%). By contrast, no severe irAEs were identified among patients treated with osimertinib followed by PD-(L)1 (0 of 29, 95% CI 0% to 14%) or PD-(L)1 followed by other EGFR-TKIs (afatinib or erlotinib, 0 of 27, 95% CI 0% to 15%). IrAEs occurred at a median onset of 20 days after osimertinib (range 14-167 days). All patients with irAEs required steroids and most required hospitalization. CONCLUSION: PD-(L)1 blockade followed by osimertinib is associated with severe irAE and is most frequent among patients who recently received PD-(L)1 blockade. No irAEs were observed when osimertinib preceded PD-(L)1 blockade or when PD-(L)1 was followed by other EGFR-TKIs. This association appears to be specific to osimertinib, as no severe irAEs occurred with administration of other EGFR-TKIs.

Variation in expenditure for common, high cost surgical procedures in a working age population: implications for reimbursement reform.

BACKGROUND: In the move toward value-based care, bundled payments are believed to reduce waste and improve coordination. Some commercial insurers have addressed this through the use of bundled payment, the provision of one fee for all care associated with a given index procedure. This system was pioneered by Medicare, using a population generally over 65 years of age, and despite its adoption by mainstream insurers, little is known of bundled payments' ability to reduce variation or cost in a working-age population. This study uses a universally-insured, nationally-representative population of adults aged 18-65 to examine the effect of bundled payments for five high-cost surgical procedures which are known to vary widely in Medicare reimbursement: hip replacement, knee replacement, coronary artery bypass grafting (CABG), lumbar spinal fusion, and colectomy. METHODS: Five procedures conducted on adults aged 18-65 were identified from the TRICARE database from 2011 to 2014. A 90-day period from index procedure was used to determine episodes of associated post-acute care. Data was sorted by Zip code into hospital referral regions (HRR). Payments were determined from TRICARE reimbursement records, they were subsequently price standardized and adjusted for patient and surgical characteristics. Variation was assessed by stratifying the HRR into quintiles by spending for each index procedure. RESULTS: After adjusting for case mix, significant inter-quintile variation was observed for all procedures, with knee replacement showing the greatest variation in both index surgery (107%) and total cost of care (75%). Readmission was a driver of variation for colectomy and CABG, with absolute cost variation of $17,257 and $13,289 respectively. Other post-acute care spending was low overall (≤$1606, for CABG). CONCLUSIONS: This study demonstrates significant regional variation in total spending for these procedures, but much lower spending for post-acute care than previously demonstrated by similar procedures in Medicare. Targeting post-acute care spending, a common approach taken by providers in bundled payment arrangements with Medicare, may be less fruitful in working aged populations.

[Polymorphism of brain derived neurotrophic factor and recovery of functions after ischemic stroke]. / Polymorphismus des "brain derived neurotrophic factor" und Erholung nach Schlaganfall.

BACKGROUND: After ischemic stroke, many factors influence the restitution of functions. In particular they include the patient age, the initial stroke severity and the presence of cognitive and neuropsychological deficits. In this study we investigated whether a polymorphism in the gene encoding for brain derived neurotrophic factor (BDNF) influences improvements of motor functions and everyday activities. METHODS: Patients with subacute ischemic stroke (n = 67) were examined at the beginning of an inpatient neurological rehabilitation, after 4 weeks of treatment and after 6 months. The Barthel index (BI) and the Rivermead motor assessment (RMA) were used to measure motor functions and everyday activities. Patients were allocated to three groups (valine [Val]/valine, val/methionine [Met] and Met/Met) depending on the BDNF polymorphism at codon 66. RESULTS: The 3 groups (Val/Val, n = 34 patients, Val/Met, n = 26 and Met/Met, n = 7) showed significant improvements in BI and RMA after 4 weeks and after 6 months as compared to the preceding measurements. The BI and RMA were positively correlated. The three groups did not differ with respect to the extent of improvement. CONCLUSION: After ischemic stroke, motor functions and everyday activities improved continuously over a period of at least 6 months. The BDNF polymorphism did not influence this development.

Morbidity of early spine surgery in the multiply injured patient.

INTRODUCTION: The optimal timing of surgery for multiply injured patients with operative spinal injuries remains unknown. The purported benefits of early intervention must be weighed against the morbidity of surgery in the early post-injury period. The performance of spine surgery in the Afghanistan theater permits analysis of the morbidity of early surgery on military casualties. The objective is to compare surgical morbidity of early spinal surgery in multiply injured patients versus stable patients. MATERIALS AND METHODS: Patients were retrospectively categorized as stable or borderline unstable depending on the presence of at least one of the following: ISS >40, ISS >20 and chest injury, exploratory laparotomy or thoracotomy, lactate >2.5 mEq/L, platelet <110,000/mm(3), or >10 U PRBCs transfused pre-operatively. Surgical morbidity, complications, and neurologic improvement between the two groups were compared retrospectively. RESULTS: 30 casualties underwent 31 spine surgeries during a 12-month period. 16 of 30 patients met criteria indicating a borderline unstable patient. Although there were no significant differences in the procedures performed for stable and borderline unstable patients as measured by the Surgical Invasiveness Index (7.5 vs. 6.9, p = 0.8), borderline unstable patients had significantly higher operative time (4.3 vs. 3.0 h, p = 0.01), blood loss (1,372 vs. 366 mL, p = 0.001), PRBCs transfused intra-op (3.88 vs. 0.14 U, p < 0.001), and total PRBCs transfused in theater (10.18 vs. 0.31 U, p < 0.001). CONCLUSIONS: The results indicate that published criteria defining a borderline unstable patient may have a role in predicting increased morbidity of early spine surgery. The perceived benefits of early intervention should be weighed against the greater risks of performing extensive spinal surgeries on multiply injured patients in the early post-injury period, especially in the setting of combat trauma.

The psychological burden of disease among patients undergoing cervical spine surgery: Are we underestimating our patients' inherent disability?

BACKGROUND: Studies have utilized psychological questionnaires to identify the psychological distress among certain surgical populations. RESEARCH QUESTION: Is there an additional psychological burden among patients undergoing surgical treatment for their symptomatic degenerative cervical disease? MATERIALS AND METHODS: Patients>18 years of age with symptomatic, degenerative cervical spine disease were included and prospectively enrolled. Correlations and multivariable logistic regression analysis assessed the relationship between these mental health components (PCS, FABQ) and the severity of disability described by the NDI, EQ-5D, and mJOA score. Patient distress scores were compared to previously published benchmarks for other diagnoses. RESULTS: 47 patients were enrolled (age: 56.0 years,BMI: 29.7kg/m2). Increasing neck disability and decreasing EQ-5D were correlated with greater PCS and FABQ(all P<0.001). Patients with severe psychological distress at baseline were more likely to report severe neck disability, while physician-reported mJOA had weaker associations. Compared to historical controls of lumbar patients, patients in our study had greater levels of psychological distress, as measured by FABQ (40.0 vs. 17.6; P<0.001) and PCS (27.4 vs. 19.3;P<0.001). DISCUSSION AND CONCLUSION: Degenerative cervical spine patients seeking surgery were found to have a significant level of psychological distress, with a large portion reporting severe fear avoidance beliefs and catastrophizing pain at baseline. Strong correlation was seen between patient-reported functional metrics, but less so with physician-reported signs and symptoms. Additionally, this population demonstrated higher psychological burden in certain respects than previously identified benchmarks of patients with other disorders. Preoperative treatment to help mitigate this distress, impact postoperative outcomes, and should be further investigated. LEVEL OF EVIDENCE: Level III.

Incidence and epidemiology of spinal cord injury within a closed American population: the United States military (2000-2009).

STUDY DESIGN: Cohort study. OBJECTIVES: The objective of this study was to characterize the incidence of spinal cord injury (SCI) within the population of the United States military from 2000-2009. This investigation also sought to define potential risk factors for the development of SCI. SETTING: The population of the United States military from 2000-2009. METHODS: The Defense Medical Epidemiology Database was queried for the years 2000-2009 using the International Classification of Diseases, Ninth Revision, Clinical Modification codes for SCI (806.0, 806.1, 806.2, 806.3, 806.4, 806.5, 806.8, 806.9, 952.0, 952.1, 952.2, 952.8, 952.9). The raw incidence of SCI was calculated and unadjusted incidence rates were generated for the risk factors of age, sex, race, military rank and branch of service. Adjusted incidence rate ratios were subsequently determined via multivariate Poisson regression analysis that controlled for other factors in the model and identified significant independent risk factors for SCI. RESULTS: Between 2000 and 2009, there were 5928 cases of SCI among a population at-risk of 13,813,333. The raw incidence of SCI within the population was 429 per million person-years. Male sex, white race, enlisted personnel and service in the Army, Navy or Marine Corps were found to be significant independent risk factors for SCI. The age groups 20-24, 25-29 and >40 were also found to be at significantly greater risk of developing the condition. CONCLUSIONS: This study is one of the few investigations to characterize the incidence, epidemiology and risk factors for SCI within the United States. Results presented here may represent the best-available evidence for risk factors of SCI in a large and diverse American cohort.

A Global Geomorphologic Map of Saturn's Moon Titan.

Titan has an active methane-based hydrologic cycle1 that has shaped a complex geologic landscape2, making its surface one of most geologically diverse in the solar system. Despite the different materials, temperatures, and gravity fields between Earth and Titan, many surface features are similar between the two worlds and can be interpreted as products of the same geologic processes3. However, Titan's thick and hazy atmosphere has hindered the identification of geologic features at visible wavelengths and the study of surface composition4. Here we identify and map the major geologic units on Titan's surface using radar and infrared data from the Cassini orbiter spacecraft. Correlations between datasets enabled us to produce a global map even where data sets were incomplete. The spatial and superposition relations between major geologic units reveals the likely temporal evolution of the landscape and gives insight into the interacting processes driving its evolution. We extract the relative dating of the various geological units by observing their spatial superposition in order to get information on the temporal evolution of the landscape. Dunes and lakes are relatively young, while hummocky/mountainous terrains are the oldest on Titan. Our results also show that Titan's surface is dominated by sedimentary/depositional processes with significant latitudinal variation, with dunes at the equator, plains at mid-latitudes and labyrinth terrains and lakes at the poles.

Basal promoter of the rat connexin 32 gene: identification and characterization of an essential element and its DNA-binding protein.

The connexin 32 (Cx32) gene, a member of a multigene family, is expressed preferentially in the liver. The basal promoter complex of the rat Cx32 gene was previously localized to a 146-bp region (map positions [mp] -179 to -34) immediately upstream of the first exon. To investigate the biochemical factors contributing to the basal promoter activity, nuclear protein-DNA complexes within this region (mp -177 to -106) were investigated by using a DNA mobility shift assay. Three DNA-protein binding activities, termed Cx32-B1, Cx32-B2, and Cx32-B3, were identified with nuclear protein extracts from hepatoma cell lines, HuH7 and FAO-1. However, only Cx32-B2 binding activity was detected in nuclear protein extract from normal rat liver tissue. This activity was significantly more abundant in rat liver tissue than in hepatoma cell lines and tissues from various other organs. By using methylation interference footprinting, the Cx32-B2 complex was localized to the region between mp -152 and -127 and a DNA probe containing this region bound to a 60-kDa protein in rat liver nuclear extracts. Mutation of two nucleotides in the Cx32-B2 binding site abrogated the formation of the Cx32-B2 protein-DNA complex and significantly reduced the transcriptional activity of the Cx32 promoter. These results indicate that the Cx32-B2 complex is an essential component of the rat Cx32 basal promoter and is likely a major factor in the preferential expression of this gene in the liver.

VHL induces renal cell differentiation and growth arrest through integration of cell-cell and cell-extracellular matrix signaling.

Mutations in the von Hippel-Lindau (VHL) gene are involved in the family cancer syndrome for which it is named and the development of sporadic renal cell cancer (RCC). Reintroduction of VHL into RCC cells lacking functional VHL [VHL(-)] can suppress their growth in nude mice, but not under standard tissue culture conditions. To examine the hypothesis that the tumor suppressor function of VHL requires signaling through contact with extracellular matrix (ECM), 786-O VHL(-) RCC cells and isogenic sublines stably expressing VHL gene products [VHL(+)] were grown on ECMs. Cell-cell and cell-ECM signalings were required to elicit VHL-dependent differences in growth and differentiation. VHL(+) cells differentiated into organized epithelial sheets, whereas VHL(-) cells were branched and disorganized. VHL(+) cells grown to high density on collagen I underwent growth arrest, whereas VHL(-) cells continued to proliferate. Integrin levels were up-regulated in VHL(-) cells, and cell adhesion was down-regulated in VHL(+) cells during growth at high cell density. Hepatocyte nuclear factor 1alpha, a transcription factor and global activator of proximal tubule-specific genes in the nephron, was markedly up-regulated in VHL(+) cells grown at high cell density. These data indicate that VHL can induce renal cell differentiation and mediate growth arrest through integration of cell-cell and cell-ECM signals.

Reduction in exposure to arsenic from drinking well-water in Bangladesh limited by insufficient testing and awareness.

This study considers potential policy responses to the still very high levels of exposure to arsenic (As) caused by drinking water from shallow tubewells in rural Bangladesh. It examines a survey of 4,109 households in 76 villages of Araihazar upazila conducted two years after a national testing campaign swept through the area. The area is adjacent to the region where a long-term study was initiated in 2000 and where households are periodically reminded of health risks associated with well-water elevated in As. Results confirm that testing spurs switching away from unsafe wells, although the 27% fraction who switched was only about half of that in the long-term study area. By village, the fraction of households that switched varied with the availability of safe wells and the distance from the long-term study area. Lacking follow-up testing, two years only after the campaign 21% of households did not know the status of their well and 21% of households with an unsafe well that switched did so to an untested well. Well testing is again urgently needed in Bangladesh and should be paired with better ways to raise awareness and the installation of additional deep community wells.

Detection of breast cancer micrometastases in axillary lymph nodes by using polymerase chain reaction.

Breast cancer micrometastases in axillary lymph nodes have been detected by serial sectioning and immunohistochemistry, and shown to have prognostic significance. We have used polymerase chain reaction (PCR) to see whether we could further improve the detection rate of micrometastases. Fifty-seven axillary lymph nodes from patients with breast cancer were examined histologically to assess the proportion of tumor involvement. Immunohistochemical staining with the use of an anti-keratin 19 antibody confirmed the histological findings. Reverse transcription PCR was then performed on extracted RNA by using K19 primers, and all 18 histologically involved nodes yielded the expected 460-base pair product. Of 39 histologically negative nodes, 4 (10%) gave K19 bands detectable with ethidium staining and a further 10 (28%) gave K19 bands after Southern hybridization. To further increase the detection sensitivity a two stage amplification was performed by using nested primers, and K19 product was found in lymph nodes from patients without cancer, as well as in all the nodes from cancer patients. This was shown to be genuine low level expression from endogenous mRNA template, and not derived from amplification of a K19 pseudogene. Reducing the number of PCR cycles in the two amplification steps did not allow sufficient discrimination between normal nodes and those involved nodes in which K19 expression was only detectable after Southern hybridization. The optimal "cut-off" point to distinguish involved nodes from normal nodes remained at the level of 40 cycles of PCR and Southern hybridization. PCR, using K19 as a tumor marker, has been demonstrated in this study to improve the detection of micrometastases in axillary lymph nodes in patients with breast cancer: sensitivity is limited by the specificity of the tumor marker.

The von Hippel-Lindau tumor suppressor gene protects cells from UV-mediated apoptosis.

The familial cancer syndrome, von Hippel-Lindau (VHL) disease, characterized by a predisposition to renal cell carcinoma and certain other tumor types, is caused by mutational inactivation of the VHL tumor suppressor gene. Loss of VHL gene function is detected also in the vast majority of sporadic renal cell carcinomas. Previous reports have determined a protective role for VHL in response to serum withdrawal and glucose deprivation. In this study, the effect of UV irradiation on VHL-negative and VHL-positive renal carcinoma cells was examined. VHL-negative 786-O renal carcinoma cells underwent apoptosis following UV irradiation. In contrast, reintroduction of wild-type VHL expression protected 786-O cells from UV-mediated cell death. p53 and Bax levels were equivalent in VHL-negative and VHL-positive 786-O cells. Strikingly, cyclin-dependent kinase inhibitors p21 and p27 underwent proteasome-dependent degradation in VHL-negative 786-O cells following UV treatment. However, p21 and p27 protein levels were stable in VHL-positive cells. Also, levels of the anti-apoptotic proteins, Bcl-2 and Bcl-xL were elevated in VHL-positive cells, consistent with the protection from apoptotic stimuli. UV treatment led to increased S phase in VHL-negative, but not VHL-positive cells. Thus, following UV irradiation, diminution of p21 and p27 levels resulted in a hyperproliferative state in VHL-negative cells, leading to apoptosis. These results suggest that loss of VHL function promotes apoptosis and may provide selective pressure toward cells that are able to escape apoptosis, leading to tumorigenesis.

Gestational diabetes mellitus. A survey of perinatal complications in the 1980s.

Neonatal morbidity was assessed in the offspring of 878 mothers with gestational diabetes mellitus (GDM), 132 mothers with pre-GDM, and 380 control subjects. Compared with the control group, the GDM group had a higher incidence of complications, including macrosomia, hypoglycemia, hyperbilirubinemia, hypocalcemia, polycythemia, and major congenital anomalies (P less than 0.05). Although our GDM patients were stringently managed with diet or diet plus insulin, as indicated, and maintained almost euglycemic values, these neonatal complications could not be eliminated. Our data may be consistent with observations published during the last decade that even subtle degrees of maternal hyperglycemia can have a detrimental effect on perinatal outcome. Most neonatal complications readily respond to therapy if diagnosed and treated early and promptly. Macrosomia can have a detrimental effect on delivery (trauma) and later long-term implications during childhood. Tight metabolic control with diet and, when indicated, insulin treatment may be advantageous in reducing fetal birth weight. Criteria of how tight the metabolic control should be remain to be accurately defined.

The detection of micrometastases in the peripheral blood and bone marrow of patients with breast cancer using immunohistochemistry and reverse transcriptase polymerase chain reaction for keratin 19.

The aim of this study was to determine whether reverse transcriptase polymerase chain reaction (RT-PCR) for keratin 19 (K19) provides additional information when combined with immunohistochemistry when used to detect micrometastases in blood and bone marrow in patients with primary breast cancer. We studied 78 patients with breast cancer who had no evidence of distant metastases. We collected blood and bone marrow, separated the mononuclear fraction and carried out RT-PCR and immunohistochemistry for K19. RT-PCR was done by two 40-cycle rounds using nested primers. In initial experiments, RT-PCR was shown to be capable of detecting one tumour cell in one million normal bone marrow cells, which was at least 10 times more sensitive than immunohistochemistry, while retaining specificity. Five per cent of the peripheral blood and 22% of the bone marrow samples contained K19 positive cells by immunohistochemistry staining. Using RT-PCR, these proportions increased to 25% and 35%, respectively. This represents a significantly greater detection frequency (P < 0.001 and P = 0.03, respectively). RT-PCR for K19 is a more sensitive method for detecting micrometastases in patients with primary breast cancer when compared with immunohistochemistry.

A survey of radiation oncologists regarding their radiation physics instruction.

The American Association of Physicists in Medicine, Committee on Training of Radiologists conducted a survey of radiation oncologists requesting information regarding their radiation oncology physics training. General questions were asked of the oncologist regarding their radiation oncology practice such as number of oncologists, number of new patients treated, and the size and type of facility in which the practice is located. The oncologist also responded to questions regarding their educational background. The survey requested the radiation oncologists to answer questions regarding the adequacy and importance of their training in specific areas of radiation physics. The responders indicated that the importance of most physics topics in their clinical practice corresponded to the level of their understanding. The survey indicated that for most radiation oncologists their physics instruction was an important and interesting part of their residency program.