Assessment of Factors Associated With the Evaluation of Children for Leukemia Predisposition Syndromes: A Retrospective Single-center Study.

Five to 10% of children with cancer are thought to have a cancer predisposition syndrome (CPS). Referral guidelines for leukemia predisposition syndromes are limited and vague, requiring the treating provider to determine whether patients should have a genetics evaluation. We evaluated referrals to the pediatric cancer predisposition clinic (CPP), the prevalence of CPS in those who elected to pursue germline genetic testing, and assessed for associations between a patient's medical history and the diagnosis of a CPS. Data were obtained via chart review of children diagnosed with leukemia or myelodysplastic syndrome between November 1, 2017, and November 30, 2021. A total of 22.7% of pediatric leukemia patients were referred for evaluation in the CPP. Of the participants evaluated with germline genetic testing, the prevalence of a CPS was 25%. Our study was able to find a CPS in different malignancies, including acute lymphoblastic leukemia, acute myeloid leukemia, and myelodysplastic syndrome. We did not find associations between a participant with an abnormal CBC before diagnosis or hematology visit and the diagnosis of a CPS. Our study supports that a genetic evaluation should be available to all children with leukemia as medical and family history alone is not predictors of a CPS.

Evaluation of barriers to referral for cancer predisposition syndromes in pediatric oncology patients in the United States.

Cancer predisposition syndromes (CPS) are underdiagnosed in the pediatric population, though the diagnosis of a CPS has important implications for the child and their family. CPS are often diagnosed by geneticists or oncologists with expertise in CPS following a malignancy. This requires a member of the care team, most commonly, the treating oncologist to suspect a CPS and refer the patient for CPS assessment. An online survey was distributed to members of the Children's Oncology Group to elucidate current referral practices and barriers to referral for patients suspected to have a CPS. Of the 183 respondents, 86.1% was pediatric oncologists and most (68.5%) used formal guidelines to aid in assessment. Most respondents indicated they would rarely refer patients with tumors highly associated with CPS for genetic assessment. Participants were more likely to refer patients with malignancy and additional features of a CPS than for a specific type of cancer, despite the use of guidelines. Parent knowledge of family history was considered the most challenging barrier to obtaining a family history, though a thorough pedigree was not consistently elicited. Providers indicated the most significant barrier to referral for CPS assessment was priority given the patient's immediate care needs. Identification of these barriers provides direction to focus efforts to increase referrals. Provider education about CPS, clear referral guidelines, and implementation of or increased collaboration with a genetic counselor in the pediatric oncology clinic may encourage CPS assessment and enable oncologists to focus on the patient's immediate care needs.

Alcohol and tobacco lower the age of presentation in sporadic pancreatic cancer in a dose-dependent manner: a multicenter study.

OBJECTIVES: The objective of this study was to examine the association between tobacco and alcohol dose and type and the age of onset of pancreatic adenocarcinoma (PancCa). METHODS: Prospective data from the Pancreatic Cancer Collaborative Registry were used to examine the association between age of onset and variables of interest including: gender, race, birth country, educational status, family history of PancCa, diabetes status, and tobacco and alcohol use. Statistical analysis included logistic and linear regression, Cox proportional hazard regression, and time-to-event analysis. RESULTS: The median age to diagnosis for PancCa was 66.3 years (95% confidence intervals (CIs), 64.5-68.0). Males were more likely than females to be smokers (77% vs. 69%, P=0.0002) and heavy alcohol and beer consumers (19% vs. 6%, 34% vs. 19%, P<0.0001). In univariate analysis for effects on PancCa presentation age, the following were significant: gender, alcohol and tobacco use (amount, status and type), family history of PancCa, and body mass index. Both alcohol and tobacco had dose-dependent effects. In multivariate analysis, alcohol status and dose were independently associated with increased risk for earlier PancCa onset with greatest risk occurring in heavy drinkers (HR 1.62, 95% CI 1.04-2.54). Smoking status had the highest risk for earlier onset pancreatic cancer with a HR of 2.69 (95% CI, 1.97-3.68) for active smokers and independent effects for dose (P=0.019). The deleterious effects for alcohol and tobacco appear to resolve after 10 years of abstinence. CONCLUSIONS: Alcohol and tobacco use are associated with a dose-related increased risk for earlier age of onset of PancCa. Although beer drinkers develop pancreatic cancer at an earlier age than nondrinkers, alcohol type did not have a significant effect after controlling for alcohol dose.

The O-Ag Antibody Response to Francisella Is Distinct in Rodents and Higher Animals and Can Serve as a Correlate of Protection.

Identifying correlates of protection (COPs) for vaccines against lethal human (Hu) pathogens, such as Francisella tularensis (Ft), is problematic, as clinical trials are currently untenable and the relevance of various animal models can be controversial. Previously, Hu trials with the live vaccine strain (LVS) demonstrated ~80% vaccine efficacy against low dose (~50 CFU) challenge; however, protection deteriorated with higher challenge doses (~2000 CFU of SchuS4) and no COPs were established. Here, we describe our efforts to develop clinically relevant, humoral COPs applicable to high-dose, aerosol challenge with S4. First, our serosurvey of LVS-vaccinated Hu and animals revealed that rabbits (Rbs), but not rodents, recapitulate the Hu O-Ag dependent Ab response to Ft. Next, we assayed Rbs immunized with distinct S4-based vaccine candidates (S4ΔclpB, S4ΔguaBA, and S4ΔaroD) and found that, across multiple vaccines, the %O-Ag dep Ab trended with vaccine efficacy. Among S4ΔguaBA-vaccinated Rbs, the %O-Ag dep Ab in pre-challenge plasma was significantly higher in survivors than in non-survivors; a cut-off of >70% O-Ag dep Ab predicted survival with high sensitivity and specificity. Finally, we found this COP in 80% of LVS-vaccinated Hu plasma samples as expected for a vaccine with 80% Hu efficacy. Collectively, the %O-Ag dep Ab response is a bona fide COP for S4ΔguaBA-vaccinated Rb and holds significant promise for guiding vaccine trials with higher animals.

Restraint and Social Isolation Stressors Differentially Regulate Adaptive Immunity and Tumor Angiogenesis in a Breast Cancer Mouse Model.

The ability of stress to induce immune suppression is widely recognized, but the mechanisms underlying the effects of stress on the adaptive immune system during tumor progression are not completely understood. To study the effect of stress on the immune system in vivo, we used a preclinical immunocompetent mouse model bearing 4T1 mammary adenocarcinoma cells. Mice were randomized into 4 groups, including social isolation (SI), acute restraint stress (aRRS), chronic restraint stress (cRRS), or no stress (NS). We found that SI significantly decreased the number of tumor-bearing mice still alive at the end of protocol (28 days), compared to NS mice. Although we did not detect significant changes in primary tumor volume, we observed a significant increase in the endothelial marker CD31 in primary tumors of SI mice and in lung metastases in SI and RRS mice. Survival decline in SI mice was associated with significant decreases in splenic CD8 cells and in activated T cells. From a mechanistic standpoint, RRS increased expression of FOXP3, CXCL-10, and granzyme B in mouse tumors, and the effects were reversed by propranolol. Our data demonstrate that various forms of stress differentially impact adaptive immunity and tumor angiogenesis, and negatively impact survival.

Dual HER2 gene protein assay: focused study of breast cancers with 2+ immunohistochemical expression.

OBJECTIVES: The combined gene protein assay (GPA) can simultaneously assess HER2 gene copy number and protein on a single slide using bright-field microscopy. METHODS: GPA was compared with a fluorescence in situ hybridization (FISH) assay on 50 invasive breast carcinomas with a 2+ score on immunohistochemistry (IHC). RESULTS: The cases were categorized into positive, equivocal, or negative for HER2 gene amplification using the 2013 American Society of Clinical Oncology/College of American Pathologists criteria. This resulted in 82% agreement (41 of 50) between FISH and GPA. In addition, 25 known IHC 3+ breast carcinomas analyzed by GPA showed protein overexpression and clusters of HER2 gene consistent with unequivocal amplification, and 22 known IHC-negative cases were negative for HER2 gene amplification by GPA. CONCLUSIONS: Although GPA is an alternative to both IHC and FISH, it may be an unnecessary test for IHC 0/1+/3+ cases. The clinical utility of GPA appears to be similar to other in situ hybridization assays (ie, adjudicator of HER2 status for IHC 2+ cases).

A Role for Histone H2B Variants in Endocrine-Resistant Breast Cancer.

Acquired resistance to aromatase inhibitors (AIs) remains a major clinical problem in the treatment of estrogen receptor-positive (ER+) breast cancer. We and others have previously reported widespread changes in DNA methylation using breast cancer cell line models of endocrine resistance. Here, we show that the histone variant HIST1H2BE is hypomethylated in estrogen deprivation-resistant C4-12 and long-term estrogen-deprived (LTED) cells compared with parental MCF-7 cells. As expected, this hypomethylation associates with increased expression of HIST1H2BE in C4-12 and LTED cells. Both overexpression and downregulation of HIST1H2BE caused decreased proliferation in breast cancer cell lines suggesting the need for tightly controlled expression of this histone variant. Gene expression analysis showed varied expression of HIST1H2BE in a large panel of breast cancer cell lines, without restriction to specific molecular subtypes. Analysis of HIST1H2BE messenger RNA (mRNA) expression in ER+ AI-treated breast tumors showed significantly higher expression in resistant (n = 19) compared with sensitive (n = 37) tumors (p = 0.01). Using nanostring analysis, we measured expression of all 61 histone variants in endocrine-resistant and endocrine-sensitive tumors. We found significant overexpression of 22 variant histone genes in tumors resistant to AI therapy. In silico The Cancer Genome Atlas (TCGA) analysis showed frequent amplification of the HIST1 locus. In summary, our studies show, for the first time, that overexpression of histone variants might be important in endocrine response in ER+ breast cancer, and that overexpression is at least in part mediated via epigenetic mechanisms and amplifications. Future studies addressing endocrine response should include a potential role of these currently understudied histone variants.

Bright-field HER2 dual in situ hybridization (DISH) assay vs fluorescence in situ hybridization (FISH): focused study of immunohistochemical 2+ cases.

OBJECTIVES: To compare the INFORM HER2 bright-field dual in situ hybridization (DISH) DNA probe cocktail assay with the PathVysion fluorescence in situ hybridization (FISH) assay on 103 invasive breast carcinomas with a 2+ score on immunohistochemistry (IHC). METHODS: The cases were categorized as positive, equivocal, or negative for HER2 gene amplification using the 2007 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) HER2:CEP17 ratio criteria and also based on mean HER2 gene copies/cell. The third criterion used a HER2:CEP17 ratio of 2 to categorize cases as positive or negative. RESULTS: The agreement between FISH and DISH was 85% using the 2007 ASCO/CAP ratio criterion, 79% using the mean HER2 gene copies/cell criterion, and 92% using the 2.0 cutoff HER2:CEP17 ratio criterion. In addition, 20 known IHC 3+ breast carcinomas analyzed by DISH showed clusters of the HER2 gene consistent with unequivocal amplification. CONCLUSIONS: Despite some technical and interpretational issues associated with DISH, it compares favorably with FISH in this group of challenging breast cancer cases.

Invasive lobular carcinoma cell lines are characterized by unique estrogen-mediated gene expression patterns and altered tamoxifen response.

Invasive lobular carcinoma (ILC) is a histologic subtype of breast cancer that is frequently associated with favorable outcomes, as approximately 90% of ILC express the estrogen receptor (ER). However, recent retrospective analyses suggest that patients with ILC receiving adjuvant endocrine therapy may not benefit as much as patients with invasive ductal carcinoma. On the basis of these observations, we characterized ER function and endocrine response in ILC models. The ER-positive ILC cell lines MDA MB 134VI (MM134) and SUM44PE were used to examine the ER-regulated transcriptome via gene expression microarray analyses and ER ChIP-Seq, and to examine response to endocrine therapy. In parallel, estrogen response was assessed in vivo in the patient-derived ILC xenograft HCI-013. We identified 915 genes that were uniquely E2 regulated in ILC cell lines versus other breast cancer cell lines, and a subset of these genes were also E2 regulated in vivo in HCI-013. MM134 cells were de novo tamoxifen resistant and were induced to grow by 4-hydroxytamoxifen, as well as other antiestrogens, as partial agonists. Growth was accompanied by agonist activity of tamoxifen on ER-mediated gene expression. Though tamoxifen induced cell growth, MM134 cells required fibroblast growth factor receptor (FGFR)-1 signaling to maintain viability and were sensitive to combined endocrine therapy and FGFR1 inhibition. Our observation that ER drives a unique program of gene expression in ILC cells correlates with the ability of tamoxifen to induce growth in these cells. Targeting growth factors using FGFR1 inhibitors may block survival pathways required by ILC and reverse tamoxifen resistance.

Impact of progesterone receptor semiquantitative immunohistochemical result on Oncotype DX recurrence score: a quality assurance study of 1074 cases.

Decreased or absent progesterone receptor expression in invasive breast carcinoma is a marker for an adverse prognosis. As part of an ongoing quality assurance study, this study evaluated the relationship between Oncotype DX recurrence score and progesterone receptor immunohistochemical result within each Nottingham tumor grade in 1074 cases of invasive breast carcinoma for which an Oncotype DX recurrence score was available. In addition to a statistically significant association between Nottingham grade and Oncotype DX recurrence score categories (P < 0.001), an inverse relationship was identified between progesterone receptor expression measured by modified H-score semiquantitation and Oncotype DX recurrence score that was independent of Nottingham tumor grade. The Oncotype DX recurrence score relies heavily on parameters already available from routine pathologic examination, and consideration of progesterone receptor status may aid in selection of patients most likely to benefit from ancillary testing.

Proteomic Analysis of Matched Formalin-Fixed, Paraffin-Embedded Specimens in Patients with Advanced Serous Ovarian Carcinoma.

OBJECTIVE: The biology of high grade serous ovarian carcinoma (HGSOC) is poorly understood. Little has been reported on intratumoral homogeneity or heterogeneity of primary HGSOC tumors and their metastases. We evaluated the global protein expression profiles of paired primary and metastatic HGSOC from formalin-fixed, paraffin-embedded (FFPE) tissue samples. METHODS: After IRB approval, six patients with advanced HGSOC were identified with tumor in both ovaries at initial surgery. Laser capture microdissection (LCM) was used to extract tumor for protein digestion. Peptides were extracted and analyzed by reversed-phase liquid chromatography coupled to a linear ion trap mass spectrometer. Tandem mass spectra were searched against the UniProt human protein database. Differences in protein abundance between samples were assessed and analyzed by Ingenuity Pathway Analysis software. Immunohistochemistry (IHC) for select proteins from the original and an additional validation set of five patients was performed. RESULTS: Unsupervised clustering of the abundance profiles placed the paired specimens adjacent to each other. IHC H-score analysis of the validation set revealed a strong correlation between paired samples for all proteins. For the similarly expressed proteins, the estimated correlation coefficients in two of three experimental samples and all validation samples were statistically significant (p < 0.05). The estimated correlation coefficients in the experimental sample proteins classified as differentially expressed were not statistically significant. CONCLUSION: A global proteomic screen of primary HGSOC tumors and their metastatic lesions identifies tumoral homogeneity and heterogeneity and provides preliminary insight into these protein profiles and the cellular pathways they constitute.

The effect of 96-hour formalin fixation on the immunohistochemical evaluation of estrogen receptor, progesterone receptor, and HER2 expression in invasive breast carcinoma.

We studied the impact of 96 hours of formalin fixation on estrogen receptor (ER), progesterone receptor (PR), and HER2 testing by comparing immunohistochemical results from core biopsy specimens fixed under current American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines with results for corresponding resection samples fixed for 96 hours. Samples enriched with cases showing weak to moderate receptor expression on core biopsy were included in the study. Cases were scored using ASCO/CAP guidelines. Of the 47 cases, only 1 case (2%) showed a qualitative change in result. However, this change was a positive ER result (H score, 1) on the 96-hour fixed resected sample compared with a negative ER result (H score, 0) for the core biopsy. Minimal changes in semiquantitative H scoring were noted for ER and PR that were likely due to tumor heterogeneity and/or intraobserver variability as the variation occurred in both directions. ER, PR, and HER2 immunohistochemical results should be considered valid for cases fixed up to 96 hours.

Interobserver agreement among pathologists for semiquantitative hormone receptor scoring in breast carcinoma.

The American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines recommend reporting of hormone receptor test results in a semiquantitative manner. This study used 74 resected estrogen receptor (ER)-positive invasive breast cancers to determine reproducibility of semiquantitative scoring of hormone receptors using the H-score method. Four pathologists independently scored each slide. Agreement among observers was analyzed via Fleiss κ statistics on ER and progesterone receptor (PR) categorical scores. Intraclass correlation coefficient (ICC) was used to estimate the interobserver agreement for ER and PR H-scores on a continuous scale (0-300). There was 100% agreement for categorical ER results (κ = 1) and 97% agreement (κ = 0.823, P < .001) for categorical PR results. For quantitative H-scores, ICC agreement was 0.85 (95% confidence interval [CI] = 0.79-0.90) for ER and 0.87 (95% CI = 0.82-0.92) for PR. Because the H-score provides a continuous measure of tumor hormone receptor content, we suggest universal adoption of this method.

Racial disparities in resource utilization for cystectomy.

OBJECTIVES: To examine the association of race with mortality and resource use among patients requiring cystectomy for bladder cancer, given the known racial differences with regard to bladder cancer incidence and survival. METHODS: Using the Nationwide Inpatient Sample (a nationally representative data set), 22,088 patients who underwent cystectomy for bladder cancer from 1988 to 2000 were identified using the International Classification of Disease, Ninth Revision, codes. The outcomes included in-hospital mortality, length of stay (LOS), and discharge status. Multivariable models were developed to perform risk-adjusted analyses and identify factors associated with these outcomes. RESULTS: The overall mortality rate after cystectomy was 2.9%. Unadjusted analyses revealed significant racial differences with respect to in-hospital mortality, LOS, and discharge disposition. Whites had a mortality rate of 2.8% compared with 4.2% for blacks and 3.9% for Hispanics (P = 0.006). Whites had a prolonged LOS 24.9% of the time compared with 38.2% for blacks and 24.6% for Hispanics (P < 0.001). The rate at which whites were discharged to subacute care facilities was 9.9% compared with 11.2% for black patients and 7.7% for Hispanics (P < 0.001). After adjusting for confounding factors, blacks were more likely to experience in-hospital mortality and prolonged LOS (odds ratios 1.66 and 2.10, respectively) compared with whites, although no significant differences were observed for Hispanics. No significant racial differences were noted for discharge status after risk adjustment. CONCLUSIONS: Black patients undergoing cystectomy for bladder cancer had greater mortality and greater LOS than did white patients. Additional study using detailed clinical data is necessary to identify the underlying causes of these differences.

Use of multiple imputation to correct for nonresponse bias in a survey of urologic symptoms among African-American men.

The Flint Men's Health Study is an ongoing population-based study of African-American men designed to address questions related to prostate cancer and urologic symptoms. The initial phase of the study was conducted in 1996-1997 in two stages: an interviewer-administered survey followed by a clinical examination. The response rate in the clinical examination phase was 52%. Thus, some data were missing for clinical examination variables, diminishing the generalizability of the results to the general population. This paper is a case study demonstrating the application of multiple imputation to address important questions related to prostate cancer and urologic symptoms in a data set with missing values. On the basis of the observed clinical examination data, the American Urological Association Symptoms Score showed a surprising reduction in symptoms in the oldest age group, but after multiple imputation there was a monotonically increasing trend with age. It appeared that multiple imputation corrected for nonresponse bias associated with the observed data. For other outcome measures-namely, the age-adjusted 95th percentile of prostate-specific antigen level and the association between urologic symptoms and prostate volume-results from the observed data and the multiply imputed data were similar.