Treatment recommendations for primary liver malignancies, including hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), are complex and require a multidisciplinary approach. Despite surgical options that are potentially curative, options for nonsurgical candidates include systemic therapy, radiotherapy (RT), transarterial chemoembolization (TACE), and radiofrequency ablation (RFA). Stereotactic Body Radiation Therapy (SBRT) is now in routine use for the treatment of lung cancer, and there is growing evidence supporting its use in liver tumors. SBRT has the advantage of delivering ablative radiation doses in a limited number of fractions while minimizing the risk of radiation-induced liver disease (RILD) through highly conformal treatment plans. It should be considered in a multidisciplinary setting for the management of patients with unresectable, locally advanced primary liver malignancies and limited treatment options. Recently, the combination of immunotherapy with SBRT has been proposed to improve antitumor effects through engaging the immune system. This review aims at shedding light on the novel concept of the combination strategy of immune-radiotherapy in liver tumors by exploring the evidence surrounding the use of SBRT and immunotherapy for the treatment of HCC and CCA. Primary liver cancer is the seventh most common cancer world-wide, with around 841,080 newly diagnosed cases in 2018 . The twoHCC accounts for 75 to 85% of primary liver cancers world-wide . Its preHCC is the second most frequent cause of cancer death in men and the sixth leading cause of cancer death in women , 8. AlthThe pathogenesis of IHC seems to be related to chronic inflammation and the resulting oxidative stress created in bile ducts . IHC conThe 5-year survival in IHC patients is less than 10%. The dismal prognosis is due to advanced stages at time of diagnosis, limited treatment options, and very high rates of recurrence and metastases . SurgicaTreatment recommendations for primary liver tumors are complex and require a multidisciplinary approach. Despite surgical options that are potentially curative, options for nonsurgical candidates include systemic therapy , external-beam radiation therapy (EBRT), TACE, and percutaneous tumor ablation . Here we focus on evolving RT and immunotherapy approaches.Historically, EBRT has not played a substantial role in the treatment of liver malignancies secondary to the limited tolerance of the whole liver to radiation. Over the past decade, the approach to liver cancer patients has been affected by a paradigm shift that has revolutionized RT –39. ConsIt is well established that RT has direct cytotoxic effects on cancer cells and can generate a robust antitumor immune response through effects on both the tumor and its microenvironment. This occurs via a variety of mechanisms including enhanced tumor antigen presentation and upregulated major histocompatibility complex (MHC) class I expression . The higEmerging clinical data suggest that RT may have systemic effects that go far beyond the locally irradiated target , 48. SucSBRT is a highly specialized form of EBRT that delivers high doses of precisely targeted radiation in a few fractions to a tumor and minimizes radiation dose to adjacent normal tissue structures , 51. It NCT01730937) by the Radiation Therapy Oncology Group.SBRT is now in routine use for the treatment of lung cancer, and there is growing evidence supporting its use in primary liver tumors. Data reveal 1-year local control (LC) rates exceeding 90% following the use of SBRT in HCC  and hepaNCT03402607].Percutaneous tumor ablation, typically by RFA or MWA, is usually performed in patients with early stage, unresectable HCC. A retrospective study from the University of Michigan highlighted the relative efficacy of using SBRT as compared to percutaneous tumor ablation in patients with unresectable HCC . FreedomA large single-institution comparison of TACE and SBRT outcomes was performed on 209 patients with small HCC tumors (2.3-2.9cm) . 84 patiNCT02470533] is based on the hypothesis that the time to progression is more favorable after SBRT than after TACE in HCC patients ineligible for surgery or RFA. Results are expected to be available in April 2020. SBRT, therefore, represents a noninvasive, potentially curative modality that can be utilized in the definitive treatment of patients with HCC and as a bridge for patients potentially eligible for transplantation. While the 2016 NCCN guidelines listed EBRT as a viable option for the treatment of advanced HCC, current 2018 NCCN guidelines implement the role of SBRT in both resectable and nonresectable cases planned for transplantation  and comparing SBRT to TACE [NCT02182687 and NCT02470533].In order to evaluate the combination of SBRT and TACE, a retrospective study of patients with HCC that are larger than 3cm in size was conducted . PatientDespite its relatively recent adoption into clinical practice, SBRT use in liver tumors was first described in the early 1990s in a pilot study by Blomgren et al. . Nine paOne retrospective study by Sanuki et al. included 185 liver tumors ranging in diameter from 0.8 to 5cm and used SBRT doses ranging from 30 to 40Gy in 5 fractions . 3-year A phase I trial included 41 patients (31 HCC and 10 IHC) with unresectable tumors that have a median liver tumor size of 173mL . After cA phase II trial from Princess Margaret Cancer Centre included patients with CTP Class A disease and used SBRT doses ranging from 24 to 54Gy in six fractions . ResultsAt the University of Michigan, an individualized dose allocation strategy using hyperfractionation was developed for liver cancer treatment and was evaluated by a phase II study. It included 46 IHC patients who were treated with conformal hyperfractionated RT with concurrent hepatic arterial fluorodeoxyuridine. Compared with historical controls, patients had significantly improved OS with a median survival of 13.3 months .Therefore, based on initial promising data, SBRT seems to be a safe, effective, and noninvasive treatment option for carefully selected patients with unresectable tumors that are not amenable to other treatments.Although there is evidence that RT alone provides the necessary signals for the cross-priming of cytotoxic T lymphocytes against tumor antigens, the adjuvant effect of RT appears to be relatively weak, and abscopal responses to RT alone are extremely rare. Also, despite the antitumor effect induced by RT, tumors often develop resistance via immune-escape mechanisms that promote recurrence , 65. In In order to overcome limitations created by the PD-L1/PD-1 interaction and to reduce the rate of tumor recurrence in primary liver tumors, novel therapies are required. Recent discoveries in tumor immunology, paralleled by technological advances in RT, have provided a promising role for combining SBRT with immunotherapy to augment and sustain the proimmunogenic antitumor effects seen with SBRT alone.Since immune checkpoint inhibitors (ICIs) were first reported in 2010 and 2012, they have translated into a significant OS advantage in comparison to established therapies in metastatic melanomas and nonsmall-cell lung cancer (NSCLC) . Their r(1) Anticytotoxic T-Lymphocyte Associated Antigen-4 Agents. CTLA-4 receptor is exclusively expressed on regulatory T-cells, naïve T-cells, and activated T-cells, and it acts as a regulator of immune cells [ne cells –74. Throne cells , 76. It The anti-CTLA-4 antibody, Tremelimumab, was evaluated in a phase I trial that included 21 patients with inoperable HCC tumors . 17.6% a (2) Antiprogrammed-Death Ligand-1 and Antiprogrammed-Death -1 Agents. PD-1 receptor belongs to the CD28 superfamily and is expressed on regulatory T-cells, B-cells, and myeloid derived suppressor cells (MDSCs) [ (MDSCs) . It tran (MDSCs) , 80. Mos (MDSCs) , 82. For (MDSCs) .PD-L1 is undetectable in most normal tissues and is inducible by inflammatory cytokines, especially types I and II interferon (IFN) . It is fMany phase I/II trials have shown promising outcomes with the use of the humanized anti-PD-1 antibody, Nivolumab, in patients with advanced melanoma, lung cancer, and renal cell carcinoma (RCC) . This waResults also showed that all participants, whether infected by HBV or HCV or not, encountered tumor size reduction . One patThe report also indicated that Nivolumab monotherapy had a favorable safety profile in HCC similar to that seen in other types of cancer . In factMany ongoing trials are assessing the efficacy of combining Nivolumab with gemcitabine/cisplatin on one hand, or Ipilimumab on the other, as a first-line therapy in advanced, unresectable CCA . A singlNCT02054806) that evaluated the role of Pembrolizumab in advanced CCA, almost 34% (n=8) of patients with positive PD-L1 expression had PR or SD [A phase II trial evaluated whether testing for mismatch-repair (MMR) deficiencies in treatment-refractory cases of IHC might be of help in identifying those who might benefit from PD-1 pathway blockade . Out of PR or SD . It remaOngoing trials will provide us with more details about the role of targeted immunotherapy in primary liver tumors. Antibodies targeting PD-1, PD-L1, or CTLA-4 are expected to be approved and implemented in the setting of HCC and IHC in the very near future.Immunotherapy has emerged as an attractive therapy for refractory cancers. Although results obtained in melanoma and NSCLC patients were beyond what is achievable with conventional therapies, it is expected that such responses will only occur in a subset of HCC or IHC patients who have a high tumor mutational burden . In ordeRT results in the immunogenic death of tumor cells. It primes tumor-specific T-cells and induces the production of IFN-beta by T-cells, thereby enhancing MHC class 1 expression on parental and resistant cancer cells . This reβ blockade but not with TGFβ blockade alone [Many preclinical studies demonstrate a synergistic effect when RT and CTLA-4 or PDL-1 inhibitors are combined . In a stde alone . Exposurde alone . Tumors Local RT and systemic PD-L1 blockade augment T-cell responses not only in the primary tumor but also at distant sites . This reCombining RT with anti-PD-1 antibody treatment has consistently produced abscopal effects on secondary tumors that are distant from the irradiated primary site in mouse models of melanoma, colon cancer, RCC, and breast cancer , 107. InThe timing of RT relative to immunotherapy may be another important consideration when combining RT with immunotherapy . This qu(1) Amelioration of Cancer-Cell Type I Interferon. It is now established that the cyclic GMP-AMP Synthase (cGAS) - Stimulator of Interferon Genes (STING) pathway plays an important role in improving the antitumor immune response triggered by RT and immunotherapy. It induces IFN which is required to achieve optimal DC recruitment and cross-priming of effector T-cells [ T-cells –114. Cro T-cells . This is T-cells . Data su T-cells .Deng et al. demonstrated that these DCs activate the cGAS-STING signaling axis following the exposure of tumor cells to RT which results in the accumulation of irradiated tumor cell derived DNA. cGAS senses these RT-generated double-stranded DNA fragments and catalyzes the reaction between GTP and ATP responsible for the formation of the second messenger, cyclic GMP-AMP (cGAMP). The latter binds to the STING adaptor protein, triggering the phosphorylation of interferon regulatory factor 3 (IRF-3) by TANK-binding kinase 1 (TBK-1). IRF-3 then transports to the nucleus where transcription of inflammatory genes is triggered and an increase in type I IFN and other immune modulatory molecules results. Type I IFNs induced by RT have been shown to mediate the antitumor immune response and increase the frequency of CD8+ T-cells in tumor-draining lymph nodes .In a preclinical study on mice bearing a B16 melanoma, those that were cGAS-deficient had a lower response to anti-PD-L1 treatment than wild-type controls . cGAS kn(2) Reduction of Tumor Microenvironment Immunosuppression. Many studies show that the reduction in MDSC levels observed with the combination therapy might be critical in achieving an abscopal effect. In an experiment performed by Deng et al. on a mouse flank tumor model, substantial tumor regression was noted in the combination treatment group and was thought to be due to the concomitant increase in cytotoxic T-cell infiltration and the dramatic reduction in MDSCs [in MDSCs . In a pain MDSCs . Demariain MDSCs . RT primin MDSCs , 120. Kain MDSCs . For insin MDSCs . In addiin MDSCs . This inin MDSCs , 123.In an attempt to elicit greater antigen release and further improve the efficacy of immunotherapeutic agents, many preclinical studies tested the effect of combining them with hypofractionated regimens, particularly SBRT. The trials to date have used a variety of different doses and fractionations ranging from 15 to 75Gy in 1 to 15 fractions. The choice of optimal radiation dose and fractionation schema is related to the resulting abscopal effects.A number of studies have demonstrated dose dependence of abscopal effect. Inferior abscopal effects have been noted with a single 20Gy dose of radiation as compared with regimens of 8Gy in three or 6Gy in five fractions , 124. InGene expression analysis of cells from the irradiated tumors revealed an IFN type I gene signature following exposure to 8Gy in 3 fractions but not following a single dose of 20 or 30Gy . This woThe promising preclinical data on the combination therapy in mouse tumor models have resulted in a number of analyses reporting the safety and efficacy of this strategy in humans. In a retrospective study by Hubbeling et al., no significant difference was reported between RT-related adverse events observed in metastatic NSCLC patients who received prophylactic cranial RT combined with PD-1/PD-L1 inhibitors as compared to events observed in patients who received only cranial RT . Park etTo examine the feasibility and efficacy of RT combined with immune checkpoint blockade, several studies have been conducted as summarized in In another prospective clinical trial, 22 patients with stage IV melanoma were exposed to palliative RT (8Gy in 3 fractions or 4Gy in 10 fractions) five days following treatment with 4 cycles of Ipilimumab . RT to 1In another clinical trial by Tang et al., 35 patients were treated with SBRT (12Gy in 4 fractions or 6Gy in 10 fractions) either concurrently (1 day after the first dose of Ipilimumab) or sequentially (1 week after the second dose) [One of the largest prospective phase I studies (Abstract 20) to determine the safety and efficacy of SBRT in combination with Pembrolizumab in patients with metastatic solid tumors who progressed on standard treatment included doses ranging from 30Gy in 3 fractions to 50Gy in 5 fractions. Pembrolizumab therapy was initiated 7 days after the final SBRT treatment. According to data presented at the 2018 ASCO-SITC Clinical Immune-Oncology Symposium, SBRT prior to Pembrolizumab treatment was well tolerated, and the OR rate was 13.5% in the 68 patients who had imaging follow-up. Some abscopal responses were seen, whereby 26.9% of patients had a reduction of at least 30.0% in any single nonirradiated lesion and 13.5% of patients had a reduction of at least 30.0% in the aggregate sum of nonirradiated lesions . A phaseCurrently, at least 12 ongoing prospective clinical trials are evaluating the safety and efficacy of the combination of RT and immunotherapy in metastatic NSCLC . SimilarAs such, current data related to combination therapy in primary liver tumors are based on results from either preclinical animal models, which are inherently limited in their applicability to the clinical setting, or preliminary results from ongoing trials. Although results seem promising, implementation in clinical practice would be premature, as robust hypothesis-testing clinical trials are required to determine appropriate approaches of integrating these modalities.While SBRT alone and immunotherapy alone have shown promise as effective therapies in patients with primary liver tumors, the combination of SBRT and PD-L1, PD-1, or CTLA-4 blockade has not been tested in these tumors . It is e


Differences in breast cancer survival by race and ethnicity are often assumed to be a fairly recent phenomenon, and are hypothesized to have arisen due to gaps in receipt of screening or therapy. The emergence of these differences in calendar time have implications for identification of their origin. We sought to determine whether breast cancer survival differences by race or ethnicity arose in tandem with the advent of screening or therapeutic advances.A cohort of women diagnosed with invasive breast cancer from 1975–2009 in 18 population-based registries were followed for five-year breast cancer cause-specific survival. Differences in survival according to race/ethnicity and estrogen receptor status were quantified in Cox proportional hazards models, with estimation of hazard ratios (HR), 95% confidence intervals (CI), and absolute risk differences. For 2010, we also assessed differences in survival by breast cancer subtypes defined by hormone receptor and Her2/neu status.Among over 930,000 women, initial differences in five-year breast cancer-specific survival by race became apparent among 1975–1979 diagnoses and continued to be evident, with stronger disparities apparent in those of Black vs. White Non-Hispanic (WNH) race and among estrogen-receptor positive vs. negative disease. Within breast cancer subtype, all included race/ethnic groups experienced disparate survival in comparison with WNH women for triple-negative disease. Black women had a consistent gap in absolute survival of .10-.12, compared with WNH women, from 1975–1979 through all included time periods, such that 5- year survival of Black women diagnosed in 2005–09 lagged more than 20 years behind that of WNH women.Survival differed by race for diagnoses that predate the introduction of mammographic screening and most therapeutic advances. Absolute differences in survival by race and ethnicity have remained almost constant over 40 years of observation, suggesting early origins for some contributors. Breast cancer survival disparities by race and ethnicity have retrospectively been identified using cancer registry and mortality data, with differences in outcome generally believed to have started in the mid-to-late1980’s have altAs early data regarding survival outcomes are an untapped resource, we evaluated temporal patterns in breast cancer survival disparities, including by three race/ethnic groups, in Surveillance Epidemiology End Results (SEER) data beginning in 1975. We sought to identify temporal changes in survival differences, including in tumor marker subsets, that might yield fresh insights regarding their origin.We conducted a retrospective analysis of a cohort of incident invasive female breast cancer cases diagnosed in 18 population-based sites of the National Cancer Institute-funded Surveillance Epidemiology End Results (SEER) cancer registry system. EligiblSEER utilizes certified tumor registrars to collect information regarding patient demographics, diagnosis, and first course of treatment . Included in this analysis is information ascertained from the medical chart regarding age, sex, race, and Hispanic ethnicity, date of diagnosis, and tumor characteristics . Since 1990, status of the hormone receptors for estrogen and progesterone  has also been collected, and in 2010, the tumor marker Her2/neu was added. The three tumor markers allow tumors diagnosed in 2010 to be classified according to four breast cancer subtypes: including hormone receptor positive (HR+), Her2 negative (HER2-) , HR+ Her2 positive (Her2+) , HR- Her2+ (Her2- overexpressing), and HR- HER2- (Triple Negative). Hispanic ethnicity was determined by the North American Association of Central Cancer Registries (NAACR) Hispanic Identification Algorithm (NHIA) as calculated by SEER. Invasive breast cancer cases were followed for breast cancer-specific death by linkage to local and state vital statistics registries, as well as the National Death Index. Vital status was confirmed by matching to the files of the Centers for Medicare and Medicaid Services. Women were followed until either death, loss-to-follow-up, or December 31, 2014, the end of the study.Cox proportional hazards models were used to calculate five-year breast cancer-specific survival, with women dying of breast cancer-specific causes within that time period considered “events” and all others censored. Hazard ratios (HR) and 95% confidence intervals (CI) were estimated with an alpha level of .05. Each five-year diagnosis group  was followed for a full 60 months (1995–1999), while breast cancer subtype diagnoses in 2010 were followed for up to 60 months (median 51 mo) through end of 2014. All analyses were adjusted for age in ten-year age groups, and for SEER registry start date. Nine registries initiated data collection in 1973–75, four in 1992, and five in 2000. Interaction terms between registry group and race/ethnicity were fit by including the main effects and their product (race/ethnicity * registry group) to determine whether hazard ratios by race differed by registry group, thus suggesting that addition of data from new registries may have altered HRs. Utilizing adjusted 5-year specific survival proportions from Cox models, absolute differences in survival were estimated, and trends with time were quantified using linear regression. Differences in trend by race/ethnicity were evaluated using generalized linear models (GLM). Analyses of estrogen receptor (ER) or progesterone receptor (PR) marker status were restricted to those of known status who were diagnosed in 1990 or later, and breast cancer subtypes to 2010 diagnoses only. The proportional hazards assumption was validated using cumulative sums of Martingale residuals. All analyses were conducted using the SEER Research Data 1975–2014 released in April 2017, based on the November 2016 submission[Overall, 930,910 women diagnosed with invasive breast cancer during 1975–2010 were included in the analysis , and of Black women had an increased risk of breast cancer specific mortality, relative to White Non-Hispanic white women, beginning in the earliest included time period (1.6-fold) . Black wHispanic women had a significantly elevated risk of breast cancer-specific mortality beginning with diagnoses in 1990–1994 (1.2-fold), relative to White Non-Hispanic women, and risk remained elevated through 2005–2009 diagnoses . While tFor American Indian/Alaskan Native women, breast cancer-specific mortality was elevated 1.4-fold compared to White Non-Hispanic women for diagnoses in 1980–1984, and again for 1990–1994 diagnoses, then American Indian/Alaskan Native women continued to have an elevated hazard ratio through the end of follow up in 2014 (2005–2009 diagnoses) . SimilarFive-year breast cancer-specific absolute survival estimates increased with time for all race/ethnic groups . NotablyCollection of estrogen receptor (ER) information began with 1990 diagnoses . Among EAmong women with ER-negative tumors, breast cancer-specific survival was also lower among Hispanic, Black, and American Indian/Alaskan Natives in all time periods compared to White Non-Hispanics . Breast The proportional hazards assumption did not hold for a few diagnosis years or race-ethnic subgroup analyses overall or in ER-positive or ER-negative analyses. Specifically, violation of the assumption only occurred in 1990 or later diagnoses, when the initiation of data collection in new SEER registries caused some subgroups to double in size, thus facilitating the detection of seemingly small quantitative differences. Survival results are split into 1–24 and 25–60 months post-diagnosis for comparison  and race/ethnicity was fit. For 1995–1999 and later years, survival was significantly greater than expected on a multiplicative scale for American Indian/Alaskan Natives in registries that initiated data collection in 1992, suggesting that addition of new SEER registries may have increased survival estimates for American Indian/Alaska Native women (data not shown).Five-year survival by race/ethnicity increased consistently throughout the observation period for White Non-Hispanic women  , Hispanic women , and American Indian/Alaska Native women . Slope did not differ by race-ethnic group . Over the entire period of observation, 5-year survival for White Non-Hispanic and Hispanic women each equally increased by .13 (from .74 to .87 for Hispanic women for instance), while American Indian/Alaska Native women gained .14 (from .72 to .86) and Black women gained .15 (from .65 to .80). ER-specific slopes were not estimated because statistically stable estimates required more than four time points.Women diagnosed in 2010 and followed through the end of 2014 offered an opportunity to evaluate five-year breast cancer-specific survival by breast subtype . Black aOur findings provide a broad perspective on the development of breast cancer survival disparities by race/ethnicity. Our results suggest that gaps in survival by race are present even in the earliest available SEER data (1975–1979), predating breast cancer screening and innovations in adjuvant therapeutics. Thus, screening and therapeutic advances alone are unlikely to entirely account for survival differences by race/ethnicity. Some results are novel, including five-year breast cancer-specific absolute survival estimates by race/ethnicity, linear trends for 1975–2009 diagnoses, recognition of a constant absolute survival difference of 10–12 points between Black and White Non-Hispanic women across the observation period, manifested as a 22 year lag in Black women’s survival attainment, hazard ratio estimates for diagnosis years prior to 1980, and statistically significant differences in breast cancer mortality among Black vs White Non-Hispanic women in Her2-overexpressing tumor subtypes, and that for all race/ethnic subgroups vs. White Non-Hispanic in triple-negative disease. Breast cancer-specific survival estimates by diagnosis year and tumor marker for American Indian/Alaskan Natives have not previously been reported, to our knowledge. Survival trends by race/ethnicity illustrate that absolute increases over the 1975–2014 period were similar for each group of women, but the baseline survival (1975–1979) differed substantially, suggesting the nature of the gap that needs to be closed.Our findings should be evaluated with consideration of the strengths and limitations of the study. SEER registry staff collect race and ethnicity as indicated in medical records, and do not have independent means to verify recorded information. Similarly, while tumor marker classification may be derived from pathology reports, some may be abstracted from medical charts. The definition of ER and PR positivity changed from 10% to 1% positive staining in 2010 , and retWhen examined across 5-year diagnosis periods, breast cancer survival estimates by race/ethnicity present several contrasting observations. Since 1990–1994 diagnoses, gaps in survival on the hazard ratio scale have become evident for Hispanic and American Indian/Alaskan Natives. One potential explanation is increasingly accurate reporting of race and ethnicity with time. Access to advances in healthcare may also be implicated. However, in 1992, additional SEER registries initiated data collection, leading to over a doubling in numbers of breast cancer cases in those race/ethnic groups compared to 1985–89 . While oAlthough the relative hazard estimates suggest an increase over time in survival disparities for Black women, an absolute risk difference of 10 to 12 percent in comparison with White Non-Hispanic women has been fairly constant for each 5-year observation period. Hazard ratio increases are apparent because the 12-point gap is measured against a White Non-Hispanic (reference group) approaching over 90% 5-year survival with time. While newly arisen factors may have contributed to the 12-point gap to a varying extent over the observation period, rather than a persistent contribution from baseline factors present in 1975–1979, the study data cannot distinguish between these hypotheses. The overall improvements in breast cancer-specific mortality, coupled with a lag, possibly of over 20 years in length, in attaining equivalent absolute survival for some presents a distinct challenge to policymakers, health care providers, and affected women.ER positive disease currently allows a wider range of treatment options and confers a more favorable prognosis. Thus, the findings raise questions regarding the source of the higher HR for race/ethnic disparities evident in less aggressive disease. Our results are consistent with those in other investigations ,8–11 incBreast cancer subtype-specific survival information should further inform efforts to close survival gaps. Her2-overexpressing disease, not previously identified as subject to differential outcomes by race/ethnicity, may warrant attention directed to appropriate therapy receipt and adherence as in other subtypes. In several studies, race/ethnic women have had poorer outcomes despite either receipt of guideline-based treatments , or stanS1 Fig(TIF)Click here for additional data file.


Additionally, aspirates from 31 euthanised dogs were collected for standardising the procedure. Each aspirate was separated into two subsamples: for cytology and RNA extraction. Additional prostate tissue samples served as control for RNA quantity and quality evaluation, and differential expression analysis. The US-FNA sampling procedure was feasible in 95% of dogs. RNA isolation of US-FNA samples was successfully performed using phenol-chloroform extraction. The extracted RNA of 56% of a subset of US-FNA samples met the quality requirements for RNA-Seq. Expression analysis revealed that only 153 genes were exclusively differentially expressed between non-malignant US-FNAs and tissues. Moreover, only 36 differentially expressed genes were associated with the US-FNA sampling technique and unrelated to the diagnosis. Furthermore, the gene expression profiles clearly distinguished between non-malignant and malignant samples. This proves US-FNA to be useful for molecular profiling.Ultrasound-guided fine-needle aspiration (US-FNA) biopsy is a widely used minimally invasive sampling procedure for cytological diagnosis. This study investigates the feasibility of using US-FNA samples for both cytological diagnosis and whole transcriptome RNA-sequencing analysis (RNA-Seq), with the ultimate aim of improving canine prostate cancer management. The feasibility of the US-FNA procedure was evaluated  However, there is still a lack of information on the molecular mechanisms underlying canine prostate diseases, which is especially relevant to the early diagnosis and management of canine prostatic tumours4. Dogs with prostate cancer (PCa) are often diagnosed at an advanced stage, which is associated with poor prognosis and limited therapeutic options5. Although the reported prevalence of prostate malignancies is low (0.2–0.6%)7, the incidence is likely to be underestimated due to the absence of reliable biomarkers and the lack of biopsies on asymptomatic dogs4. In addition, aside from humans, dogs are the only non-human species to naturally develop PCa sharing many clinical properties8 and will often have metastases10, similar to men11. This highlights the importance of characterising the transcriptomic landscape of canine PCa. Next-generation sequencing (NGS) of RNA (RNA-Seq) has enabled comprehensive insights into cancer-relevant mechanisms13 and is a well-established method for differentiating between neoplastic diseases14. Compared to conventional “gene by gene” approaches, RNA-Seq has several advantages: it is fast, cost-efficient and provides simultaneous information on several thousands of genes13. There is little doubt that RNA-Seq has the potential of becoming a routine diagnostic tool – at least in human medicine15. And even if the cost and complexity of the analysis may prevent this in veterinary practice in the immediate future17, RNA-Seq data already serve as a solid basis for fostering new diagnostic and therapeutic approaches16. Indeed, RNA-Seq has already been utilised in dogs to address clinical issues in different organs22. And while no genome-wide transcriptome analysis have been conducted for canine PCa, there is a growing body of information on the transcriptomic landscape of human PCa26. NGS allows the analysis of the transcriptome from minimal amounts of sampling material27. This is of special interest in clinical routine diagnostics, where representative sample material from tumours or organs must be obtained intra vitam. Meeting the requirements of a minimal invasive sampling procedure2, aspirates collected by ultrasound-guided fine-needle aspiration (US-FNA) biopsy are regularly employed in the cytological diagnosis of canine prostate diseases28 and other diagnostic approaches, such as flow cytometric phenotyping29. In this context, ultrasonographic parameters such as prostate size and differences in echogenicity within the parenchyma are used to identify lesions3 and guide the fine-needle to aspirate cells from representative areas30. The cells targeted and collected by the US-FNA procedure normally undergo cytological diagnosis28, but are also viable for molecular profiling32.Numerous diagnostic approaches for canine prostate diseases have been developed in the last few decades, substantially improving the detection of benign conditionsThe aim of the present study was to determine the suitability of aspirates derived from canine prostate US-FNA for molecular diagnostic. In pursuing this aim, the following questions were investigated: (a) the technical feasibility of the canine prostate US-FNA procedure as a sampling tool for cytological diagnosis and laboratory workflow; (b) the quantity and quality of the RNA isolated from US-FNA subsamples; (c) the impact of methodological contamination  in US-FNA aspirates on the transcriptome analysis; and (d) the similarities and differences in the molecular profiles of malignant and non-malignant US-FNA samples compared to those of prostate tissue samples.post mortem FNA (PM-FNA) training set  and histopathologically diagnosed as normal to hyperplastic , inflammation , prostatic atrophy , prostate carcinoma  or metastasis  from a transitional cell carcinoma of the urinary bladder or lymphoma  but failed due to the presence of clotted blood. Semi-quantitative macroscopic evaluation of the remaining 40 US-FNA samples  revealed that 65% showed moderate to strong blood contamination, 28% had weak to nearly invisible blood additions and 8% exhibited no signs of blood contamination. For this reason, RNA isolation from these samples was performed using the phenol-chloroform extraction method, which performed well in all but one US-FNA sample, resulting in a mean of 70.8 ng/µl of total RNA , RNA isolation from tissue resulted in a mean of 789.0 ng/µl of total RNA , which was significantly higher (p < 0.05) than those obtained for the US-FNA samples and PM-FNA training set .Based on clinical data, histopathological and cytological diagnosis see Fig. , 16 US-FThe RNA integrity numbers (RIN) were more consistent for tissue samples  performed on the expression values of the DEGs see Fig.  indicateMYC), catenin beta 1 (CTNNB1), prostate transmembrane protein, androgen induced 1 (PMEPA1), proliferating cell nuclear antigen (PCNA) and marker of proliferation Ki-67 (MKI67). The remaining 450 (42%) down-regulated genes included kallikrein-related peptidase 2 (KLK2), kallikrein-related peptidase 4 (KLK4), NKX3 homeobox 1 (NKX3-1) and acid phosphatase prostate (ACPP). These have been reported as genes with potential diagnostic value in the literature36 and their consistent expression in US-FNA and tissue samples supports the use of US-FNA samples for transcriptomic profiling. In addition, only 36 genes (1% of the DEGs) were associated with both non-malignant and malignant aspirates of US-FNAs  and 2 (HBA2), ENSCAFG00000029518 and ENSCAFG00000030286 as an orthologue of haemoglobin subunit delta (HBD) and haemoglobin beta (HBB) and ENSCAFG00000029224 as haemoglobin subunit mu. Therefore, molecular profiling of sampling-associated differences appears to reflect blood contamination in US-FNA samples.Moreover, hierarchical clustering of the fold-changes of the expression values of the DEGs relative to the control group confirmed this result, revealing a largely common molecular profile between malignant US-FNA and malignant tissue samples see Fig. . Specifi4. Having the opportunity to further use samples collected for routine diagnostic for molecular profiling is paramount to improve our molecular understanding of diseases16. An ideal biopsy procedure should be minimally invasive and allow rapid sampling and comprehensive clinical diagnostics, including molecular and cellular marker analysis27. In general, the FNA procedure has been shown to meet these requirements37. In particular, the present study confirms that FNA samples are a reliable sampling tool for both cytological and molecular diagnosis.Canine prostate diseases are currently unsatisfactorily characterised at molecular level, hampering early detection strategies38. In order to address the aggressiveness of human PCa using the Gleason Score, the FNA procedure has been largely replaced in routine diagnostic by multiple core needle biopsies39. Nevertheless, aspirates collected by FNA are acknowledged as samples with molecular diagnostic potential43. In dogs, the collection of canine prostate samples is challenging due to the anatomical localisation of the prostate gland44. Although histopathological biopsy is described as the gold standard for diagnosis of canine prostate diseases44 and a histopathological standard terminology is available45, the collection of tissue samples has several disadvantages, such as the need for general anaesthesia and the relatively high complication rates46. A practicable minimally invasive sampling procedure would be preferable both veterinary research and, especially, in clinical routine16. Based on the current state of knowledge, this is the first study to evaluate the feasibility of using samples of the canine prostate obtained intra vitam by US-FNA with 22-gauge needles for both cytological diagnosis and RNA-Seq.In human oncological medicine, different types of biopsies – including FNA – have been comparatively assessed as samples for NGS applications aimed at optimising the management of patients based on their molecular profiles28, the US-FNA procedure was well tolerated by 95% of the dogs in the study without the need of general anaesthesia. Complications associated with the US-FNA procedure were transient and mild and in concordance with previous studies47. The US-FNA procedure proved more challenging in neutered healthy dogs due to the dense and firm prostate tissue structure48 caused by androgen deprivation49 and resulted in generally low quantities of RNA. Nonetheless, the US-FNA procedure yielded adequate quantities of RNA in the case of neutered dogs with PCa, which typically exhibit disease-related changes of the prostate. This is particularly important since neutered dogs have an increased risk to develop canine PCa50. These findings indicate that the US-FNA procedure is feasible in diagnostic routine.In agreement with the literature51. In the present study, 88% of the cytological smears exhibited sufficient cellularity for cytological diagnosis. This was similar to the fraction of the subsamples that met the requirements for RNA-Seq: 93% of US-FNA samples. In concordance with these results, previous studies described the remaining aspirates as a viable source for advanced diagnostic procedures after the preparation of cytological smears53. In general, the results of this study support the idea of using subsamples to bridge the gap between clinical and molecular diagnosis.In this study, US-FNA samples were split into two subsamples to address conventional and molecular diagnostic questions. An adequate cellularity is crucial for cytology and RNA extraction. The strategy of splitting the aspirates into subsamples generally decreases the cellularity. Varying cellularity can be additionally explained by the non-standardisable conditions during collection and subsampling in the clinical routine. The cellularity of aspirates has been previously analysed in different tumour samples and shown to be affected by the application of suction, the number of needle passes and cell exfoliation54. In fact, in a previous study, the researchers opted for not using highly blood contaminated canine FNAs of different tumour entities for RNA extraction51. In the present study, 65% of US-FNA samples had moderate to strong blood contaminations, probably related to the aspiration technique51 and the use of intravenous needles55. Processing and pipetting of contaminated US-FNA samples was challenging and might have led to a decrease in the number of cells. However, US-FNA samples were successfully used for RNA purification by phenol-chloroform extraction. Based on the results in this study, the use of column-based isolation should be avoided without any further lysis steps or reagents. Interestingly, and consistent with the macroscopic findings, five globin genes, which can be associated with erythroid cells56, were more highly expressed in US-FNA samples compared with tissue samples. Therefore, blood contamination was detectable at molecular level, thus indicating that RNA-Seq enables intra vitam sampling associated effects to be identified. Additionally, there was no evidence that 0.1% of DEGs identified as globin genes in anyway impaired the actual diagnostic for gene expression profiling.Cytological findings on the sample quality such as necrosis and blood contamination appear to provide important information regarding the viability of molecular analysis. Large amounts of blood are known to influence the quality of cytological smears, hampering an adequate cytological diagnosis57. Of US-FNA samples, 56% met the requirements of RNA-Seq. It has been shown before that RIN values of FNA samples are not always detectable51. For precise assessment of RNA quality the Agilent RNA 600 Nano assay requires a minimum RNA concentration of 25 ng/µl58. Of the US-FNA samples, 31% (n = 5) selected for quality control were below this threshold, which could explain some of the poor RIN values. Furthermore, the RIN value and the corresponding electropherogram needs to be evaluated. In addition, the nucleases and factors associated with the FNA procedure itself, such as the needle length, can cause RNA degradation59. Moreover, this study evaluated the RNA integrity in non-malignant and malignant canine prostate tissue samples with no evidence of a difference. Similar to the results of the present study, one recent study on canine tumour FNA found no significant difference analysing the integrity of RNA from readily exfoliative tumour and non-readily exfoliative tumour FNA. However, non-malignant samples were not part of the above mentioned study51.In general, RNA-Seq demands high-quality total RNA60. To evaluate the molecular diagnostic potential of canine prostate US-FNA subsamples for further gene expression studies, seven US-FNA samples were subjected to RNA-Seq and their whole transcriptome profiles were compared to those of 18 prostate tissue samples. The main advantage in using NGS is the possibility to comprehensively characterise the transcriptome, allowing rapid profiling of neoplastic diseases for downstream diagnostics of therapeutic intervention61. The findings of the present study indicate that the sampling procedure has a minor impact on the sample’s gene expression profile, since only 36 (1%) of 3,587 DEGs were specifically identified in all US-FNA samples relative to the non-malignant prostate tissue control. Differences on the gene expression level between US-FNA and tissue samples can be explained based on the variability of the individual samples and the diagnosis. In particular, tumour heterogeneity has been described in different cancer types62, including canine PCa63. To minimise these effects and ensure representative material for RNA-Seq analysis as well as adequate diagnosis, the samples were collected under ultrasound guidance and characterised by cytological examination. The molecular profiling of aspirates collected by FNA samples in dogs has been previously performed and compared to matched tissue samples for lymph nodes using microarray technology31. Nevertheless, that study differs from the present one in that FNA samples were i) collected only from euthanised dogs (post mortem), and ii) not separated into two subsamples for cytological and molecular analysis. Overall, the authors described FNA samples as a reliable source for molecular profiling31, which agrees with the results presented here. In this context, US-FNA transcriptomics becomes increasingly attractive as a diagnostic tool. Indeed, malignant and non-malignant US-FNA samples were perfectly separable based on their molecular profiles, matching the cytological diagnosis. In addition, genes that are well known to be deregulated in cancer such as MYC, MKI67, ACPP, CTNNB1, KLK2, KLK4, PCNA, PMEPA1, NKX3-168 were also significantly deregulated (and in the same direction) in US-FNA samples, confirming the similarity of US-FNA samples and their prostate tissue counterparts. These genes and other genes with similar molecular profiles are interesting from a diagnostic point of view. In particular, genes like MYC, MKI67, CTNNB1, PCNA, NKX3.1, ACPP and kallikreins are warranted further investigation as diagnostic markers for cancer and cell proliferation. The combination of US-FNA and RNA-Seq opens up a wide array of possibilities in clinical diagnostics.One previous investigation on human PCa emphasises the importance of biopsy based gene-expression studies and successfully used prostatectomy samples for molecular profiling and additional needle core biopsies for identification of candidate genesIn conclusion, the data presented here underline the suitability of the US-FNA procedure as a sampling tool for cytological and molecular diagnostic purposes. To optimise the laboratory workflow, details on cytological examination may be helpful for identifying and processing adequate samples prior to sequencing. This study highlights the strengths of US-FNA as a sampling tool for molecular profiling of the canine PCa. Validation on a larger number of US-FNA samples is warranted to confirm these conclusions. Beyond the use of US-FNA for whole transcriptome analysis, this study provides a solid basis for approaches such as targeted sequencing or qPCR screening of single genes, opening new perspectives for biomarker discovery for early detection and follow-up diagnosis and, eventually, for developing and establishing individualised therapeutic strategies.Intra vitam aspirated cells, collected by US-FNA were referred to as US-FNA, while aspirates collected by FNA after prostatectomy from euthanised dogs were referred to as PM-FNA training set  after the PM-FNA procedure from euthanised dogs  attached to a 5 mL Luer syringe . The needle was inserted into prostate tissue and moved several times while aspirating. A fraction of the aspirated cells were used to prepare a smear for cytological examination; the remaining cells were expelled to a 1.8 mL Nunc CryoTube . The needle was then rinsed with 50 to 100 µl sterile phosphate-buffered saline (PBS) for collecting the remaining aspirate in the CryoTube. Matched prostate tissue samples were collected from macroscopically representative areas for molecular biological procedures and histopathological classification.A training set of 31 PM-FNA samples was collected from euthanised dogs during necropsy after prostatectomy  was isolated in accordance with the manufacturer’s protocol of Qiagen AllPrep™DNA/RNA Micro Kit . For lysis and homogenisation, Buffer RLT Plus and QIAshredder spin column  were used and purified RNA was eluted RNAse-free water.intra vitam collected US-FNA samples (n = 42), the method of RNA isolation was adjusted to phenol-chloroform extraction (n = 40), since column-based isolation of total RNA from two US-FNA samples failed due to clotted blood. A semi-quantitative scoring was used prior to RNA isolation to assess blood contamination in 40 US-FNA samples macroscopically: Score 0 was used for samples without macroscopically visible contaminations of blood; score 1 comprised samples with almost invisible contamination of blood; score 2 included light red coloured samples for weak but clearly visible blood contamination; score 3 included red coloured samples, describing a moderate contamination and score 4 deep red samples indicating strong blood contamination.For Phenol-chloroform extraction of RNA from remaining aspirates collected by US-FNA (n = 40) was performed using QIAzol Lysis Reagent with Qiagen miRNeasy Micro Kit  eluted RNase-free water. RNA quantity of US-FNA and PM-FNA samples was determined using the Qubit® RNA HS Assay Kit  with Qubit 2.0 Fluorometer.TM Multi-Volume Plate by Synergy™II multi-mode reader, evaluated by Gen5™ Microplate Data Analysis Software .For isolating total RNA from 10-20 mg prostate tissue samples (n = 43), AllPrep® DNA/RNA/miRNA Universal Kit  was used in accordance with the manufacturer’s protocol. Tissue samples were disrupted utilising Buffer RLT Plus and 5 mm stainless steel beads by TissyeLyser II . Total RNA was eluted in 30 µl RNase free-water and RNA quantity was measured on a Take3US-FNA and prostate tissue samples for RNA-Seq were selected in accordance with multiple criteria. First, they were required to contain a minimum of 10 ng and a maximum of 1 µg of total RNA. To ensure their comparability, the samples were further selected based on their histopathological and cytological diagnosis see Fig. . The PM-The RNA quality was measured using the RNA 6000 Nano LabChip on an Agilent Bioanalyzer 2100 , based on the 18S to 28S ribosomal ratio provided as electropherogram. The RIN value was determined with the Agilent 2100 Expert software and ranged from 1 (most degraded RNA) to 10 (most intact RNA). For samples with low RIN values, details in the electropherogram were used for further evaluation of RNA degradation.Statistical analysis of RNA quantity and quality was performed using SAS Enterprise Guide 7.1 . The Shapiro-Wilk test was applied to confirm the normal distribution of the data. The influence of neutering and diagnosis on RNA quantity in prostate tissue samples was evaluated with the two-sample t-test. Wilcoxon’s signed-rank test was used to compare the differences between US-FNA aspirates and the PM-FNA training set. A two-sample t-test was performed to analyse differences of RIN-values between non-malignant and malignant tissue samples. Significance level was defined as p < 0.05.72.Samples with a RIN value ≥ 5.5  were then subjected to NGS library preparation  with the following parameters: LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36. Trimmed reads were then mapped to the CanFam3.1 assembly of the dog genome using TopHat275 with default parameters and supplying the Ensembl GTF annotation (Ensembl release 8576) through the -G option. This resulted in an average of ~14 million pairs of mapped reads per sample.Raw sequencing reads were quality checked using FastQC77 with default parameters. The sample groups: non-malignant US-FNA, malignant prostate tissue and malignant US-FNA were each independently tested against non-malignant prostate tissue (control) for differential expression with the R/Bioconductor package DESeq278. p-values were adjusted for multiple testing using the false discovery rate (FDR). Genes with an FDR < 0.00001 in any of the three comparisons were considered as DEGs.Read counts for each protein-coding gene were calculated for each sample using the htseq-count tool of the Python package HTSeq76 not associated with any Official Gene Symbol were aligned for identifying sequence homologies based on Ensembl IDs using the online BLAST  interface provided by NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi 80).Nucleotide sequences of Ensembl gene IDsDEGs were used to perform a PCA to summarise and visualise variances across all samples. Specifically, PCA was performed on library size-normalised regularised log-transformed expression values, which were computed using the rlogTransformation function in the R/Bioconductor DESeq2 package.Fold-changes relative to non-malignant prostate tissue of DEGs were hierarchically clustered using complete linkage and Euclidean distance (genes) or a Pearson correlation-based distance measure (samples). On basis of fold-changes, a list of well-studied genes was used for further profiling of US-FNA in comparison with prostate tissue samples.82), using the 19,856 Ensembl IDs corresponding to all protein-coding genes annotated in the dog genome (Ensembl release 8576) as background and a FDR ≤ 5% cut-off for significance.DEGs identified exclusively in US-FNA samples were analysed on biological pathway. This pathway analysis was performed with DAVID (Database for Annotation, Visualization and Integrated Discovery Functional Annotation Tool


The curative effects of nanoparticle albumin-bound (nab)-paclitaxel in the first-line treatment of metastatic breast cancer (MBC) are still controversial, with even more after the removal of marketing approval of indication of bevacizumab. Five electronic databases and the related resources were searched for eligible randomized clinical trials (RCTs) without year and language restrictions to perform a meta-analysis. The studies were comparing the efficacy and safety between nab-paclitaxel chemotherapy versus solvent-based (sb)-taxanes chemotherapy such as sb-paclitaxel and docetaxel. The primary end points were overall response rate (ORR) and disease control rate (DCR). Secondary end points were progression-free survival (PFS), overall survival (OS), adverse events (AEs), and dose discontinuation rate (DDR). Five RCTs  were finally identified from 1,902 studies. When compared to sb-paclitaxel, nab-paclitaxel showed significant beneficial effects in terms of ORR , DCR , and PFS . Nab-paclitaxel also showed significantly longer OS  than docetaxel. AEs and DDR were comparable between the two arms. Using nab-paclitaxel could significantly improve efficacy with comparable toxicities in the treatment of MBC. Although nab-paclitaxel was initially developed to minimize the toxic effects of taxane treatment, several early clinical trials demonstrated that nab-paclitaxel was also more effective than the conventional solvent-based (sb) paclitaxel in the treatment of metastatic breast cancer5.Nanoparticle albumin-bound (nab) paclitaxel is solvent free and employs a novel delivery mechanism for paclitaxel to tumors6. For example, Rugo et al. showed that nab-paclitaxel was not superior to sb-paclitaxel . Results were concordant with overall survival (OS), and time to treatment failure was significantly shorter in the nab-paclitaxel arm vs the sb-paclitaxel arm6. Hematologic and non-hematologic toxicity, including peripheral neuropathy, was more prevalent in the nab-paclitaxel arm, which also had more frequent and earlier dose reductions6.However, recent randomized controlled trials (RCTs) have suggested that nab-paclitaxel chemotherapy is not as efficacious as sb-taxanes, such as sb-paclitaxel and docetaxel, and that nab-paclitaxel is often associated with more frequent adverse eventset al. conducted a first meta-analysis of randomized clinical trials to evaluate the efficacy and toxicity of nab-paclitaxel compared with sb-paclitaxel and docetaxel in the treatment of metastatic breast cancer (MBC), and they concluded that nab-paclitaxel was associated with more frequent sensory neuropathy, but only equivalent survival and possibly higher overall response for only some specific subgroups7. They also cited that nab-paclitaxel chemotherapy was more expensive than conventional sb-taxane chemotherapy7. The findings of Liu et al. seemed to be affected largely by the phase III trial of Rugo et al. In this trial, they administered bevacizumab , along with nab-paclitaxel  or sb-paclitaxel (for the control arm) to chemotherapy-naive MBC patients.After these conflicting results were generated, Liu 8. Therefore, it might be reasonable to assume that the combined bevacizumab regimen may obscure the taxane effect, meaning that the previous meta-analysis may not reveal the real efficacy and safety of nab-paclitaxel and sb-taxanes. To prevent information loss due to summarization, it is important to include all relevant and eligible randomized trials in a meta-analysis9. However, we believe that it is also reasonable to exclude data that are inappropriate.However, the US Food and Drug Administration (FDA) withdrew its approval of bevacizumab in combination with paclitaxel in the treatment of MBC in December 201010.In this updated meta-analysis, therefore, we conducted a subgroup analysis aimed to concentrate on the comparison of medications without bevacizumab which compared between nab-paclitaxel and sb-taxanes. With this approach, we can concentrate solely on the efficacy and safety of nab-paclitaxel compared with conventional sb-taxanes. We analyzed two more efficacy variables, disease control rate (DCR) and PFS, with an additional recent randomized trial10 for this meta-analysis . Our meta-analysis, therefore, effectively included 5 trials containing 1,947 patients, out of which the data of 1,554 patients were eligible for inclusion in this meta-analyses, 774 patients had been randomized to nab-paclitaxel arm and 780 patients to sb-taxanes arm .After screening 1,902 studies chosen from electronic databases and manual searches, we finally included six eligible randomized trialsThe characteristics of the included trials are shown in Table 6 were conducted with chemotherapy-naive patients. The other 3 trials10 included both chemotherapy-naive patients and pretreated patients. By reviewing original studies, we found that two trials excluded patients if they received adjuvant chemotherapy with taxanes (sb-paclitaxel or docetaxel) 12 months prior to study enrollment3, and another trial only included patients who had no history of chemotherapy after confirmation of metastasis10. We concluded that these 3 trials would not be any different from the trials with chemotherapy-naive patients.Two trials5. To avoid double counting of the placebo arm, we chose the nab-paclitaxel arm with 150 mg/m² (qw3/4) (n = 74) as the experimental arm for this analysis. This choice was driven by the fact that the original study indicated the dose was the most effective one compared with the other two nab-paclitaxel dosing regimens of 300 mg/m² (q3w) and 100 mg/m² (qw3/4). Therefore, we thought this nab-paclitaxel dose will more likely be administered in clinical treatments. Another trial had an ixabepilone arm6, but we only selected nab-paclitaxel and sb-paclitaxel arms for our meta-analysis.One trial had three investigational arms and one control arm6, while all the other four trials included single taxane-only therapy10. In three trials, the sb-paclitaxel arm was the control group6, and the docetaxel arm was the control group in the other two trials10. Three trials were performed on Western regions6, and the other two trials were conducted on Eastern regions10.One trial investigated combined therapy with bevacizumab2. Considering the fact that TTP and PFS scales are generally similar to one another11, we used TTP data as the best PFS surrogate for this meta-analysis. TTP-weeks were converted to PFS-months using the multiplier 7/30 . Another study presented PFS with 90% CI instead of 95% CI10. We converted the 90% CI into 95% CI using methods described by Tierney12. Efficacy data, grade ≥ 3 adverse events, and dose discontinuation data for both arms from the five trials are presented in Table One trial reported time to progression (TTP)-weeks instead of PFS-monthsAll 5 trials were included in the overall meta-analysis. Compared with sb-taxanes, nab-paclitaxel conferred no significant benefit on ORR and DCR (primary outcomes), PFS and OS (secondary outcomes). . These results are shown in the end part of Fig. s , while other AEs, such as neutropenia, leukopenia, fatigue, and DDR were all comparable between the two arms .  %) Figs. . DCR als%) Figs. . However%) Figs. . The com%) Figs. 2 = 90%; sensory neuropathy, OR 2.90, 95% CI 1.45–5.79, p = 0.003, I2 = 48%). Leukopenia, fatigue, and DDR were comparable between the two arms , we did further subgroup analysis to see whether nab-paclitaxel have superiority to both of the control drugs. When compared to sb-paclitaxel, nab-paclitaxel demonstrated significantly better efficacy in terms of ORR, DCR, and PFS . Nab-paclitaxel also showed superiority to docetaxel on DCR and OS . , but fatigue was significantly more frequent in the nab-paclitaxel arm . When compared to docetaxel, neutropenia and leukopenia were significantly less frequent , but sensory neuropathy was significantly more frequent  in the nab-paclitaxel arm.  compared to sb-taxanes, but not in Eastern region. Nab-paclitaxel showed significantly better results on DCR regardless of the region  than sb-taxanes.  than sb-taxanes in the Western region, but not in Eastern region with all grade ≥ 3 AEs.We conducted a meta-analysis with originally identified 5 RCTs to see whether nab-paclitaxel had a certain benefit over conventional sb-taxanes in terms of efficacy and toxicities in the first-line treatment of MBC. However, the overall analysis results showed no efficacy benefit with worse toxicities of nab-paclitaxel. So, we further conducted subgroup analyses with 4 single taxane RCTs. Through the exclusion of bevacizumab combined therapy, we could find the evidence that nab-paclitaxel was superior to sb-taxanes in terms of ORR and DCR. Concerning grade ≥ 3 AEs, the two study drugs were comparable, since neutropenia was more frequent in the sb-taxane arms, while sensory neuropathy was more frequent in the nab-paclitaxel arms. DDR was also comparable between the two study drugs.Through the further subgroup analyses within the single taxane subgroup, we could find the evidence that nab-paclitaxel was superior to sb-paclitaxel in terms of ORR, DCR, and PFS. Nab-paclitaxel was also superior to docetaxel on OS. However, the subgroup analyses by study region revealed that these affirmative effects of nab-paclitaxel proved to be justifiable only in the Western region.8, in the middle of the enrollment period for the Rugo et al. study (Oct. 2008-Nov. 2011)6, because of the failure to provide evidence of an increase in PFS or OS when nab-paclitaxel or sb-paclitaxel was administered combined with bevacizumab. There were concerns about an overall increase in serious AEs related to bevacizumab as well8. Following the withdrawal of FDA approval for bevacizumab indication for MBC patients, although Rugo et al. made a protocol amendment to make bevacizumab use optional, 97% of patients still received bevacizumab6. From this trial, Rugo et al. reported no significant difference in the effect on PFS (primary end point) between the nab-paclitaxel plus bevacizumab arm and the sb-paclitaxel plus bevacizumab arm. They also reported high rates of neurotoxicity (27% of patients had grade ≥ 3 sensory neuropathy), dose reductions (45% by cycle 3), and dose discontinuations (>40% by cycle 5) for the nab-paclitaxel plus bevacizumab arm6, suggesting that the combination of bevacizumab with nab-paclitaxel 150 mg/m2(qw3/4) was not ideal. Furthermore, the high rate of grade ≥ 3 hematologic adverse events for the nab-paclitaxel plus bevacizumab arm (51%) and the withdrawal of FDA approval of bevacizumab as a first-line treatment for MBC suggests that this regimen will not be pursued in subsequent trials13.The subgroup analyses conducted in our study are meaningful because the US FDA withdrew its accelerated approval of bevacizumab indication as a first-line treatment for MBC in combination with paclitaxel use in December 2010et al. noted that various Phase II studies have been conducted evaluating nab-paclitaxel as part of combination therapy with cytotoxics  and biologics  in the treatment of MBC, and although the nab-paclitaxel dosing and schedules varied among these studies, in all cases, the nab-paclitaxel combinations were highly active and well tolerated14.Even though it might be indirectly inferred from our analyses that nab-paclitaxel monotherapy may have superior efficacy over combined therapy with bevacizumab, this does not necessarily mean that nab-paclitaxel should always be administered by itself. Combination therapy with nab-paclitaxel to increase efficacy is an attractive option for some patients, particularly those with a high tumor burden or those who require rapid disease control. Minckwitz 13, the DCR data of a certain drug would be more helpful than the ORR data for a certain group of patients. Moreover, the Southwest Oncology Group reported that DCR might be a better predictor of survival than response rate alone among patients with non-small-cell lung cancer, based on the fact that, for any given treatment, many more patients typically achieve stable disease (SD) or have progressive disease (PD) than achieve a response. They also reported that, although ORR (CR + PR) at week 8 was associated with longer survival , DCR (CR + PR + SD) was better for predicting survival 15.In this analysis, we included DCR as one of the end points. We thought that DCR could be an important criterion influencing the choice of chemotherapy drug for MBC patients. ORR is the sum of complete response (CR) and partial response (PR), while DCR is the sum of additional stable disease (SD) to ORR. So, ORR may be an important index to the patient who hopes “to be getting better,” while DCR may be a critical index to the patient who hopes “not to be getting worse.” Since the 5-year survival of MBC patients is only around 25%For similar reasons, we also analyzed DDR due to unacceptable toxicity or serious AEs as an end point. Considering that 5-year survival is very low among MBC patients, it can be hypothesized that the formal declaration of dose discontinuation by the medical clinic would have a seriously bad impact on patient’s quality of life. DDR due to toxicity would be helpful during the management of MBC patients when choosing between nab-paclitaxel and sb-taxane therapy. In our meta-analysis, DDR was comparable between the two study drugs.In our regional subgroup analyses, nab-paclitaxel and sb-taxanes were almost comparable in efficacy and toxicities in the Eastern region (only DCR was significantly higher in the nab-paclitaxel arm in the Eastern region). More studies are needed to clarify the reasons of this regional difference, including the genetic characteristics of each regional region.10 and q3w3 of taxane schedule. We also were not able to coordinate the efficacy differences in dosage between docetaxel 100 mg/m2 5 and 75 mg/m2 10. It was because of the small number of eligible RCTs. For the right comparison of nab-paclitaxel with sb-taxane and for the right discrimination of a specific drug’s superiority or inferiority, it should be determined first what dose and what schedules are the best for each study drug, and then we need the actual curative effect data based on their “best” dose and schedules. However, the search of the optimum dose and schedules of nab-paclitaxel and sb-taxanes is still ongoing13. Another limitation is that the trial of Rugo (CALGB) did show a PFS and an OS benefit in the TNBC subgroup, however, a subgroup analysis was not performed on the subset of TNBC patients separately. It was because, among 5 RCTs, only 2 studies presented data about the TNBC subset (Rugo 2015 and Tamura 2017). And the results on PFS were just comparable between the study drugs .OS data for the TNBC patients was presented only in one study (Tamura 2017), which was also comparable between the study drugs . Lastly, there existed very high heterogeneities among individual effect sizes, aside from the dose and schedule issue which makes it difficult to interpret the results of this meta-analysis confidently. This “heterogeneity problem” cannot be solved easily, since generally, the number of RCTs are so small in this field, as Higgins et al. previously pointed out16.Our meta-analysis has some limitations. We were not able to take into account the efficacy differences between weekly (qw3/4)Nevertheless, the high heterogeneity provided an opportunity to probe the consistency of the treatment effects across the single or combined therapy approaches used. Further, all the trials made statements of randomization and presented suitable data for this meta-analysis. Therefore, we deemed methodological quality of these studies not to be a source of heterogeneity.17.We searched PubMed, Cochrane Library, Ovid MEDLINE, Web of Science, and ClinicalKey, without year and language restrictions. We used (breast neoplasm(s) OR breast cancer) AND  AND (paclitaxel OR sb-paclitaxel) AND (docetaxel OR taxoids) as a searching algorithm. The last search was updated on May 14, 2018. Reference lists from the relevant full texts were checked to identify other potentially eligible studies. We also manually searched the references of review articles and abstracts from American Society of Clinical Oncology Annual Meetings. Trials were potentially eligible regardless of the study phase, dose schedule, and study region. We followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines with respect to our search strategy and selection processTwo reviewers independently screened the titles and abstracts of all studies identified in the literature search to verify compliance with the inclusion criteria. Disagreements between the two reviewers were resolved by consensus involving a third reviewer. The same reviewers performed data extraction and quality assessment of the included studies.9 summary of our study is shown in Table From each eligible trial, we extracted the following items: study and article characteristics , study region , study drug , efficacy outcomes, adverse event outcomes, and dose discontinuation rate (DDR) due to unacceptable toxicities or adverse events (AEs) for both arms. The PICOSThe primary end points were overall response rate (ORR) and DCR. Secondary end points were PFS, OS, AEs, and DDR.12 or NORMSINV function18.For binary outcomes , odds ratios (ORs) were calculated for each eligible study. For time-to-event outcomes (PFS and OS), hazard ratios (HRs) were calculated for each eligible study to estimate the relative risk among MBC patients receiving nab-paclitaxel vs sb-taxanes. When 95% CI data for PFS or OS were not immediately available from the original studies, we derived them using methods described by Tierney19 and quantified using I2 statistics16. In our study, when high heterogeneities were observed, we conducted subgroup analysis to explore the possible explanations for the observed heterogeneities. After the overall analysis was conducted comparing nab-paclitaxel with bevacizumab vs sb-taxanes with bevacizumab, subgroup analyses were conducted comparing nab-paclitaxel vs sb-taxane without bevacizumab in each group, which compared nab-paclitaxel vs sb-paclitaxel or nab-paclitaxel vs docetaxel. Another subgroup analysis were also conducted to compare nab-paclitaxel vs sb-taxanes based on study regions such as in western region and in eastern region each. All p-values were two-tailed. All the statistical analyses were performed using Revman software V.5.320. We assessed the methodological quality of the eligible trials using Cochrane’s Risk of Bias (RoB) tool21. The study protocol was exempted from the requirement for ethical approval by the Ewha Womans University Ethics Committee (IRB No: 163–2).We then synthesized the individual OR and HR data across studies using fixed effects (Mantel-Haenszel) or random effects (DerSimonia and Laird) model to obtain pooled effect sizes. Since there were many high heterogeneous variables in our analyses, we mainly adopted random effects model. The presence of statistical heterogeneity was first assessed using Cochran’s Q test (considered significant for p < 0.10)Nab-paclitaxel is an effective anti-tumor drug in the first-line treatment of MBC. Using nab-paclitaxel instead of sb-taxanes could significantly improve ORR and DCR with comparable toxicities and DDRs.


There are very few reports of eczema and other prosthetic-related allergic skin complications following arthroplasty. We aimed to assess the risk of eczema after joint replacement.We performed a retrospective population-based cohort study in 2024 joint replacement patients using the Longitudinal Health Insurance Database. For comparison, 8096 controls were selected, with 4 control subjects for each joint replacement patient matched for age, sex, and index year, to assess eczema risk. We examined 14-year cumulative eczema incidence associated with age, sex, immunity, disease history, and joint replacement location.Eczema rates in the joint replacement patients were 38% higher than in the control group . Compared with the control group, joint replacement patients showed a 1.35-fold increased risk of eczema according to the multivariable Cox model . Knee replacement patients had higher eczema risk compared with the control group . Stratified by study period, the joint replacement cohort had a higher eczema risk after the 3-month follow-up.Our study revealed that joint arthroplasty increased risk of eczema in this 14-year follow-up study, and this was not related to personal atopic history or gender. Among these reactions, eczema is the most-reported hypersensitivity reaction observed after arthroplasty and may be associated with nickel, chromium, or cobalt allergies, and non-metals, such as silicon and bone cement components.–15 However, the risk of eczema after joint replacement has only been reported in case reports, small patient cohorts, or meta-analyses.,15–26 Unfortunately, there has only been 1 study reporting on metal allergy in knee arthroplasty, which was conducted in a Danish population. This suggests a need for nationwide population-based studies with long-term follow-up periods to provide a comprehensive overview of the overall incidence and risk factors. Therefore, the aim of this study was to evaluate whether joint arthroplasty is associated with an increased risk of eczema and to identify the medical or demographic risk factors over a 14-year follow-up period.Artificial joint arthroplasty has excellent clinical results for the treatment of end-stage arthritis. Joint prosthesis composite materials are foreign to the body, and include plastic polymers, bone cement (methyl methacrylate), and metals, such as chromium, cobalt, and titanium alloys. Since the first reported case of allergy after arthroplasty in 1966,22.1This was a retrospective cohort study using the Longitudinal Health Insurance Database (LHID), which contains 1 million beneficiaries randomly selected from the Taiwan National Health Insurance Programme in 2000. This program is a compulsory insurance program and the LHID includes all de-identified data, including medial claims and treatments as well as both outpatient and inpatient visits, for each beneficiary from 1996 to 2013. The China Medical University and Hospital Institutional Review Board approved this study. To define diseases and treatments from the LHID, we used the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) and ICD-9-CM operation codes.2.2(1)Joint or revision replacement  history(2)History of eczema (3)Spine- or fracture-associated operation (ICD-9-CM operation code 79 and 81)(4)Cellulitis (ICD-9-CM 682.5-682.9)(5)Osteomyelitis (ICD-9-CM 730)(6)History of Pyogenic arthritis (ICD-9-CM 711)(7)History of end-stage renal disease (ICD-9-CM 585.6)Patients with joint replacements  following the Taiwan Ministry of Health and Welfare guidelines from 2000 to 2010 were selected (N = 8277). The date of joint replacement was defined as the index date. The exclusion criteria were as follows:The control group was comprised of patients without any joint replacements listed in the LHID. The exclusion criteria were identical for the control group. Approximately 4 controls for each joint replacement patient were randomly selected, according to age , sex, and index year. Figure 2.3All study subjects were followed from the index date until the development of eczema, as diagnosed by a dermatologist on 3 unique visits. Those who did not develop eczema were followed until the end date of 2013 or until they withdrew from the program, whichever came first. The baseline comorbidities considered in this study included asthma (ICD-9-CM 493), allergic conjunctivitis , allergic rhinitis (ICD-9-CM 477), and immunity disorders including lupus erythematosus (ICD-9-CM 710.0) and rheumatoid arthritis (ICD-9-CM 714.0).2.4The scientific committee of the China Medical University Hospital and the ethical committee in the China Medical University Hospital waived approval for the human protocol for this investigation and each author certifies that all investigations conformed with ethical principles of research. This work was performed at the China Medical University Hospital, Taichung, Taiwan.2.5t test was used to test differences in mean ages between the 2 cohorts. The results are shown as the mean and standard deviation. The rate per 1000 person-years was counted as the sum of eczema development divided by the sum of person-years during the study period. We used a Cox proportional hazard regression analysis to estimate the hazard ratio (HR) and compared the 95% confidence interval (CI) for eczema between joint replacement and control cohorts. A multivariable Cox model was adjusted for age, sex, and comorbidity. For sensitivity analysis, the age-, sex-, and comorbidity-stratified analyses were assessed. The association between developing eczema and the location of the joint replacement was estimated. We also estimated the combined effect for eczema between joint replacement and comorbidity. Because this study violated the Cox proportional hazard assumption via a scaled Schoenfeld residuals test (P = .03), we analyzed the association between eczema and joint replacement stratified by follow-up time. To plot the cumulative incidence in the 2 cohorts, we used a Kaplan–Meier analysis and a log-rank test to test the difference between the 2 cohorts. All the analyses were performed with SAS 9.4 software  and all statistical tests were two-sided. A P value of <.05 was considered statistically significant. We used SPSS V18 software  to plot the cumulative incidence.A Chi-Squared test was used to determine differences among age , sex, and comorbidities between joint replacement and control cohorts. The variables are presented as number of cases and as a percentage of the total sample. Student 3In total, 2024 joint replacement patients and 8096 control patients were selected for this study. There were no significant differences in age or sex between the joint replacement group and the age- and sex-matched controls. The mean age was 66.4 ± 12.1, with more women (63.6%) than men (36.4%) in the joint replacement cohort (Table P < .001) .Eczema can occur following implantation in orthopedic prosthesis and has been associated with nickel, chromium, or cobalt composites in the implants.,31–33 A major difficulty in understanding skin conditions following implant is the lack of a universally feasible testing method, such as skin patch test or lymphocytes transformation testing (LTT), that results in underreporting. Another difficulty is the paucity of clinical studies providing clear data on a connection between metal sensitivity and implant outcomes.,35 Our study revealed that eczema incidence rate was 57.90 per 1000 person-years and the adjusted overall HR was 1.35 after a 14-year follow-up. Our results are consistent with other reports in the literature; a systemic review revealed that the prevalence of metal allergy was higher after joint arthroplasty compared with the control group ).Of these materials, chromium is considered most likely to cause dermatitis after joint arthroplasty. We observed a higher percentage of allergic and immunity disorders in the joint replacement group in our study. Men and women had approximately the same increase in risk of developing eczema after joint replacement. The result did not meet our expectations because the overall prevalence of metal hypersensitivity in the general population is estimated to be between 10% and 15% and is higher in women than in men.,37 Our study also showed that age influenced the risk of eczema. In a study of 493 trauma patients with an average age of 39 years, Swiontkowski et al found that the prevalence of metal sensitivity was considerably lower than the rates reported in the general population, leading the authors to suggest that metal sensitivity may be less prevalent in younger populations.The distribution of our study group demography is the same as in previous literature, with more women than men and most patients aged over 65 years. Further research is needed to isolate eczema risk in upper limb arthroplasty.After further stratifying the risk of eczema after arthroplasty by anatomical site, there was a relatively higher risk in the hip and knee arthroplasty group than in the control group compared with the upper limb comparison, likely due to the relatively small sample size for upper limb replacement (n = 4) in our study. Although upper limb joints are not weight bearing, abundant metal particles in macrophages in some tissue from resected, failed reverse total shoulder arthroplasties have been previously described. Our results also indicate that eczema risk increased with time. Over time, prosthesis wears with use and the resulting particles accumulate around the joint, thereby activating the host immune response. The immune system responds to implant debris by forming myeloid progenitor cells and lymphoid stem cells, which are responsible for innate (non-specific) and adaptive (specific) immune reactivity, respectively. Cell-mediated delay type hypersensitivity with TH1 cells has been studied in the literature.,41 The interplay between the resulting chemokine and cytokine expression and subsequent activation of innate and adaptive immunity is partially understood, but is limited due to a lack of basic understanding of a few central chemokines, including MCP-1, IL-8, and MIP-1.Duration since join replacement plays a role in the relationship between artificial prostheses and the development of eczema. Our study observed an increased eczema risk after 2–3 months, when the healing process is expected to finish. This increased risk of eczema after a period of time comes from clinical observations in the literature.,43–45 The impact of pre-existing metal sensitivity on clinical outcomes, as demonstrated by preoperative history or patch testing, remains controversial.,46 The reason that these methods cannot accurately predict allergic reactions may be explained in part by these reactions, which do not associate allergies with a single material or alloy, but rather a combination of innate and acquired immunity responses. Physicians should be aware of skin lesion complaints following joint replacement, even years after surgery. Self-reported skin allergies are an important first step in diagnosing and evaluating implant allergies. Eczema reactions do not necessarily indicate poor functional outcomes that require surgical revision.,45 However, revision surgery is suggested if skin reactions lead to recurrent erythematous swelling and poor wound healing. Several studies have reported that the eczema condition has been resolved after revision to prostheses with ceramic-based components,18,20–23 and uncemented prostheses.,26Due to increasing awareness of eczema risk after joint replacement, many predictive methods have been developed, such as the patch test and lymphocyte transformation test. These are important methods in diagnosing and evaluating implant allergies. However, the current viewpoint of these tests is that there is no association between post-operative allergic symptoms.Our study had several limitations. Firstly, we relied on National Health Insurance Research Databases ICD 9 code to reach the diagnosis of eczema. No eczema location information in ICD 9 compared with ICD 10. Besides, the etiology of eczema is multiple included asteatotic, atopic, venous insufficiency, or contact-induced. Consequently, the incidence rate of contact dermatitis may be overestimated. This study revealed the HR is reliable as both groups exhibited the same eczema etiology and well-controlled for comorbidity. Secondly, information regarding arthroplasty prior to the year 2000 was unavailable, and thus, may have been misclassified in both cohorts. Third, joint replacement may be linked with osteoporosis due to steroid treatment of allergic or immune-related conditions which were associated with joint replacement in the study and could be, in turn, associated by themselves with eczema. As the result, it can partially explain in our study, joint replacement patients were likely to have more comorbidities including asthma, allergic conjunctivitis, and immunity disorders. Due to treatment effect of steroid for eczema, the hazard ratio may be underestimated. However, the main advantage of this study is the use of population-based data, which is highly representative of the general population.5Our data indicate that physicians may notice eczema risk long after joint replacement surgery. Although the immune mechanism of eczema following joint replacement is complex and not well understood, the trend among surgeons is to choose a hypoallergenic prosthesis or a biological joint preserving procedure in addition to developing novel allergy tests for select patients. Communication and collaboration between surgeons and dermatologists can identify the risk of eczema, as well as possible implant complications, in patients following joint arthroplasty.www.uni-edit.net) for editing and proofreading this manuscript.This study was supported in part by This study is supported in part by Taiwan Ministry of Health and Welfare Clinical Trial Center (MOHW108-TDU-B-212-133004), China Medical University Hospital (DMR-107-071/CRS-108-035), Academia Sinica Stroke Biosignature Project (BM10701010021), MOST Clinical Trial Consortium for Stroke (MOST 107-2321 -B-039 -004-), Tseng-Lien Lin Foundation, Taichung, Taiwan, and Katsuzo and Kiyo Aoshima Memorial Funds, Japan. We would like to thank Uni-edit (Conceptualization: Po-Yuan Wu, Chun-Hao Tsai.Data curation: Chih-Hsin Muo, Chun-Hao Tsai.Formal analysis: Chih-Hsin Muo, Chun-Hao Tsai.Investigation: Chun-Hao Tsai.Methodology: Chih-Hsin Muo, Chun-Hao Tsai.Resources: Chun-Hao Tsai.Software: Chih-Hsin Muo.Supervision: Chun-Hao Tsai.Validation: Po-Yuan Wu, Chun-Hao Tsai.Visualization: Po-Yuan Wu.Writing – original draft: Po-Yuan Wu, Chih-Hsin Muo.Writing – review & editing: Po-Yuan Wu, Chun-Hao Tsai.