Background
Thrombotic microangiopathy (TMA) is a rare syndrome combining diffuse microvessel thrombosis and mechanical haemolytic anaemia, often associated with thrombocytopenia [
1]. It can result in severe, life-threatening organ dysfunction, especially affecting the kidneys and central nervous system. TMA is a causally heterogeneous syndrome related to several conditions including thrombotic thrombocytopenic purpura (TTP) and haemolytic-uraemic syndrome (HUS), which are primarily caused by a functional deficiency of ADAMTS 13 (an enzyme involved in the degradation of Von Willebrand Factor) activity and Shiga toxin or complement dysregulation, respectively. TMA may also occur following exposure to certain drugs [
1], including bevacizumab and gemcitabine, two antineoplastic agents that have been approved for metastatic breast cancer. TMA has also been observed in patients with solid tumours: cancer-related microangiopathic haemolytic anaemia (MAHA) is a rare paraneoplastic syndrome, first described as a clinicopathological entity in 1979 by Antman et al. [
2]. Pathogenesis of cancer-related MAHA remains unknown; three mechanisms might be involved: (i) mechanical lysis of red blood cells, related to tumour micro-emboli in micro-vessels; (ii) inflammatory syndrome following activation of endothelial cells by circulating tumour cells; (iii) activation of the coagulation cascade (high tissue factor expression by endothelial and tumour cells; mucins secretion by tumour cells; von Willebrand Factor release caused by long-lasting bone marrow metastasis) [
3‐
9]. Over the last 40 years, single cases or small retrospective series of cancer-related MAHA have been reported, with very poor survival [
4‐
7]. Apart from lymphomas, most cases were reported in patients with adenocarcinoma, while very few cases have been reported with squamous cell carcinomas. In 2012, Lechner et al. performed a literature search and compiled 168 published cases of cancer-related MAHA; gastric and breast adenocarcinomas were the two most common primary tumour types in patients with cancer-related MAHA, accounting for 26% and 21% of compiled cases, respectively [
10]. To date, despite a handful of case reports [
5,
11‐
14], breast cancer-related MAHA remains a very poorly known condition. We therefore undertook a multicentre retrospective study to specifically identify breast cancer-related MAHA characteristics, outcomes and prognostic factors.
Methods
This study was approved by the Institut Curie review board; a waiver of informed consent was granted because of the retrospective nature of the work.
Eligibility criteria
The presence of schistocytes (> 0.5%) and either low haptoglobin or cytopenia (anaemia, thrombocytopenia or both) were mandatory for a diagnosis of MAHA, in agreement with current guidelines [
15,
16]. Other eligibility criteria were patients with histologically proven breast cancer and MAHA diagnosed between 1995 and 2019 in participating centres. Ineligibility criteria were MAHA attributed to a cause other than breast cancer determined by treating physician and patients treated with either gemcitabine or bevacizumab at the time of or during the 6 months prior to MAHA diagnosis.
Case search
In July 2018, a call for participants was sent to 13 French cancer centres, outlining the study’s objectives, and listing the data that needed to be collected for the study. Participating centres were encouraged, whenever possible, to automatically screen their patient files by means of computerised searches using the following key words (in French): “microangiopathie(s) thrombotique(s); micro-angiopathie(s) thrombotique(s); micro angiopathie(s) thrombotique(s); schistocyte(s)”. Computerised screening of laboratory registries was also performed, searching for blood counts with elevated schistocytes. Senior medical oncologists manually reviewed all cases retrieved by computerised search to confirm the diagnosis of breast cancer-related MAHA. In centres in which computerised screening was deemed unfeasible, physician-based case declaration was also accepted. Case collection was closed in February 2019.
Statistics
Data requested from participating centres are listed in Additional file
1, Supp Mat 1A. The PRONOPALL score, a validated prognostic score in oncology patients [
17], was obtained from collected data. Because of the rarity of breast cancer-related MAHA, no data apart from those required for the diagnosis of MAHA were considered to be mandatory. The call for participants indicated that the study would investigate patient characteristics, response to treatment, outcomes and prognostic factors. In the absence of robust breast cancer-related MAHA data in the literature, no hypothesis could be formulated concerning the number of cases needed to achieve any of the study’s objectives; we did not hierarchise objectives into primary or secondary and this exploratory study did not have a predefined power.
The patients’ clinical characteristics are expressed as numbers and proportions; the Chi-squared test or Fisher’s exact test was used to compare categorical variables. Median follow-up was estimated using the Kaplan-Meier method.
Overall survival (OS) was determined from the date of MAHA diagnosis until the date of death or last follow-up. Survival curves were established by the Kaplan-Meier method. Biological and clinical factors were tested using a log-rank test in univariate analysis. The survival time variable was binarised into 2 categories: “death before or at 28 days” versus “death after 28 days”. The Hmisc package was used for imputation of missing data using the “aregImpute” function and to perform univariate and multivariate logistic regressions using “fit.mult.impute” with “lrm” as modelling function [
18,
19]. Factors considered useful according to clinical considerations or with
p value less than 0.2 in univariate analysis were included in a stepwise top-down procedure using the Akaike information criterion (AIC) and the likelihood ratio test as a criterion for variable selection. The accuracy of the final model was verified by controlling calibration and discrimation with the RMS package. The model with the lower value of Brier Score and the higher value of R
2 was selected as the final model to ensure the best discrimination. Calibration was assessed by visual examination of the calibration plot generated after bootstrap resampling. A prognostic score was constructed and weighted with β-coefficients estimation in the final model. The discriminatory capacity of the score, which represents the probability of dying within 28 days after the diagnosis of MAHA, was estimated by calculating the sensitivity and specificity of the score with their 95% confidence intervals (95% CI). A ROC curve was displayed and the area under the curve (AUC) was calculated using the ROCit package [
20]. The 95% CI of the AUC was estimated by bootstrap.
All statistical analyses were performed with R software (version 3.6.2). This report was written in accordance with the REMARK guidelines.
Discussion
To our knowledge, this is the first large cohort study of breast cancer-related MAHA addressing the clinical and laboratory characteristics at MAHA diagnosis and identifying survival prognostic factors. Fewer than 60 individual cases of breast cancer-related MAHA have been reported in the literature, mostly corresponding to single case reports [
23]. The limited clinical and laboratory data available in these reports and a very likely publication bias (biased toward patients with exceptional survival) limit the value of case report compilations.
Firstly, our study confirms that breast cancer-related MAHA is very rare: we identified 54 cases over the last 20 years (1995–2018) in seven of the largest breast cancer centres in France. Patients with a schistocyte count higher than 0.5% were included in our study, while some guidelines recommend a 1.0% cut-off for the diagnosis of mechanical haemolysis [
24,
25]. However, only six of our patients had a schistocyte count between 0.5 and 1.0%, and neither their clinical characteristics nor their outcome was significantly different from those of the other patients.
Our study did not estimate the breast cancer-related MAHA incidence rate, as the number of metastatic breast cancer patients treated over the same time period is unknown. However, for benchmarking purposes, in a single participating institution (
Institut Curie, Paris), 2 patients were diagnosed with breast cancer-related MAHA and 91 patients were diagnosed with breast cancer meningeal carcinomatosis between 2000 and 2007 [
26]. Meningeal carcinomatosis has an estimated cumulative incidence of < 5% in metastatic breast cancer patients [
26,
27]. A cumulative incidence of ~ 0.1% is therefore likely for breast cancer-related MAHA among metastatic breast cancer patients. However, the short survival observed in our study suggests that many patients may die before MAHA is even diagnosed.
Secondly, regarding primary tumour characteristics, a new finding of our study is the high prevalence of breast adenocarcinoma with either lobular histology or overt lobular component (44.2%) compared to previous reports describing the metastatic breast cancer population (10–14%) [
22,
28]
. To our knowledge, this association has not been previously demonstrated: while four of the eight cases of breast cancer-related MAHA reported by Regierer et al. were lobular adenocarcinoma, the histological subtype was missing in the 36 cases compiled in the compilation of published cases by Lechner et al. [
10,
14]. Interestingly, lobular breast adenocarcinoma and gastric adenocarcinoma, described as the leading cause of cancer-associated MAHA, share many phenotypic and genotypic traits in common, such as low E-Cadherin [
29]. High mucin expression may also play a direct role in the pathogenesis of cancer-related MAHA, as it triggers platelet aggregation independently of tissue factor secretion [
9,
30]. Regarding immunohistochemical profile, oestrogen receptor-positive/HER2-negative, HER2-positive and triple-negative subtypes frequency were similar to that observed in the general metastatic breast cancer population [
22].
In accordance with previous MAHA reports, all patients had stage IV disease and many presented multiple metastatic sites [
31‐
34] with laboratory signs of bone marrow involvement (myelemia, erythroblastemia) and/or cytologically-proven bone marrow metastasis [
10,
33‐
37,
38]. Degradation fibrin markers (such as D-Dimers) were not available for most patients. However, coagulation disorders observed for 6 patients suggest possible DIC, according to ISTH-DIC criteria [
21]. One of them presented with a low fibrinogen (< 1 g/L) and high D-Dimers, suggesting hyperfibrinolysis. To diagnose DIC in cancer, best strategy should be a longitudinal biological parameters monitoring including platelets, PT, fibrinogen and D-Dimers [
39]. Unfortunately, due to the retrospective nature of our study, we were not able to perform it. Those are serious limitations for defining DIC in our cohort. Noteworthy, DIC can be responsible for biological disorders such as hemolysis, thrombocytopenia and schistocytes formation [
40]. Then, it is almost impossible to know whether TMA is the origin or the consequence of coagulopathy. Establishing DIC frequency in a CR-MAHA population is challenging and, in practice, hard to determine [
41].
Moreover, in keeping with prior reports focused on CR-MAHA [
34], kidney and neurological disorders were rare, compare to other MAHA’s causes.
OS was very poor with a median OS of 4.0 weeks, shorter than that reported in some previous studies on cancer-associated MAHA [
10,
34,
42]. Although a difference in survival specifically related to breast cancer-related MAHA cannot be ruled out, this difference compared to previous studies could be primarily attributed to our study method: most cases were retrieved by a systematic in silico search, while previous reports may be subject to declaration (to a MAHA registry [
34]) or positive publication [
10] biases.
To the best of our knowledge, no survival prognostic factors have yet been identified for breast cancer-related MAHA. In our study, altered performance status, abnormal prothrombin time, and elevated total bilirubin were the three strongest independent prognostic factors, while low haemoglobin had a more marginal impact. These factors could be used to distinguish patients likely to benefit from urgent antineoplastic therapy (the only effective treatment for CR-MAHA [
34,
35,
38,
43‐
45]) from those who should preferably be referred for palliative care. Of note, the proposed algorithm was not validated on an external series, due to the rarity of breast cancer-related MAHA. Other limitations of our study include its retrospective nature, limited sample size and a lack of a systematic TMA diagnosis strategy including ADAMTS13 activity dosage to formally exclude idiopathic TTP. To prevent those bias, prospective studies should thus be performed to explore the incidence of CR-MAHA in metastatic breast cancer patients.
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