Analysis of hospitalization and death for COVID-19 in breast cancer patients
In order to test the hypothesis that hormonal therapies may influence COVID-19 susceptibility and severity of symptoms, we mined real-world clinical data from the BC population of Emilia Romagna region assessing the frequency of hospitalization and death for COVID-19 in patients treated with tamoxifen, AIs, fulvestrant and anti-HER2 therapies (trastuzumab and pertuzumab) the latter used as a control group as non-hormonal therapy treated BC patients. Out of a total of 24628 BC patients (24252 females, 376 males), 195 (187 females, 8 males) were hospitalized for COVID-19 in 2020. The rate of hospitalization for patients was higher than the overall rate of hospitalization for COVID-19 observed for residents in Emilia Romagna (0.79% vs 0.57%; 25448/4474292 residents) during 2020. As expected, age was an important determinant of hospitalization (OR, 1.06; 95% CI, 1.04 to 1.07; p<0.001) and death (OR, 1.12; 95% CI, 1.08 to 1.15; p<0.001) for COVID-19 in the BC patients cohort and its distribution differed among therapy groups (p<0.001). Statistical analysis applying an age-adjusted logistic model to the cohort stratified by therapy (single agent) revealed significant decreases in the hospitalization rate in tamoxifen (0.38%; OR, 0.26; 95% CI, 0.13-0.56; p=0.001; Table 1) and AIs treated patients (0.84%, OR, 0.31; 95% CI, 0.18- 0.61; p=0.001; Table 1) as compared to 1.56% observed in the anti-HER-2 therapy reference group. In contrast, fulvestrant-treated BC patients showed no significant difference as compared to the reference group (1.65%; OR, 0.55; 95% CI, 0.23-1.31; p=0.18). The analysis of mortality for COVID-19 confirmed a significant protective role for tamoxifen (0%; OR, 0.03; 95% CI, 0-0.34; p=0.005; Table 1), as compared to anti-HER2 therapies. Strikingly, no COVID-19 fatalities on 4784 tamoxifen-treated patients occurred. The mortality by COVID-19 in AIs and fulvestrant-treated patients was not significantly different (p>0.05) as compared to anti-HER2 treated patients although AIs p-value was very close to significance (0.21%; OR, 0.23; 95% CI, 0.07-1.14; p=0.07; Table 1). Following analyses focused on the comparison of COVID-19 hospitalization and mortality among female patients treated with hormonal therapies. Tamoxifen treated female patients showed a significantly lower rate of hospitalization for COVID-19 (0.31%; OR, 0.41; 95% CI, 0.18-0.94; p=0.04; Table 2) compared to fulvestrant treated female patients used as reference group while AIs did not (0.84%; OR, 0.56; 95% CI, 0.31-1.12; p=0.10; Table 2). Interestingly, we observed a similar picture for COVID-19 mortality in tamoxifen (0%; OR, 0.09; 95% CI, 0-1.08; p=0.06; Table 2) and AIs treated female patients (0.21%; OR, 0.65; 95% CI, 0.22-3.19; p=0.54; Table 2).
Then we compared the COVID-19 hospitalization and mortality rates for female patients treated with the different hormone therapies with those expected from the regional rates. In tamoxifen female treated patients we observed only 14 hospital admissions for COVID-19 out of the 21 expected according to the regional risk matched by age suggesting that the risk of hospitalization in tamoxifen female treated patients was lower (SHR: 0.67; 95% CI, 0.61-1.32; Table 3). Conversely, none of the 4580 tamoxifen treated female BC patients died for COVID-19 while 5 were expected according to the regional mortality risk. Importantly, AIs did not show a decreased hospitalization risk (SHR: 0.98; 95% CI, 0.81-1.12; Table 3) but a significantly lower mortality risk (SMR: 0.73; 95% CI, 0.45-0.90; Table 3) compared to the region. Fulvestrant-treated female BC patients showed a higher hospitalization rate for COVID-19 as compared to the region with 10 hospital admissions on 6 expected according to the regional risk adjusted by age (SHR: 1.67; 95% CI, 0.00-2.00; Table 3). Mortality by COVID-19 in the fulvestrant treated female cohort instead was as expected, with 2 fatalities observed based on the regional risk (SMR: 0.89; 95% CI, 0.00-1.92; Table 3).
Interestingly, also anti-HER2 female treated patients showed an increased risk of hospitalization (SHR: 2.50; 95% CI, 0.00-2.72) for COVID-19 with 8 hospital admissions on 3 expected according to the regional risk adjusted by age.
Table 1. COVID-19 hospitalization and mortality rates in patients under monotherapy in Emilia Romagna region from January 1st to December 31st, 2020. OR=odds ratio, 95% CI= 95% confidence interval. Statistically significant values are indicated in bold. The logistic model applied is described in the Methods.
|
COVID-19
hospitalization
|
Therapy
|
OR
|
95% CI
|
Count
|
COVID-19 hospitalization / death (%)
|
p-value
|
Anti-HER2
|
reference
|
705
|
11 (1.56)
|
|
Tamoxifen
|
0.26
|
0.13-0.56
|
4784
|
18 (0.38)
|
0.001
|
Aromatase inhibitors
|
0.31
|
0.18-0.61
|
18533
|
156 (0.84)
|
0.001
|
Fulvestrant
|
0.55
|
0.23-1.31
|
606
|
10 (1.65)
|
0.18
|
Age
|
1.06
|
1.04-1.07
|
|
|
< 0.001
|
COVID-19
mortality
|
Anti-HER2
|
reference
|
705
|
2 (0.28)
|
|
Tamoxifen
|
0.03
|
0.00-0.34
|
4784
|
0 (0)
|
0.005
|
Aromatase inhibitors
|
0.23
|
0.07-1.14
|
18533
|
39 (0.21)
|
0.07
|
Fulvestrant
|
0.35
|
0.05-2.32
|
606
|
2 (0.33)
|
0.26
|
Age
|
1.12
|
1.08-1.15
|
|
|
< 0.001
|
Table 2. COVID-19 hospitalization and mortality rates in female patients treated with a single hormone therapy in Emilia Romagna region from January 1st to December 31st, 2020. OR=odds ratio, 95% CI= 95% confidence interval. Statistically significant values are indicated in bold. The logistic model applied is described in the Methods.
|
COVID-19
hospitalization
|
Therapy
|
OR
|
95% CI
|
Count
|
COVID-19 hospitalization / death (%)
|
p-value
|
Fulvestrant
|
reference
|
601
|
10 (1.66)
|
|
Tamoxifen
|
0.41
|
0.18-0.94
|
4580
|
14 (0.31)
|
0.04
|
Aromatase inhibitors
|
0.56
|
0.31-1.12
|
18425
|
155 (0.84)
|
0.10
|
Age
|
1.06
|
1.05-1.07
|
|
|
< 0.001
|
COVID-19
mortality
|
Fulvestrant
|
reference
|
601
|
2 (0.33)
|
|
Tamoxifen
|
0.09
|
0.00-1.08
|
4580
|
0 (0)
|
0.06
|
Aromatase inhibitors
|
0.65
|
0.22-3.19
|
18425
|
39 (0.21)
|
0.54
|
Age
|
1.12
|
1.08-1.16
|
|
|
< 0.001
|
Table 3. COVID-19 hospitalization and mortality rates in female patients treated with a single hormone therapy in Emilia Romagna region from January 1st to December 31st, 2020 and compared to the regional risks. SHR=standard hospitalization ratio SMR=standard mortality ratio, 95% CI= 95% confidence interval, Exp.= expected number of cases. Statistically significant values are indicated in bold. The logistic model applied is described in the Methods.
|
|
COVID-19 hospitalization
|
COVID-19 mortality
|
Therapy
|
SHR
|
95% CI
|
Exp.
|
COVID-19 hospitalized (%)
|
SMR
|
95% CI
|
Exp.
|
COVID-19 deaths
(%)
|
Tamoxifen
|
0.67
|
0.61-1.32
|
21
|
14 (0.31)
|
-
|
-
|
5
|
0 (0)
|
Aromatase inhibitors
|
0.98
|
0.81-1.12
|
158
|
155 (0.84)
|
0.73
|
0.45-0.90
|
54
|
39 (0.21)
|
Fulvestrant
|
1.67
|
0.00-2.00
|
6
|
10 (1.66)
|
0.89
|
0.00-1.92
|
2
|
2 (0.33)
|
Breast cancer patients cohort
|
0.97
|
0.83-1.11
|
-
|
179 (0.76%)
|
0.67
|
0.47-0.88
|
-
|
41 (0.17)
|
Next, we analyzed the distribution of hospitalization occurrence for COVID-19 normalized to drug half-life (HL), calculating the number of HL equivalents between the date of last treatment or drug administration and the hospitalization, to estimate the duration of protection or of susceptibility for each drug. Strikingly, 80% of hospitalizations were observed within the first HL since the last administration of fulvestrant (Figure 1) suggesting that a correlation between the plasmatic concentration of fulvestrant in BC patients and an increased hospitalization risk may exist. Conversely, the COVID-19 hospitalization rates increased over time after treatment discontinuation with tamoxifen and AIs showing respectively 50% and 85% of admissions occurring after the equivalent of 4 HLs (<6.25% putative drug blood concentration) (Figure 1) suggesting a fade of a protective action as drug concentration lowers.
Tamoxifen treatment protects human cells while fulvestrant makes them more vulnerable to SARS-CoV-2 infection in vitro
In order to gain insights into the mechanism of protection from SARS-CoV-2 infection mediated by tamoxifen and of increased susceptibility by fulvestrant, we performed in vitro experiments on human cells. Specifically, breast cancer cell lines expressing or not ER, MCF7 and MDA-MB-231 cells respectively, were selected as an in vitro model. First, cell susceptibility to SARS-CoV-2 was tested. Once assessed low levels of viral replication in both cell lines, we determined basal levels of ACE2, TMPRSS2 and NRP1 expression and whether 4-OH-tamoxifen (4-OHT), an active metabolite of tamoxifen, and fulvestrant affect the expression of those genes. In MCF7 cells, treatment with fulvestrant at 10nM and 1uM concentration significantly increased the expression of ACE2 (fold change>1.8; p=0.025), NRP1 (fold change>4.7; p=0.015) and TMPRSS2 (fold change>9.0; p=0.007) at 48 hours post treatment while 4-OH-T did not (Figure 2a). This effect correlates with ER expression since fulvestrant cannot modulate the expression of those genes in triple negative MDA-MB-231 cells (data not shown). Importantly, we tested whether 17β-estradiol (E2) can modulate ACE2, NRP1 and TMPRSS2 expression in MCF7 cells at physiological concentrations corresponding to pre and postmenopausal ranges (30-400 pg/ml = 0.11-1.47nM; <15 pg/ml = <0.055nM), respectively, and found both NRP1 and TMPRSS2 genes repressed at premenopausal concentrations while not significantly affected below 0.05nM E2 (Figure 2b). This suggests that the decline in E2 concentration during aging may promote their expression in tissues. Importantly, E2 treatment does not alter the expression of ACE2 while fulvestrant treatment does, suggesting that fulvestrant may exert its action also through estrogen-independent ER mechanisms. Then, we tested whether 4-OHT and fulvestrant affect cell susceptibility to SARS-CoV-2 infection. MCF7 cells, that in our conditions are largely refractory to infection, were pretreated for 72hr with 4-OHT, fulvestrant or vehicle, re-plated at the same concentration, infected with SARS-CoV-2 and cultured in the presence of the drugs. At 48hr from infection, we measured viral RNA amount inside cells (Figure 2c) and in the culture supernatant (Figure 2d) by Q-PCR and found that upon fulvestrant treatment, the quantity of viral RNA was increased about 24 times inside cells and about 4 times in the supernatant consistent with an altered susceptibility to SARS-CoV-2 infection as expected by ACE2, TMPRSS2 and NRP1 overexpression. Conversely, 4-OHT pretreatment decreased the amount of viral RNA of about 2 fold inside cells and 4.2 fold in the supernatant (p=0.002) suggesting that tamoxifen interferes with SARS-CoV-2 replication. Coherently, infectious particles could be retrieved only from the supernatant of fulvestrant-treated MCF7 cells and not from the 4-OHT- or vehicle-treated cells (Figure 2e).