To our knowledge, this is the first study to follow a group of breast cancer patients with preplanned analyses to examine changes in gut microbiome during their first year of treatment in relation to type of chemotherapy, taking into account changes in weight during the study period. In all three groups, the final fecal sample was collected more than 100 days after the end of chemotherapy (for neoADJ and ADJ groups) or radiation (for noC group), representing a recovery period. We found striking differences in gut alpha diversity changes by treatment, increases in all four alpha diversity measures in the neoADJ group that was not observed in the ADJ or noC groups. Increases in alpha diversity were also observed in the weight loss but not in the weight gain groups. However, there were no significant changes in alpha diversity in relation to risk of recurrence/metastases. There were notable changes in taxa abundance that differed by treatment; one taxa, p Bacteroidetes (g_Alistipes) showed changes in abundance that reached the Bonferroni threshold of p<0.0007, while changes in abundance of 12 taxa showed p values that ranged from <0.001 to 0.0007 without or with adjustment of weight changes. In contrast, there were far few changes in taxa differences between the weight loss and weight gain groups, but one taxa, p_Firmicutes (g_Lachnobacterium) showed changes that were consistent in all three statistical analyses.
Although average weight changes were modest during this first year of treatment, our finding of a significant increase in the alpha diversity measures in the neoADJ group may be related in part, to weight loss in the neoADJ group (-1.48 kg) but weight gain in the ADJ (+1.01 kg) and noC (+0.33 kg) groups. Alpha diversity measures have been used to assess health habits including body composition, and low gut alpha diversity has been associated with obesity in some studies [24, 25]. In a study of 26 cancer patients (7 with breast cancer) who were treated with cytotoxic, targeted chemotherapy, or a combination of chemotherapy with immununotherapy, gut microbiome Shannon index was higher in responders than in nonresponders, who also displayed higher abundance of Alistipes, a genus member of the Rikenellaceae family within the Bacteroidales order . In a study of non-small cell lung cancer patients treated with immune checkpoint inhibitors, responders showed higher diversity of gut microbiome as well as an enrichment of Alistipes . In our analysis, Alistipes emerged to be important in both Wilcoxon rank sum and negative binomal analyses but its abundance decreased in the neoADJ group but not in the ADJ group after completion of treatment. The significance of our finding on Alistipes is unclear but this genus has been found to be correlated with both healthy phenotypes as well as having pathogenic roles  in colorectal cancer  and liver diseases . It has been suggested that decrease in Alistipes contributes to the decrease in short chain fatty acids which have anti-inflammatory properties.
We also found suggestive differences in changes in taxa of select Erysipelotrichaceae genera (Catenibacterium, Eubacterium and Clostridium), abundance increased in the neoADJ but decreased in the non-neoADJ groups. In a small study of patients with breast or gynecological cancers, Erysipelotrichaceae abundance also increased but this was mainly among women who gained weight following treatment . The immunogenic properties of some members of the Erysipelotrichaceae family may lead to gut inflammation and weight gain . Our findings on changes in taxa abundance of Verrucomicrobia (g_Akkermansai), in particular, a reduction in abundance in the neoADJ group but an increase in the non-neoADJ group adds to the literature of the importance of this butyrate-producing bacteria [31, 32]. In a study of breast cancer patients treated by neoadjuvant chemotherapy, breast tumor tissue microbiome profile was impacted by treatment but a comparable group of patients treated with adjuvant chemotherapy was not included in this study . Nevertheless, results from this  and our study suggest that type of chemotherapy may impact breast and gut microbiome changes.
Our finding of a difference in the abundance of Lachnobacterium between the weight gain and weight loss groups needs confirmation. There is scant information on this genus. A recent Swedish cross-sectional study found that high intake of sugar and sweet bevarages was significantly inversely associated with abundance of Lachnobacterium . However, another cross-sectional study found abundance levels of Lachnobacterium was higher in obese subjects than normal weight subjects and higher among individuals with low physical activity than those with high physical activity .
Strengths of this pilot study include the longitudinal collection of gut microbiome data on 33 breast cancer patients at multiple (baseline, during, and at the completion) time points during the first year of treatment with either neoADJ, ADJ, or noC treatment. In addition to the detailed information on breast cancer treatment, tumor characteristics, and lifestyle information that was updated at each clinic visit, body composition was assessed using DXA at baseline and at the completion of study. Our results on gut microbiome changes were analyzed using complementary statististical methods, Wilcoxon rank sum test and negative binomial mixed models for longitudinal microbiome data with adjustment for select covariates including changes in weight. We also considered multiple comparisons and used a Bonferroni-adjusted type I error rate to evaluate p-values. Participants included whites and nonwhites, reflecting the catchment area of USC. However, we are limited by a modest sample size and the no chemotherapy group was based on only 4 patients. Although we conducted results separately for the three treatment groups, our main analysis was based on comparing neoADJ to non-neoADJ groups (i.e, ADJ + noC). Because of the inherent differences in timing of treatment between neoADJ, ADJ, and no chemotherapy groups, we were not able to collect fecal samples at a standardized interval and the period of enrollment and length of follow-up were not identical in the three groups (Figure 1). Nevertheless, the baseline fecal samples were collected before initiation of chemotherapy for the neoADJ and ADJ groups or radiation for the no chemotherapy group and the final fecal samples were collected when there was a recovery period of at least 100 days after completion of chemotherapy or radiation. Because this was funded as a pilot study, we only monitored patients during the first year of treatment and did not collect information on additional treatment (e.g., hormone therapy).
In conclusion, this pilot longitudinal study found significant increases in gut microbiome alpha diversity measures in the neoADJ group but not in the non-neoADJ group and also intriguing changes in select Bacteroidetes and Firmicutes taxa. The dynamic nature of the gut microbiome in association with chemotherapy and weight changes highlight the need to better understand the significance of these findings and how to harness this information to identify a gut microbiome profile that would have lasting beneficial effects among women with breast cancer. Given the very modest sample size of this pilot study, we view these taxa changes as potentially informative and worthy of investigation in future studies with larger sample sizes of breast cancer patients and with longer duration of follow-up.