ALC is associated with survival in ovarian cancer and many other cancers, but the mechanistic basis of this association is poorly understood. We hypothesized that HGSC patients with high intrinsic ALC values might mount stronger anti-tumor immune responses, reflected by increased TIL, decreased tumor burden and prolonged survival. Contrary to this hypothesis, we found that intrinsic ALC values (measured ≥ 2 years prior to the diagnosis of cancer) have no bearing on tumor burden or progression-free survival upon the subsequent development of HGSC. Instead, we found that ALC declines upon development of HGSC to levels proportionate to tumor burden. Accordingly, ALC values recorded at the time of diagnosis are strongly associated with prognosis. ALC values increase after cytoreductive surgery but rarely return to intrinsic levels, indicating long-term impairment of lymphoid homeostasis. Finally, we showed that ALC is not associated with the presence of CD8+ or CD20+ TIL. Collectively, our findings demonstrate that ALC and TIL are independent immunological parameters associated with outcome in HGSC. In the case of ALC, this appears to reflect an association with disease burden rather than an immunological mechanism.
To determine patients' intrinsic ALC, we obtained values that were recorded two or more years prior to HGSC diagnosis. While the natural history of HGSC remains poorly understood, the latency between disease initiation and clinical diagnosis is estimated to be 3-4 years based on biomarker and modeling studies [24, 25]. Thus, with a two-year threshold, some patients in our cohort presumably harbored small occult cancers at the time their pre-diagnostic ALC values were measured. Nevertheless, we note that the mean pre-diagnostic ALC for our cohort (1.9 Giga/L) was similar to the mean for healthy women (1.9-1.95 Giga/L)  and in most cases did not decline until ≤ 1 year prior to HGSC diagnosis. A related caveat is that pre-diagnostic ALC values might have been collected for reasons such as acute illness (e.g., infection) and hence might not reflect "intrinsic" ALC values in all cases. That said, for several patients we were able to obtain serial ALC values over several years. In general, these values were stable over time until declining ≤ 1 year prior to HGSC diagnosis.
What mechanism(s) might underlie the decline in ALC upon the development of HGSC or other cancers? Similar to the development of lymphopenia during sepsis, lower ALC values might reflect a response to systemic inflammation. Ovarian cancer is often accompanied by systemic inflammation, as evidenced by increased neutrophil counts (Figure 4). Moreover, several markers of inflammation have been associated with increased tumor burden and/or adverse outcome in ovarian cancer, including high neutrophil-to-lymphocyte ratio ; high monocyte count ; elevated C-reactive protein and hypoalbuminaemia ; and elevated IL-6 and IL-8 levels in ascites fluid [28, 29]. Inflammation could depress ALC values by several possible mechanisms. In mice, IL-6 has been shown to induce Id1 expression in uncommitted hematopoietic progenitors, thereby promoting myelopoiesis over lymphopoiesis . Consistent with this, lymphopenia correlates strongly with increased serum levels of IL-6, as well as soluble IL-2 receptor and TNF receptor in soft tissue sarcomas . In addition to inflammatory cytokines, ovarian cancer and other carcinomas are also associated with elevated levels of Vascular Endothelial Growth Factor , which can inhibit T cell development .
ALC might also decline due to apoptosis of lymphocytes. For example, during sepsis, lymphopenia is associated with apoptosis-induced depletion of lymphocytes and dendritic cells . Lymphopenia has also been attributed to lymphocyte apoptosis in pancreatitis  and measles infection . Finally, CD8+ T cells from cancer patients have been shown to undergo apoptosis in response to tumor-derived microvesicles expressing tumor antigens, Fas ligand and MHC class I . In summary, the decline in ALC observed in cancer patients may reflect both reduced production and increased apoptosis of lymphoyctes. It is noteworthy that ALC rarely recovers to pre-diagnostic levels in HGSC (Figure 4 and data not shown). Similar results were seen in head and neck cancer, even in patients with no evidence of disease two or more years after treatment . Thus, cancer can lead to the long-term impairment of lymphoid homeostasis, a condition that may need to be addressed for immunotherapy to be effective.
In contrast to our initial hypothesis, we failed to find an association between ALC and TIL (Figure 5). Although we only evaluated CD8+ and CD20+ TIL, these two subsets are strongly associated with patient survival and hence are most relevant to our hypothesis [4–9]. Whether ALC values are associated with other mechanisms of anti-tumor immunity, such as innate or humoral responses, remains to be determined. Even so, these other immune mechanisms have yet to show the same prognostic significance as TIL. As for the issue of why TIL densities vary among patients, previous work in ovarian cancer has linked the presence of TIL to chemokine profiles in tumors , functional status of the BRCA DNA repair pathway , and other factors (reviewed in ). It appears these mechanisms have a greater influence than ALC in regulating TIL responses.
Our results have implications for cancer immunotherapy, in particular strategies designed to non-specifically increase lymphocyte numbers in cancer patients . For example, administration of IL-2 causes transient lymphocytosis (increased ALC), which correlates with tumor response in metastatic melanoma  and renal cancer . Administration of IL-2 prior to surgery can prevent lymphopenia and provide possible survival benefit in pancreatic , colorectal , and gastric cancer . In the latter study, IL-2 administration increased both ALC and TIL, suggesting these two immune parameters can be enhanced together. IL-7 can also be used to increase circulating lymphocytes and has the advantage of being less toxic than IL-2. For example, cancer patients treated with IL-7 experienced marked increases in peripheral CD4+ and CD8+ T cells, resulting in a rejuvenated circulating T-cell profile . Finally, ALC can also be increased through cell therapy. In the setting of autologous hematopoietic stem cell transplantation (AHSCT), the extent of ALC or T cell recovery has been positively associated with survival in myeloma and lymphoma, breast cancer, and ovarian cancer [15, 21, 22]. Based on these results, researchers at the Mayo Clinic are investigating whether increasing the lymphocyte content of the AHSCT cell product can improve clinical outcomes in lymphoma [7, 15].
Although the above approaches may have merit, it is noteworthy that in the present study the extent of ALC recovery after standard treatment had no bearing on prognosis (at least in suboptimally de-bulked patients), suggesting that enhancing ALC alone does not confer survival benefit. This implies that interventions that simply increase ALC may not be sufficient to elicit effective tumor immunity. A more promising goal may be to develop strategies that not only increase ALC but, in the process, skew lymphocyte recovery in favour of a tumor-reactive repertoire.