Researchers at University of California San Diego School of Medicine have found evidence that helps explain why patients who are young and/or female have especially low response rates to some types of cancer immunotherapy.
Their findings suggest that since the typically robust immune systems of young and female patients are better at getting rid of tumor cells, the cells left behind are not as readily visible to the immune system to begin with, rendering some types of immunotherapy ineffective.
The study is published August 17, 2020, in Nature Communications.
“Now that we know why some patients don’t respond as well to immunotherapy, we can begin developing more informed approaches to treatment decisions – for instance, developing predictive algorithms to determine a person’s likely response before initiating immunotherapies that may have a high probability of not working or working poorly for them,” said senior author Hannah Carter, Ph.D., associate professor of medicine at UC San Diego School of Medicine.
Cancerous or infected cells wave molecular flags that tell the immune system to clear them away before the problem gets out of control.
The flag poles – molecules of the Major Histocompatibility Complexes (MHC) – are displayed at the surface of most cells in the body.
MHCs hold up antigen flags – bits of just about everything from inside the cells – and display them to immune cell surveyors that are constantly checking for damaged or infected cells.
Since tumor cells carry a lot of mutations, they show up frequently among these flags, allowing the immune system to detect and eliminate them.
But some tumor cells evade the immune system by also throwing up a stop sign molecule that keeps the immune system from recognizing the MHC flags.
And here’s where immune checkpoint inhibitors come in: This type of cancer immunotherapy uses antibodies to make the tumor cell once again visible to the patient’s immune system.
So why would a person’s age or sex influence how well immune checkpoint inhibitors work?
Sex and age differences have long been observed when it comes to immune response.
For example, females have twice the antibody response to flu vaccines and are far more susceptible to autoimmune diseases.
Similarly, human immune systems tend to weaken as we age. But if females and younger people have stronger immune responses in most cases, you might expect cancer immunotherapy to work better for them, not worse.
To get to the bottom of this conundrum, Carter’s team looked at genomic information for nearly 10,000 patients with cancer available from the National Institutes of Health’s The Cancer Genome Atlas, and another 342 patients with other tumor types available from the International Cancer Genome Consortium database and published studies.
They found no age or sex-related differences in MHC function.
What they did find was that, compared to older and male patients with cancer, younger and female patients tend to accumulate more cancer-causing genetic mutations of the sort that MHCs can’t present to the immune system as efficiently.
Carter said this is likely because robust immune systems of the young and female are better at getting rid of cells displaying well-presented mutant self-antigens, leaving behind tumor cells that rely more heavily on the poorly presented mutations. This selective pressure is known as immuno-editing.
“So if a tumor cell doesn’t present highly visible, mutated self antigens to begin with, checkpoint inhibitor drugs can’t help reveal them to the immune system,” she said.
“This shows an important thing, that the interplay between the cancer genome and the adaptive arm of the immune system is not a static one,” said co-author Maurizio Zanetti, MD, professor of medicine at UC San Diego School of Medicine and head of the Laboratory of Immunology at UC San Diego Moores Cancer Center.
“Two simple but important variables, age and sex, influence this interplay. The study also emphasizes the master role of the MHC in dictating the outcome of this interplay, reaffirming its central role in the evolution of disease, cancer included, at the level of the individual and population.”
Carter cautions that their findings for “younger” patients don’t necessarily apply to children since, genetically speaking, pediatric tumors are very different from adult tumors.
In addition, she noted that, like most genomics databases, those used in this study contain data primarily from people of Caucasian descent, and more diversity is needed to confirm that the findings can be generalized to all populations.
“Cancer isn’t just one disease, and so the way we treat it can’t be one-size-fits-all,” she said. “All checkpoint inhibitors can do is remove the generic block that tumors put up to hide from the immune system.
The more we learn about how interactions between tumors and immune systems might vary, the better positioned we are to tailor treatments to each person’s situation.”
Breast Cancer Immunotherapy
The first cancer immunotherapy treatments were based on the use of humanized monoclonal antibodies with the ability to bind and neutralize a targeted altered molecule expressed by cancer cells and on which their survival and proliferation depends.
The approval in September 1998 of trastuzumab (Herceptin®, Genentech, Inc., South San Francisco, CA, United States) represented the release of the first antibody for the treatment of metastatic breast cancer patients with HER2 (Receptor tyrosine-protein kinase ERBB2, CD340) overexpression and/or gene amplification, which represented a milestone in the treatment of breast cancer.
After trastuzumab, other different anti-HER2 monoclonal antibodies including lapatinib (Tykerb®, GlaxoSmithKline, Brentford, United Kingdom), neratinib (Nerlynx®, Puma Biotechnology, Los Angeles, CA, United States), gefitinib (Iressa®, AstraZeneca, Cambridge, United Kingdom), or afatinib (Giotrif®, Boehringer Ingelheim Pharmaceuticals, Inc., Ingelheim am Rhein, Germany)  as monotherapy or in combination with conventional treatments have contributed to increasing the number of therapeutic options for breast cancer patients.
Although the use of monoclonal antibodies targeting altered proteins has definitely improved the outcome of cancer patients, modest response rates (Table 4) and resistance development  remain as the major impediments for treatment success and require the search for new approaches apart from combined therapies, among which antibody-drug conjugates (ADC) such as the recently FDA approved ado-trastuzumab emtansine (Kadcyla®, Genentech, Inc., South San Francisco, CA, United States)  and T cell bispecific antibodies stand out among the most promising strategies for breast cancer patients .
Table 4 – Approved humanized monoclonal antibodies for breast cancer treatment.
|Monoclonal Antibody||Response Rates (Monotherapy)||Most Common Treatment-Related Adverse Events|
|Trastuzumab||35% (95% CI, 24.4% to 44.7%) and none in patients with 3+ and 2+ HER2 overexpression by immunohistochemistry, respectively . Further, 34% (95% CI, 23.9% to 45.7%) and 7% (95% CI, 0.8% to 22.8%) in patients with and without HER2 gene amplification by fluorescence in situ hybridization analysis, respectively . Approximately 15% of patients relapse after therapy .||Chills (25%), asthenia (23%), fever (22%), pain (18%), nausea (14%), cardiac dysfunction (2%) .|
|Pertuzumab||3% to 7.6% complete response and 16.7% partial response in previously trastuzumab-treated breast cancer patients [51,52].||Diarrhea (48.3%), Nausea (34.5%), vomiting (24%), fatigue (17%), asthenia (17%), back pain (10%) .|
|Lapatinib||24% in trastuzumab-naïve and less than 10% in trastuzumab-refractory breast tumors .|
Partial response in 39% (95% CI, 30% to 48%) of patients with relapsed or refractory HER2-positive inflammatory breast cancer .
|Diarrhea (59%), fatigue (20%), nausea (20%), rash (18%), anorexia (16%), dyspnoea (14%), vomiting (13%), back pain (11%) .|
|Neratinib||Pathological complete response in 56% of HER2-positive but HR- breast cancer patients compared to 33% in the control group. Further, 84% response rate in HER2-positive and hormone receptor-positive compared to a 59% response rate in HER2+ and hormone receptor-negative .||Diarrhea (83.9%), nausea (37.9%), abdominal pain (28.4%) .|
|Gefitinib||No complete or partial responses observed in previously treated patients with advanced breast cancer .||Diarrhea (45.2%), skin rash (12%) .|
|Afatinib||Partial response in 10% and progressive disease in 39% of extensively pretreated HER2-positive patients metastatic breast cancer progressing after trastuzumab. No complete response observed .||Diarrhea (24.4%), skin rash (9.8%) .|
As a result of the latest studies in this field and in line with the encouraging long-term success of checkpoint inhibitors in the treatment of different tumors, distinct research groups have focused their efforts in developing analog treatments for breast cancer patients.
In fact, as a result of the findings from the Phase III double-blind IMpassion130 trial (ClinicalTrials.gov ID NCT02425891), which reported a 40% reduced risk of disease progression or death in patients receiving atezolizumab plus nab-placlitaxel or placebo [58,59,60], in March 2019, the FDA approved the first checkpoint inhibitor immunotherapy drug, the anti-PD-L1 antibody atezolizumab (Tecentriq ®), in combination with chemotherapy (Abraxane®) for the treatment of triple-negative, metastatic breast cancer patients with positive PD-L1 protein expression .
However, despite this great milestone, modest complete response rates (7.1%, 95% CI, 4.9–9.9 and 10.3%, 95% CI, 6.3–15.6 in PD-L1 positive subgroup) and immune-mediated serious adverse events such as pneumonitis, hepatitis, colitis, and endocrinopathies that can cause treatment discontinuation  remain as notable impediments for the success of this treatment and justify the search of new therapeutic strategies.
Mechanisms of Immune Evasion in Breast Cancer
As stated above, tumor immune evasion can occur as a result of defective tumor-directed T-cell activation, deficient activated T-cell infiltration into the tumor microenvironment, or because of the tumor cell resistance to cytotoxic action of the immune cells .
Breast Tumor Microenvironment
Immunogenicity is defined as the ability to induce a humoral and/or cell-mediated adaptive immune response.
In fact, both the burden of tumor mutations and the load of neo-epitopes represent two of the factors that are linked to response to checkpoint inhibitors in different malignancies like melanoma or lung cancer .
However, although tumor neoantigens that are produced as a result of breast cancer cells’ genomic instability can be recognized by the immune system and induce T-cell responses and antitumor immunity [62,64], the immunogenicity of breast cancer can be rather heterogeneous, depending to a large extent on the specific subtype of breast cancer .
In the particular case of HER2-positive breast tumors, gene profiling studies have shown that highly suspicious calcifications are associated with decreased immune system activity and ERBB2 overexpression .
For this reason, breast calcifications would be useful not only in the radiological assessment of breast lesions , but also in the management of breast cancer patient candidates for immunotherapy.
On the other hand, although estrogen receptor-negative and HER2-positive have shown evidence of immunogenicity , these types of inflammatory breast tumors are rare (1–5% of cases)  when compared to triple negative breast tumors, which are unique among breast cancer subtypes in having strong antigen expression  and high stromal and tumor-infiltrating lymphocytes, parameters with a strong prognostic and predictive significance to immunotherapy and chemotherapy [62,63,70,71].
Accordingly, triple negative breast tumors with high infiltration of tumor-associated macrophages have been found to have a higher risk of metastasis and lower rates of disease-free survival and overall survival, having been proposed as potentially useful prognostic markers for triple negative breast cancer patients [72,73].
Except for these immunogenic subtypes, breast tumors have historically been classified as immunologically silent  or “cold” tumors, characterized by the presence of low mutation and neoantigen burden and few effector tumor infiltrating lymphocytes, factors proposed as prognostic markers , and metastasis to lymph nodes correlation .
Since non-inflamed tumors represent a significant impediment to the success of T-cell-based immunotherapies, different studies have aimed their efforts towards developing new strategies to increase the presence of immune infiltrates and hence, to improve patient prognosis.
Among these, in addition to directly causing cell damage , the use of local tumor hyperthermia has proven to be a valuable tool as an immunotherapy strategy for cancer  by boosting immune cell activation and increasing the sensitivity of tumor cells to anti-tumor immune responses by different mechanisms, including:
Enhancing the expression of tumor surface HLA class I polypeptide-related sequence A (MICA) and HLA type I, which promote tumor cell sensitivity to lysis by NK cells and CD8+ cells, respectively .
Increasing the release of heat shock proteins, which leads to NK cells activation as well as to APCs activation and antigen presentation to CD8+ cells .
Increasing the release of tumor cells exosomes, which apart from containing chemokines, transfer potential tumor antigens to APCs and subsequent CD8+ activation .
Promoting changes in the tumor vasculature, which facilitates better trafficking of immune cells between the tumor and draining lymph nodes .
In this context, different studies are reporting promising results for hyperthermia as complementary treatment to surgery, chemo, radio, and immunotherapy in breast cancer patients [75,77,78,79].
However, convincing data about the benefit of the combination of hyperthermia with checkpoint inhibitors for breast cancer treatment should be provided by multicenter clinical trials in which related side-effects are also evaluated .
Likewise, radiation has also shown to increase mutational load of tumors, optimize antigen presentation, and decrease immune suppressors in the tumor microenvironment, priming the tumor for immunotherapy , which justifies additional studies in these fields.
Besides the presence of high tumor infiltrating lymphocytes, recognition of tumor cells is a critical step for the success of the immune response. In this regard, although estrogen has an immunoenhancing impact on the immune system  with an apparent effect in all major innate and adaptive immune cells , high levels of estrogens may interfere with HLA-II expression and IFN-γ signaling, with significant implications for tumor immune escape .
Estrogens are also well known to be a risk factor for breast cancer by enhancing the expression of genes involved in tumor cell survival and proliferation as well as growth factors including vascular endothelial growth factor (VEGF) , epidermal growth factor (EGF), insulin growth factor (IGF), fibroblast growth factor (FGF) [69,84], and their receptors .
Since estrogen presence in tumor microenvironment can also play a significant immunosuppressive role by promoting tolerance of weakly immunogenic tumor cells , the use of antiestrogen therapies in combination with aromatase inhibitors could be a rational strategy to enhance the response to immunotherapies.
However, although adjuvant hormonal therapy combined with HER2-targeted agents in hormone receptor-positive and HER2-positive breast cancer patients already represents a standard treatment, recent studies have shown that estrogen deprivation promotes transcriptional programs that favor immune evasion and increases PD-L1 expression in metastasis arising from breast cancer patients receiving adjuvant hormonal therapy for their local disease .
For this reason, the use of hormone-therapies in combination with PD-1/PD-L1 blocking immunotherapies should be thoroughly investigated.
On the other hand, and for the reasons mentioned above, the application of conventional monoclonal antibodies targeting one or more growth factors would be a useful adjuvant to enhance the efficacy of breast cancer immunotherapy by improving APCs function [86,87].
Changes in Breast Tumor Cells
Instead of loss of the targeted protein, resistance to cancer immunotherapies, such as monoclonal antibodies, is frequently due to the activation of alternate pathways  like immunosuppressive checkpoint pathways.
Among these, and largely due to the FDA approval of atezolizumab, blockade of the PD-1/PD-L1 pathway constitutes one of the most promising strategies for breast cancer immunotherapy.
Despite this important addition to the number of therapeutic options available for metastatic breast cancer patients, it is important to note that the objective response rate achieved by atezolizumab was 53% versus 33% for the placebo group  and that to date, it has been approved for the treatment of the triple-negative subtype, which only constitutes 10–15% of breast carcinomas , with positive PD-L1 protein expression, which occurs in approximately 20% of breast cancers (mainly HER2-positive and triple negative) .
Similarly to the PD-L1 pathway, different randomized clinical trials are currently evaluating the effect of PD-1 inhibitors as monotherapy or in combination with conventional and non-conventional treatments [62,65] in breast cancer patients with results that although modest, are encouraging.
In this respect, even though PD-L1 status remains the core predictor for anti-PD-1/PD-L1 therapies and patient selection , the validity of PD-L1 expression as a prognostic marker remains controversial  and justifies the need to develop new immunotherapy biomarker panels as well as new strategies to improve response rates.
Another major impediment to immunotherapy success in breast cancer patients is the selection of apoptosis-resistant cells, which constitutes one of the hallmarks of cancer .
Since both chemo- and immunotherapies directly or indirectly activate the cellular apoptosis machinery, tumor sensitivity to anti-cancer treatments will significantly depend on the level of expression of anti-apoptotic proteins  in general and, more specifically, on the existence of a pro-survival profile characterized by an increased ratio between anti-/pro-apoptotic proteins [24,90,91].
Provided that antiapoptotic proteins such as clusterin (APO-J) [91,92], BCL-2, BMF  as well as different pro-survival kinases  are frequently altered in metastatic breast cancer, the use of profiling techniques or systematic mapping of anti-apoptotic gene dependencies would be justified in order to effectively select those patients that could better benefit from combined treatments of protein inhibitors and immunotherapy.
In this regard, different studies have already evidenced the need to use therapies with a combination of inhibitors targeting different anti-apoptotic proteins in order to achieve better clinical benefits and avoid the activation of alternate pro-survival pathways [8,23,24].
HLA-I expression on the surface of breast tumor cells, which is positively correlated with tumor-infiltrating lymphocytes, is essential for an effective cytotoxic response  and the subsequent success of T-cell mediated immunotherapies.
For this reason, loss or changes in HLA-I expression, which is another of the hallmarks of cancer , also represent a significant impediment for breast cancer immunotherapy.
Total loss of HLA-I is found in 37% of in situ breast carcinomas, 43% of the primary tumors, and 70% of the lymph node metastases . Since HLA-I expression in these tumors is related with a pro-death phenotype characterized by an increased proapoptotic BAX/antiapoptotic BCL2 ratio , preliminary studies for patients’ selection would be justified in order to ensure the success of immunotherapies in breast cancer patients.
In the case of triple negative breast tumors, HLA-I expression is variable, contributing when altered to the development of an immunosuppressive tumor microenvironment and immune escape .
However, since the activation of the HLA-II presentation pathway occurs in approximately 30% of triple negative breast cancer patients , associated with the presence of tumor infiltrating lymphocytes and improved prognosis [95,96], the expression of both receptors are factors that must be taken into consideration prior to the application of immunotherapy treatment.
With respect to HER2 overexpressing tumors, although this receptor-tyrosine kinase represents a valuable target for T-cell based immunotherapies, these tumors may escape cytotoxic T lymphocyte-mediated lysis by downregulating HLA-I, since the expression of both receptors is inversely correlated with breast cancer cells [69,97].
Similarly, in normal and cancerous breast tissues, HLA-I expression is inversely correlated with the expression of estrogen receptors, which may be related to the low level of tumor-infiltrating lymphocytes , and hence, with the failure of the T-cell cytotoxic response.
It is worth emphasizing at this point that provided that agents targeting different protein kinases such as Mitogen Activated Protein Kinase (MAPK) or HER2 may increase HLA-I expression in breast cancer cells [97,98], the use of kinase inhibitors would be a valuable strategy to increase the antitumor effects of T-cell based immunotherapies.
Similarly, strategies aimed at inducing HLA-II expression in tumor cells may be valuable tools to increase patient response and prognosis to such therapies .
Reference link : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6966503/
More information: Andrea Castro et al, Strength of immune selection in tumors varies with sex and age, Nature Communications (2020). DOI: 10.1038/s41467-020-17981-0