Abstract

Introduction

The immune system may play a pivotal role in mediating long-term survival in women diagnosed with early stage breast cancer. The benefits of conventional breast cancer therapies such as chemotherapy (1), human epidermal growth factor receptor 2 (HER2)-targeted therapy (2–4), and radiotherapy (5) are at least partially dependent on an adaptive immune response against the cancer. The response to chemotherapy and overall survival can be predicted by the quantity of tumor infiltrating lymphocytes (TILs) in tumor biopsies (6). However, not all breast cancers appear to trigger an antitumor immune response. Some breast tumors are densely infiltrated by lymphocytes, whereas most are devoid of lymphocytes by routine H&E staining. Tumors with abundant lymphocytes grow despite immune engagement, whereas tumors with few or no lymphocytes grow in absence of immune engagement. Patients with “TIL-low” tumors, also described as “immunologically ignorant”(7) or “non-inflamed” tumors (8), are more prone to chemotherapy resistance (lower pathologic complete response rates to neoadjuvant therapy) and poor overall survival (6, 9, 10), suggesting that endogenous immune engagement may be a key survival determinant.

Immune therapies have the potential to facilitate de novo immune engagement when an endogenous immune response has not occurred. To achieve this, an optimal immune therapy would facilitate four critical components of a de novo adaptive response: tumor antigen release, tumor antigen presentation, diminished immune suppression, and tumor antigen–specific T-cell activation. The first components, tumor antigen release and presentation, can be facilitated by tumor cryoablation (cryo, using an image-guided probe to lyse tumor cells). The latter components, T-cell activation and T-regulatory cell depletion, can be induced by a therapeutic antibody against the T-cell co-inhibitory molecule CTLA-4 (cytotoxic T-lymphocytic antigen 4) (11, 12). In mice, the combination of these two immunotherapies, coined “cryo-immunotherapy”, generated intratumoral T-cell expansion specific to tumor-associated antigens (TAAs), systemic tumor regression, and improved overall survival (11).

During cryo, the tumor is treated with repetitive freeze/thaw cycles, inducing crystallization, mechanical cellular damage, microvascular injury, ischemia, cell death, and massive protein release (13). Freezing is most efficient at the center of the probe, leading to cellular necrosis, release of inflammatory cellular contents, and activation of the innate immune system via activation of Toll-like receptor and other innate receptors. At cryoablation margins (lower isotherms), apoptotic cell death occurs, which is associated with phagocytosis and antigen presentation. Thus, cryotherapy may produce an optimal microenvironment for T-cell activation against a broad array of tumor-associated antigens.

Blocking antibodies to CTLA-4 activate T cells by binding and inhibiting CTLA-4, a T-cell surface protein that is upregulated with TCR signaling and limits T-cell activation. The CTLA-4 blocking antibody ipilimumab (ipi) improves overall survival in metastatic melanoma (14, 15) and has been approved by the United States Food and Drug Association for this condition. CTLA-4 blockade is thought to decrease the threshold required for T-cell activation following exposure to antigen (16), increase the quality and duration of T-cell activation, and deplete suppressive T-regulatory cells within the tumor microenvironment (17). However, immune checkpoint antibody therapy alone may not be sufficient to induce an effective antitumor immune response in all tumors. In metastatic melanomas treated with pembrolizumab, another immune checkpoint antibody against programmed death 1 (PD-1), tumors with low TILs were unlikely to respond to therapy, whereas tumors with robust TILs were more likely to respond (18). Therefore, especially in TIL-low tumors, a combination approach may increase likelihood of mounting an effective antitumor immune response.

In a post-hoc analysis of a recent study (19) evaluating cryo-immunotherapy in early stage breast cancer, we sought to determine whether cryo-immunotherapy induced broad and robust T-cell activation and proliferation within the tumor microenvironment. Because breast cancer core biopsies are small and less conducive to comprehensive immunologic profiling by flow cytometry, and because routine histologic or immunohistochemical quantitation of T-cells is difficult when tumor microarchitecture is disrupted by cryo, we used a novel high-throughput TCR β (TCRB) DNA sequencing technique capable of quantifying T cells from DNA extracted from a small (25 micron) tumor sample.

TCRB DNA sequencing can be used to quantify expansion of individual clones, which may serve as a surrogate marker for therapy-associated induction of antigen-specific T-cell activation. During T-cell development in the thymus, the complementarity-determining region 3 (CDR3) region of the TCRB gene undergoes recombination and nucleotide addition/deletion steps to generate a vast pool of T-cell clones, each with a unique TCR conferring antigen specificity. If a T-cell binds its cognate antigen and becomes activated, it undergoes clonal proliferation, allowing for an adaptive immune response against the target antigen.

In this study, TCRB DNA sequencing was performed on pre- and post-cryo-immunotherapy human breast cancer specimens to study the effects of cryo-immunotherapy on T-cells in the tumor microenvironment. The intent was two-fold: to evaluate TCRB sequencing as a novel biomarker in early stage breast cancer, and to provide proof-of-concept that TCRB sequencing may be used to monitor the intratumoral and peripheral blood effects of immunotherapy in a pre-operative early stage breast cancer trial, highlighting analytical limitations as well as opportunities to integrate TCRB sequencing into immunotherapy clinical trials.

Methods

Clinical Trial Design

In a pilot study conducted at Memorial Sloan Kettering Cancer Center (MSKCC) between 2012–2013 (MSKCC IRB#11-202, NCT01502592), 18 women with operable early stage breast cancer who had elected mastectomy were treated with: cryo alone (group A, n = 6), single-dose ipi 10mg/kg IV alone (group B, n = 6), or combination cryo plus ipi 10mg/kg IV (group C, n = 6) (Table 1 and Fig. 1). In one subject from group A, a cryo probe malfunction occurred resulting in incomplete tumor freezing; this subject was replaced, however the baseline core biopsy data (which was unaffected by the probe malfunction) was included in the exploratory biomarker analyses (group A total, n = 7).

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Figure 1

Study design.

Table 1

Tumor characteristics and TCR deep sequencing metrics, by patient

The reported metrics describe the research core biopsy, obtained at the time of cryoablation (arms A&C) and shortly after ipilimumab (arms B&C). Due to limited specimen availability, experiments were only repeated when sequencing did not meet quality control standards (refer to Methods)

Group	ID	Subtype	Diff	Days duration between		H&E	Bx T-cell Receptor Deep Sequencing Metrics			Density	Clonality
				Ipi➔Bx	Bx➔Surgery	TIL Score (%)	Sample Source	Absolute # Sequences	# Unique Sequences
Cryoablation	1	HR+	mod		10	<5	FFPE	2193838	16895	30	0.11
	2	HR+	mod		8	<5	Frozen	1209573	7909	7	0.20
	3	HR+	poor		8	<5	Frozen	1453469	14020	5	0.12
	4	HR+	mod		8	85	FFPE	867803	49283	24	0.23
	5*	HR+	mod		9	<5	FFPE	652336	2974	5	0.13
	6	HR+	well		7	n/a	Frozen	55054	647	2	0.12
	7	HR+	well		7	<1	FFPE	82343	3467	5	0.12
Ipilimumab	8	HER2+	poor	8	4	80	FFPE	5278898	67770	19	0.14
	9	TNBC	poor	1	7	<5	FFPE	205971	755	3	0.13
	10	HR+	well	3	7	<5	Frozen	694364	2651	0	0.41
	11	HR+	poor	5	8	n/a	FFPE	164476	697	2	0.17
	12	HER2+	poor	1	7	40	FFPE	8916191	33101	16	0.23
	13	TNBC	poor	1	8	10	FFPE	368214	13966	15	0.11
Cryoablation + Ipilimumab	14	HR+	mod	3	8	<5	FFPE	2714728	11751	6	0.15
	15	TNBC	poor	1	7	40	FFPE	3479585	14414	10	0.16
	16	HR+	poor	2	6	5	FFPE	235779	9486	13	0.13
	17	HR+	poor	4	7	30	FFPE	429163	12035	7	0.14
	18	HR+	mod	2	10	<5	FFPE	1397054	5412	5	0.12
	19	HR+	mod	5	7	<5	Frozen	583965	7382	4	0.10
median				2.5	7	<5		781084	10619	6	0.14

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^*In patient 5, cryoablation probe malfunction precluded interpretation of therapy effect, however the core biopsy was still evaluable.

Legend: Diff- differentiation; Ipi- ipilimumab; Bx- biopsy; n/a- not available for analysis

The treatment schedule was designed to optimize antigen exposure time without disrupting the average 2-week lead time between diagnosis and standard-of-care mastectomy. Following consent, ipi (groups B & C) was administered a median of 2.5 days (range 1–8) prior to core biopsy, cryo (groups A & C) was administered at the time of core biopsy, and mastectomy was performed a median of 7 days (range 4–10) following core biopsy. In all subjects, core biopsy and mastectomy specimens were formalin-fixed and paraffin embedded (FFPE), and cryopreserved if additional tumor was available. Peripheral blood mononuclear cells (PBMCs) collected on the day of core biopsy and mastectomy date were cryopreserved.

Results

TCRB sequencing measures T-cell infiltration in early-stage breast cancer biopsies

In previous series of early stage breast cancer patients, TIL counts as measured by routine H&E staining are non-normally distributed, with most tumors exhibiting sparse or no TILs (as measured by ≤10% of stroma, 69% of triple negative tumors in a recent report), and the minority of tumors exhibiting dense TILs (comprising upwards of 80% of the stromal tissue examined). (21) In our dataset using the standardized H&E TIL scoring methodology proposed by the San Antonio TIL Working group, we found the same non-normal distribution (Table 1), with the majority of tumors having TIL count ≤ 10% (6).

As an alternative to H&E, we characterized the degree of T-cell infiltrates in core biopsies using two TCRB sequencing methodologies. The first methodology, absolute T-cell count, is a direct measure of the number of detectable T-cell DNA sequences within the specimen. The second methodology, T-cell density, is a relative measure of T-cell DNA relative to total DNA (including tumor and stromal DNA), which may be more stable to variations in sequencing depth. In our series, both absolute T-cell count and T-cell density exhibited a non-normal distribution similar to H&E TIL count. Absolute T-cell sequence counts ranged from 5.5 × 10⁴ to 8.9 × 10⁶ sequences per sample (mean 1.98 × 10⁶, standard deviation, SD, 2.68 × 10⁶), and T-cell densities ranged from 0 to 30% (mean 5.7%, SD 8.1%). T-cell density was significantly correlated with TIL count by H&E (r = 0.64, P = 0.007). There was a trend towards correlation between absolute T-cell count and H&E (r = 0.46, P = 0.06).

We conducted TCRB sequencing of normal adjacent breast tissue specimens (at least 3cm away from tumor) obtained during mastectomy. In subjects who did not receive ipi (i.e. patients undergoing cryo alone, n = 7), the T-cell densities and absolute T-cell counts of normal breast tissue were lower and more uniformly distributed compared to tumor (mean/SD T-cell density: tumor, 5.7%/8.1%; normal, 2.3%/1.1%, P = 0.002). In subjects who received ipi (i.e., ipi alone and ipi/cryo arms), normal breast tissue appeared to have more dense and less uniformly distributed T-cell infiltrates, similar to the tumoral specimens (mean/SD T-cell density: tumor 7.9%/8.5%). Samples with few or no apparent TILs by H&E (≤ 5%) exhibited a median of 301,603 T-cell sequence counts by TCRB sequencing (range: 42,126 to 3.6 × 10⁶).

The small size of this study precluded definitive assessment of the influence of tumor/patient characteristics on core biopsy T-cell infiltrate quantity. However, Fig. 2 illustrates the potential influence of various factors on baseline T-cell infiltrate quantity. Both tumor differentiation and tumor subtype appeared to influence T-cell infiltrate quantity, with higher grade lesions and TNBC or HER2⁺ lesions having denser infiltrates. Ipi was administered prior to the core biopsy (median 2.5 days prior), and hypothetically this may have influenced T-cell infiltrates in this biopsy. However, subjects who received ipi more than 3 days prior to biopsy (n = 6) did not demonstrate appreciable differences in infiltrate quantity compared to subjects not receiving ipi or receiving ipi within 2 days of biopsy (Fig. 2).

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Figure 2

Influence of patient and tumor characteristics on core biopsy T-cell infiltrates

T-cell density and clonality are assessed by TCR DNA sequencing, with group medians depicted as solid lines. Unless specified, differences across groups are not statistically significant. T-cell density in untreated normal breast tissue was lower compared to tumor; HER2+ and TNBC subtypes appear to have denser infiltrates compared to luminal-type (i.e. HR⁺/HER2⁻); tumor grade is associated with T-cell density; age is not associated with T-cell density or clonality; and subjects receiving ipi greater than 3 days preceding biopsy did not have appreciable differences in T-cell density or clonality compared to subjects not receiving ipi, or receiving ipi within 0–2 days. HER2⁺, human epidermal growth factor receptor 2 positive; TNBC, triple negative breast cancer; mod, moderate; Ipi- ipilimumab.

Induction of T-cell polyclonality and clonal repertoire remodeling

TCR sequencing can also assess the clonal nature of infiltrating T-cells, i.e. whether the infiltrates are comprised of a few high-frequency clones (oligoclonal) versus many unique low-frequency clones (polyclonal). In our dataset, the number of unique T-cell clonal populations within a sample correlated with the absolute T-cell count (r=0.76, P = 0.0002). To assess clonal diversity independent of this observed correlation, we calculated the Shannon entropy of the specimen and normalized it to the number of unique clones within the sample. This previously described “clonality” metric ranges from 0 to 1 and is relatively insensitive to sample size, with 1 representing a purely monoclonal population, versus 0 representing a purely polyclonal population (22). Using this metric, clonality of the core biopsy specimens ranged from 0.10 to 0.41 (mean 0.16, SD 0.07), which was similar in degree and variation to normal breast tissue T-cell clonality (mean 0.18, SD 0.07). In this small dataset, T-cell clonality did not appear to be associated with subject age, tumor pathologic grade, or HR/HER2 subtype (Fig. 2).

To determine the intratumoral effects of cryo and/or single-dose ipi, we conducted T-cell repertoire analysis on matched core biopsies and mastectomies. Effects on absolute count varied widely within treatment groups (data not shown). Cryo decreased T-cell density in the majority (5/6) of subjects, conversely ipi alone increased T-cell density in the majority (5/6) of subjects, whereas no trend was observed following combination therapy (Fig. 3). We then evaluated the effect of therapy on clonality. 100% of cryoablated tumors (n = 12), irrespective of ipi, experienced a shift towards polyclonality (median clonality change: −.04, P = 0.005), whereas no trend in clonality was observed following ipi alone. These findings are graphically depicted in figure 3, which illustrates the change in T-cell clonality (x-axis) and the change in T-cell density (y-axis) for each patient, according to treatment arm (Fig. 3).

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Figure 3

Effect of therapy on intratumoral T-cell density and clonality by TCR deep sequencing

T-cell density and clonality are depicted as fold-change, comparing core biopsy to mastectomy. The x-axis depicts fold-change in T-cell clonality, with positive values indicating a shift towards oligoclonal T-cell infiltrates; the y-axis depicts fold-change in T-cell density, with positive values indicating a shift towards denser T-cell infiltrates. Each line/dot demonstrates an individual patient treated on protocol, and numbers indicate patient identifier (as listed in table 1).

Next, we evaluated whether ipi and/or cryo remodels the clonal repertoire, i.e., whether new clones are introduced or the distribution of clones is modified. Morisita’s overlap is a previously described (23) metric that measures the proportion of shared T-cell sequencing reads over 2 samples, normalizing for biases introduced by sample size. This overlap metric ranges from 0 to 1, with low overlap scores indicating repertoire remodeling and potential influx of novel T-cell clones. Relative to the ipi alone group, we found a trend of lower Morisita’s overlap in cryo-treated groups (P = 0.08), suggesting greater remodeling of the intratumoral clonal repertoire following cryo. In summary, these findings suggest that ipi alone expands intratumoral lymphocytes, cryo alone contracts intratumoral lymphocytes and induces polyclonality and repertoire remodeling, and the combination induces polyclonality and repertoire remodeling with less lymphocytic contraction.

Induction of high-magnitude intratumoral T-cell clonal expansions

Certain T-cell clones, when exposed to released antigens following cryo, might expand more robustly with the addition of ipi. Therefore, we calculated the pre-/post-treatment change in abundance of each individual T-cell clone found in either the core biopsy or mastectomy specimen. We then quantified the number of clones that expanded with therapy by certain absolute sequence count thresholds, and normalized this number by the total clone count. Using this technique with the threshold set to ≥1 copy (thus measuring the proportion of clones expanding by any magnitude) we found that the greatest percentage of T-cell clones expanded with ipi alone (median % clones expanding: 18% or 4454 clones, cryo; 62% or 41399 clones, ipi, 36% or 4307 clones, cryo+ipi). However, if we repeated this with the threshold set to 10, we found that the greatest percentage of T-cells expanded with cryo plus ipi (median % clones expanding: 14% or 2822 clones, cryo; 23% or 11322 clones, ipi, 31% or 3658 clones, cryo+ipi). At higher thresholds (10² or 10³), the differences appear more pronounced (Fig. 4), indicating that more high-magnitude T-cell expansions were observed in the tumors treated with combination ipi plus cryo, relative to monotherapy.

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Figure 4

Effect of therapy on clonal expansion

Each graph depicts the proportion of intratumoral T-cell clones expanding by the specified threshold (i.e. expansion by ≥1 copy, >10 copies, >100 copies, or >1000 copies), comparing core biopsy to mastectomy. Each solid line represents an individual patient, with dotted lines representing group medians. Legend: Cryocryoablation; Ipi-ipilimumab.

Independent intratumoral and peripheral TCR repertoire landscapes

Assuming that TILs are exposed to tumor-associated antigens and therefore enriched for tumor-reactive T-cell clones, the more contracted intratumoral clonal repertoire could be used to identify and monitor tumor-reactive clones within the peripheral blood. We tested the hypothesis that intratumoral T-cell expansions might be mirrored in peripheral blood by categorizing intratumoral clonotypes into groups by degree of intratumoral expansion, using the previously defined thresholds (>10 copies, >100 copies, and >10³ copies). We then evaluated the degree of expansion of each of these clonotype groups within the peripheral blood over time, comparing baseline to 30-day post-treatment followup. Most of the clones expanding within the tumor were undetectable in the peripheral blood, either before or after therapy. Regardless of the degree of intratumoral clonal expansion, only a low percentage of clonotypes were also expanding in the peripheral blood, and was not influenced by the degree of intratumoral expansion (median proportion of peripherally expanding clones across all groups: > 10 copies, 6%; > 100 copies, 7%).

We conducted graphical analyses of intratumoral versus peripheral blood clonotype expansion, plotting the absolute intratumoral expansion/contraction on the x-axis, versus peripheral expansion/contraction on the y-axis (Fig. 5). Using this technique, we found no correlation between intratumoral clonal expansion and peripheral blood clonal expansion; however, we observed several general trends across the three treatment groups. First, cryo alone appeared to induce intratumoral contraction of the majority of T-cell clones, with very little effect on peripheral blood clones (as represented by the asymmetry across the y-axis and symmetry across the x-axis). Next, ipi alone appeared to induce expansion of clonotypes both in the peripheral blood and in the tumor (as represented by a dominance of clones in the double-positive quadrant). Finally, combination ipi/cryo induced a mixed contraction and expansion of intratumoral clones, as represented by symmetry across the y-axis. Furthermore, the median degree of expansion with ipi/cryo was shifted rightward, indicating that a greater proportion of clones experienced high-magnitude expansions, compared to ipi alone.

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Figure 5

Relationship of intratumoral versus peripheral blood T-cell clonal expansion

Each graph represents the T-clonal repertoire of a representative patient of each of the three treatment groups. Each point represents a unique T-cell clone detectable in the core biopsy, mastectomy, and/or peripheral blood. The x-axis depicts the absolute change in intratumoral clonal frequency count, comparing core biopsy to mastectomy. The y-axis depicts the absolute change in peripheral blood clonal frequency count, comparing pretreatment) to post-treatment timepoints. For example, points in the upper right quadrant depict T-cell clones that have increased in frequency both within the tumor and in the peripheral blood.

Conclusion

The two goals of this post-hoc analysis were to explore the potential utility of TCRB sequencing as an immune-based biomarker in early stage breast cancer, and to characterize the effect of cryo-immunotherapy on intratumoral and peripheral blood T-cell proliferation. We first compared TCRB sequencing to the current gold-standard immune biomarker in early stage breast cancer, TIL count by H&E staining. Recently, a standardized method of assessing TILs by H&E has been developed, and was validated to be predictive and prognostic in triple negative breast cancer (6). Despite its utility and simplicity, the H&E TIL score provides only a semi-quantitative assessment on immune infiltration, and poorly discerns the highest-risk patients (with the highest risk low-TIL group exhibiting relapse rates of only 30% in a recent series) (21). TCRB sequencing is an attractive alternative because it quantifies T-cells (the immune cell type most attributable protective immunity) and characterizes T-cell clonal diversity, which might be useful to further stratify risk. In our dataset, we found that the T-cell density, a method of quantifying T-cells by TCRB sequencing, correlates with the gold standard TIL assessment by H&E. Furthermore, the distribution of TIL quantity (with most tumors having low T-cell density), and the association of T-cell density with poorly differentiated and HER2+/TNBC tumors, closely resembles the H&E TIL quantity metric (21, 24). One primary difference, however, is that TCR sequencing could quantify up to millions of T-cell DNA sequences even in samples with no TILs or low TILs by H&E. Some of these sequences may arise from non-tumor-bearing breast tissue or vasculature within the sequenced sample, nevertheless these data suggest that TCR sequencing could be a potentially more sensitive method of measuring intratumoral T-cells. Although not addressed by this dataset, it is conceivable that a more precise quantification of T-cells could be helpful in stratifying the highest-risk tumors amongst tumors with low TILs by H&E. The correlation between H&E TIL score and T-cell density was imperfect: discordant scores may be related to intratumoral heterogeneity, or the preponderance of non-T-cell lymphocytes in some specimens, which would result in a higher H&E TIL score but a lower T-cell density.

Our second goal was to demonstrate how TCRB sequencing could be used to characterize the effects of immunotherapy in serially collected intratumoral and PBMC specimens. We used two TCRB sequencing metrics, T-cell density and T-cell clonality, to characterize the effects of cryo and/or ipi over time in tumors. T-cell clonality describes whether T-cell populations are oligo-clonal (i.e. reacting to the one or a few antigens) or polyclonal (reacting to many different antigens). In one trial, melanomas with oligoclonal T-cell infiltrates were more likely to respond to immunotherapy with anti-PD-1 (pembrolizumab) (18), potentially because the dominant T-cell clones were tumor-specific and amenable to re-invigoration with anti-PD-1. In our dataset, cryo alone appeared to deplete T-cells but induced a polyclonal shift (Fig. 3). The observed decrease in T-cell density is consistent with previous observations that cryo induces thermal necrosis and apoptosis (of both tumors and TILs) (25). The polyclonal shift, and the low overlap between pre- and post-treatment clones, suggests that new T-cell clones are infiltrating the tumor bed, and are reactive to a broader variety of antigens compared to the pretreatment resident T-cells. The polyclonal shift, however, argues against the presence of dominant, high-magnitude clonal expansions associated with a robust response against the tumor.

In the ipi ± cryo arms, ipi was administered a median of 2.5 days before the research biopsy ± cryo, and surgical mastectomy was performed a median of 7 days thereafter. This provided a unique opportunity to assess serial changes following ipi ± cryo, with the limitation that early post-ipi changes (i.e., during the short window between ipi and biopsy) would not be captured in this analysis. Ipi appeared to expand tumor-infiltrating T-cells, which is consistent with previously described observations that immunologic checkpoint blockade could induce intratumoral T-cell expansion (7). Cryo+ipi had a mixed effect on T-cell density, suggesting that neither ipi nor cryo were dominant in their effects on T-cell density.

We hypothesized that cryo+ipi may generate more T-cell expansions relative to cryo alone, however, as illustrated in figure 3, cryo+ipi produced a polyclonal shift similar to cryo alone. Therefore, we more closely evaluated intratumoral clonal T-cell expansion by comparing the concentration of individual T-cell clones before and after therapy. By this approach, we observed that cryo+ipi was associated with a greater number of high-magnitude clonal expansions relative to monotherapy. If confirmed, this finding would suggest that cryo+ipi mediates rapid proliferation of small subset of clones. This analytic method of clonotype expansion was more useful in this dataset than the clonality metric, for discerning differences across treatment groups. Although clonal TIL expansion does not necessarily indicate an antitumor response, one would expect clonal TIL expansion in the setting of T-cell responses against tumor-associated antigens. Therefore, we argue that this metric may be a candidate biomarker for assessing therapy-associated clonal proliferation in pre-operative immunotherapy clinical trials.

We also evaluated the utility of serial TCRB sequencing of PBMCs. In our paired analysis of blood and tumor, we found an increase in the number of peripherally expanding clones associated with combination therapy, but found little evidence in support of an association of intratumoral clonal change with peripheral blood clonal change. One explanation is that intratumorally expanding T-cell clones are diluted in PBMCs by the more diverse and abundant peripheral blood clonal repertoire, and may be difficult to track serially over time in PBMCs in early stage disease. However, it is conceivable that in metastatic disease, intratumorally expanding T-cell clones expand peripherally after exposure to additional tumor antigen, and therefore may be easier to detect in the blood or in satellite metastases.

Because the analysis was post hoc with no explicit statistical analyses or adjustments for multiplicity, because the histologies of the treated tumors varied and were imbalanced across groups, and because the timing of ipi was heterogeneous, we are limited in our ability to generalize our characterization of breast cancer TILs. Also, our observations regarding the clonal effects of ipi ± cryo must be confirmed prospectively in a study with more standardization of variables such as biopsy timing and specimen processing. In such a trial, more information could be derived by characterizing T-cell repertoire changes across various subsets of T-cells (for example in effector T-cells, helper T-cells, and regulatory T-cells, which could be sorted by flow cytometry), or within draining lymph nodes, which might provide additional information regarding priming and expansion of T-cells following cryo-immunotherapy.

In metastatic clinical trials, immune checkpoint blockade with anti-PD-1/anti-PD-L1 is modestly effective across all metastatic breast cancer subtypes (26–29). Therefore, we anticipate heightened investigation of novel combination strategies as a means of improving efficacy. We also anticipate increasing utilization of pre-operative clinical trials, on the basis that serial analysis of breast cancer tissue may be inform drug development and understanding of underlying mechanism. In conclusion, we have demonstrated that TCRB sequencing is feasible to conduct in early stage breast cancer using small core biopsy specimens, and that the TCRB T-cell density metric correlates with the current gold-standard immune biomarker, TIL count by H&E. We also illustrated how multi-dimensional data obtained from TCRB sequencing could elucidate the effects of immunotherapy on TILs and PBMCs, and may be informative as a biomarker in pre-operative clinical trials.

Acknowledgments

Footnotes

References